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For staining of tissues on frozen sections, paraformaldehyde (4%) fixed eyeballs or lenses were cryoprotected with a PBS-buffered 30% sucrose and embedded in SAKURA Tissue-Tek optimum cutting temperature (O.C.T.) compound . The embedded tissues were sectioned at 8 µm. Slides were then incubated overnight at 4°C with the primary antibodies: GLI3 , GRK1 , GUCY2F , and PDE6B . Secondary antibodies and Hoechst were used to visualize the stained cells.
|
39007834_p10
|
39007834
|
Histology and Immunofluorescence
| 4.072417 |
biomedical
|
Study
|
[
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0.0003296334471087903,
0.00026129797333851457
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[
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0.0013658077223226428,
0.00048391983727924526
] |
en
| 0.999996 |
The mice were anesthetized by intraperitoneal injection of 0.1 mL of a 1% pentobarbital solution per 10 grams of mouse weight. Prior to optical coherence tomography measurements, tropicamide eye drops were applied to the mice's corneas to dilate the pupils. A 100 kHz speed full-range swept-source optical coherence tomography device was used to perform optical coherence tomography of the mouse eye. (TowardPi Medical Technology Co., Ltd., Beijing, China).
|
39007834_p11
|
39007834
|
OCT
| 4.032566 |
biomedical
|
Study
|
[
0.9995294809341431,
0.00032721649040468037,
0.0001433639699826017
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[
0.9934512376785278,
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0.0005479856044985354,
0.0002390342124272138
] |
en
| 0.999994 |
RNA-seq analysis was conducted to investigate the differential gene expression profiles in TgGli3Ki/Ki knockin mice retinas and lenses ( n = 4, TgGli3Ki/Ki versus normal). The experiment was conducted as described before. 36 The obtained differentially expressed genes were further subjected to functional enrichment analysis using gene ontology and pathway databases.
|
39007834_p12
|
39007834
|
RNA-Seq Analysis
| 4.028839 |
biomedical
|
Study
|
[
0.9995686411857605,
0.00020777835743501782,
0.0002235665888292715
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[
0.9995026588439941,
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en
| 0.999997 |
For the statistics in Figures 2 – 6 , unpaired t -tests were utilized to compare the data between normal and knockin mice. Values are presented as mean ± standard error of the mean (SEM). Statistical significance is indicated as follows: P < 0.1: *, P < 0.01: **, P < 0.001: ***, and P < 0.0001: ****. For the statistics in Figure 2 , we used the t -test to compare the data between normal and TgGli3Ki/Ki knockin mice at 4 weeks, 16 weeks, and 9 months separately. The P values obtained from multiple t -tests were adjusted using the Bonferroni correction. For the statistics in Figure 5 A, for each gene, we analyzed the difference between normal and TgGli3Ki/Ki data using ANOVA.
|
39007834_p13
|
39007834
|
Statistics
| 3.980188 |
biomedical
|
Study
|
[
0.9996558427810669,
0.00014965263835620135,
0.00019444756617303938
] |
[
0.9991521835327148,
0.0004511729348450899,
0.00034073813003487885,
0.000055916014389367774
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en
| 0.999996 |
The schematic description of the creation of the mouse model is provided in Figure 1 . The TgGli3Ki/Ki transgene consists of chicken Crybb1 promoter, 3X HA, mouse Gli3 Coding sequence, and 6X FLAG. It was inserted into an intergenic H11 locus on mouse chromosome 11. We chose this site because it has been confirmed that homozygous insertions into this locus are not predicted to disrupt any endogenous genes, and the resulting mice are completely healthy and fertile. 37 , 38 All the vectors were confirmed through restriction digestion and Sanger sequencing. The sequence of the sgRNA is: “CUGAGCCAACAGUGGUAGUA.” The homozygous TgGli3Ki/Ki knockin mice were constructed in GemPharmatech Co., Ltd (Nanjing, Jiangsu, China). Briefly, the Cas9 mRNA, sgRNA, and the Gli3-donor plasmid were co-injected into zygotes. Thereafter, the zygotes were transferred into the oviduct of pseudopregnant C57BL/6JGpt females at 0.5 dpc. In addition, F0 mice was birthed after approximately 19 to 21 days of transplantation, all the offspring of C57BL/6JGpt females (F0 mice) were identified by PCR and sequencing of tail DNA. Crossing positive F0 mice with wildtype mice were done to build up heterozygous mice. Finally, cross heterozygous mice were done to obtain homozygous mice. A total of 1225 embryos were injected during the model creation process, resulting in 287 pups, out of which 13 F0 positives were recommended for breeding, with a transplantation success rate of 23.4% and an F0 positive probability of 4.5%.
|
39007834_p14
|
39007834
|
The Construction of TgGli3Ki/Ki Lens-Specific Over-Expression Mice
| 4.230906 |
biomedical
|
Study
|
[
0.9994211196899414,
0.0003848489432130009,
0.0001940262591233477
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[
0.999169111251831,
0.0004289627249818295,
0.0002978464763145894,
0.00010404715430922806
] |
en
| 0.999996 |
Eyeballs and lenses were isolated from normal and TgGli3Ki/Ki mice at the age of 4 weeks, 16 weeks, and 9 months and the photographs were taken freshly. The size of the eyeballs and lenses was estimated using quadrille paper . The multiple t -tests were used to analyze the significant difference between normal and TgGli3Ki/Ki group. Both the whole eyes and the lenses of TgGli3Ki/Ki mice are significantly smaller than that of normal mice . Additionally, the eye size of TgGli3Ki/Ki mice increases with age; the lens size grows from 4 weeks to 16 weeks, but remains stable from 16 weeks to 9 months .
|
39007834_p15
|
39007834
|
The Morphological Characterization of TgGli3Ki/Ki Mice
| 4.064158 |
biomedical
|
Study
|
[
0.9995920062065125,
0.000162595504662022,
0.0002453539054840803
] |
[
0.9994372725486755,
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0.0002743951918091625,
0.00005083454379928298
] |
en
| 0.999997 |
To dynamically measure the axial length and lens depth, we used a full-range swept-source optical coherence tomography device (with an adapted arm for securing the platform for small animals) to conduct optical coherence tomography scans on the mice's eyes. As shown in Figure 3 , the axial length and lens depth in TgGli3Ki/Ki mice is significantly smaller than that in normal mice. Both the TgGli3Ki/Ki mice and the normal mice were 12 weeks old at the time of optical coherence tomography scan. From these optical coherence tomography images, we observed opacity in the lens, suggesting an early onset of cataract in TgGli3Ki/Ki mice.
|
39007834_p16
|
39007834
|
The Morphological Characterization of TgGli3Ki/Ki Mice
| 4.109403 |
biomedical
|
Study
|
[
0.9995623230934143,
0.000243765112827532,
0.00019380917365197092
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[
0.9994550347328186,
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0.00007124997500795871
] |
en
| 0.999998 |
Hematoxylin and eosin (H&E) staining of the eye slice from paraffin sectioning showed more clearly that, different from the normal one, the eye of Gli3 KI/KI mouse is smaller and contains a smaller lens . Moreover, in the TgGli3Ki/Ki eye, the iris is completely adhered to the cornea, which explains why this TgGli3Ki/Ki strain is blind. H&E staining images of more eyeballs were included in the Supplementary Figures S1 and S2 .
|
39007834_p17
|
39007834
|
The Morphological Characterization of TgGli3Ki/Ki Mice
| 3.905534 |
biomedical
|
Study
|
[
0.9995720982551575,
0.00016510581190232188,
0.0002627049689181149
] |
[
0.9945295453071594,
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0.0002526709868106991
] |
en
| 0.999997 |
To confirm the lens-specific expression of Gli3 , we quantified GLI3 at the RNA level using qPCR assay and at the protein level using Western blot and immunofluorescence staining. As shown in Figure 5 A, at the mRNA level, the expression of Gli3 and other four genes ( Ptch1 , Ccnd1 , Ccne1 , and Smo ) in the HH signaling pathway increased significantly in the lenses of TgGli3Ki/Ki mice than in the normal lenses. In contrast, in the results from the retina samples, only Gli3 showed a significant increase, albeit with a smaller fold change than in the lens samples. Figure 5 B demonstrated that, despite multiple nonspecific bands, GLI3 expression is notably higher in the lenses of TgGli3Ki/Ki mice than in normal mice. From the immunofluorescence staining of GLI3 antibody in the lens, we observed that the GLI3 proteins predominantly expressed in the nucleus of the lens in the TgGli3Ki/Ki mice . Both lens and retinal samples were obtained from the 12-week-old mice in both groups.
|
39007834_p18
|
39007834
|
The GLI3 Expression in TgGli3Ki/Ki Mice
| 4.141832 |
biomedical
|
Study
|
[
0.9995269775390625,
0.00026154794613830745,
0.00021152418048586696
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[
0.9995269775390625,
0.00016884486831258982,
0.00024026638129726052,
0.00006401792779797688
] |
en
| 0.999997 |
To investigate changes in transcriptional patterns across the whole genome, we conducted RNA-Seq analyses on lens and retina samples obtained from both TgGli3Ki/Ki and normal mice. This assay included four groups: lens of TgGli3Ki/Ki mice (GL), retina of TgGli3Ki/Ki mice (GR), lens of normal mice (NL), and retina of normal mice (NR). Each group contained four biological replicates, with each replicate representing a pool of three lenses or retinas. The gene expression data from all four groups was presented using heatmaps . We then compared differential expressed genes (DEGs) between GL and NL, where our analysis of enriched bioprocesses and signaling pathways of DEGs revealed the “phototransduction” pathway as having the highest enrichment factor . These results suggest that this pathway was activated in the lens of TgGli3Ki/Ki mice, possibly induced by the transcription factor GLI3.
|
39007834_p19
|
39007834
|
The Phototransduction Pathway Is Activated in the Lens of TgGli3Ki/Ki Mice
| 4.112289 |
biomedical
|
Study
|
[
0.9995260238647461,
0.00026527963927946985,
0.00020870924345217645
] |
[
0.9994999170303345,
0.00017169192142318934,
0.00026547390734776855,
0.00006287411815719679
] |
en
| 0.999998 |
To validate the results of the RNA-Seq analyses, we conducted qPCR to assess the RNA levels of five crucial genes ( Gucy2e , Gucy2f , Pde6a , Pde6b , and Grk1 ) in the phototransduction pathway. We examined these genes in lens and retina samples obtained from both TgGli3Ki/Ki and normal mice. All of these genes demonstrated significant upregulation in TgGli3Ki/Ki mice lenses; whereas only Gucy2e and Pde6a presented a significant increase in TgGli3Ki/Ki mice retinas, and the foldchange is much lower than that in the lens samples . Furthermore, we performed immunofluorescence staining of GRK1, GUCY2F, and PDE6B and confirmed an increase level of these markers in the lens nuclei of TgGli3Ki/Ki mice . Figure 7 displays the immunofluorescence images of the PDE6B staining. In addition, we analyzed the enriched pathway in the TgGli3Ki/Ki mice retinas and confirmed the activation of the “NF-kappa B pathway” in the retina samples, using a method similar to that used in analyzing the lens samples in Figure 6 . We provided these data in the Supplementary Figure S4 .
|
39007834_p20
|
39007834
|
The Phototransduction Pathway Is Activated in the Lens of TgGli3Ki/Ki Mice
| 4.129063 |
biomedical
|
Study
|
[
0.999498724937439,
0.0002805952972266823,
0.0002206580393249169
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[
0.9994958639144897,
0.00014387400005944073,
0.0002906786394305527,
0.00006955460412427783
] |
en
| 0.999996 |
Microphthalmia is a developmental defect that arises from genetic mutations or environmental factors during the initial 3 months of pregnancy. Approximately 11% of blind children are diagnosed with microphthalmia, yet there are presently no available treatments. 39 Given that roughly 85% of the protein-coding genes in mice share analogous functions with those in humans, and that ocular development follows a comparable pattern, mouse models have proven valuable in elucidating the mechanisms of the causal genes in human microphthalmia.
|
39007834_p21
|
39007834
|
Discussion
| 4.032402 |
biomedical
|
Study
|
[
0.9996166229248047,
0.0001791307149687782,
0.00020425632828846574
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[
0.912060022354126,
0.008883235976099968,
0.07863466441631317,
0.00042208548984490335
] |
en
| 0.999997 |
During human embryonic eye development, on the 25th day of gestation, the optic vesicle emerges from the neural tube. One end of the optic vesicle elongates to form the optic stalk, which eventually develops into the optic nerve. The other end folds inward to form the optic cup, giving rise to the retina, iris, and ciliary body. In contrast to the optic cup, lens cells are of surface ectoderm origin and differentiate into lens epithelium (the capsule) and lens fiber cells. The ocular lens is unique among organs due to its avascular nature, transparency, and relative isolation. These characteristics make the lens an excellent model for investigating fundamental developmental questions.
|
39007834_p22
|
39007834
|
Discussion
| 4.129289 |
biomedical
|
Study
|
[
0.9991771578788757,
0.0003821899590548128,
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[
0.7224341630935669,
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0.0014203989412635565
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en
| 0.999995 |
The HH signaling pathway plays a vital role in embryonic development and is conserved throughout evolution from invertebrates to vertebrates. GLI family zinc finger proteins are mediators of SHH signaling in vertebrates. Strong transcriptional activation ability of GLI3 is one of the key mechanisms of the SHH signaling. Previous studies showed that GLI3-R plays is essential for mouse eye development and governs eye development partially via controlling WNT/β-CATENIN signaling. 18
|
39007834_p23
|
39007834
|
Discussion
| 4.254228 |
biomedical
|
Study
|
[
0.9996961355209351,
0.00012416621029842645,
0.0001797677541617304
] |
[
0.994621753692627,
0.0016980632208287716,
0.0035317291039973497,
0.00014852586900815368
] |
en
| 0.999998 |
In the current study, our objective was to investigate the role of GLI3 in lens morphology. We generated a novel mouse model that expresses GLI3 in the lens in an ectopic manner. Our observations revealed significant reductions in both lens and overall eye size during eye development compared to normal counterparts. The TgGli3Ki/Ki mice did not produce measurable results during the assessment with an infrared photorefractor, as no signals were detected. In addition, the H&E staining images revealed total synechia of the iris, leading us to speculate that these mice may be blind. These findings suggest that abnormal GLI3 levels interfere with lens development, subsequently affecting overall eye development. We hypothesize that lens size plays a crucial role in determining overall eye size. At the transcriptional level, the overexpression of Gli3 led to substantial changes in gene expression profiles in both the lens and retina. Interestingly, we found that among the DEGs in the lens, those most enriched were related to the phototransduction pathway. This result is particularly intriguing as previous reports did not directly associate Gli3 with this pathway, and lens cells are not typically involved in the physiological process of phototransduction. In human diseases, mutations in GLI3 have been associated with various disorders, including Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS), preaxial polydactyly type IV, and postaxial polydactyly types A1 and B. Ocular hypertelorism, characterized by widely spaced eyes, is a commonly observed phenotype in these diseases. Our study suggests that mouse GLI3 affects multiple aspects of ocular development beyond just the manifestation of hypertelorism.
|
39007834_p24
|
39007834
|
Discussion
| 4.403918 |
biomedical
|
Study
|
[
0.9993129968643188,
0.0004811110266018659,
0.00020579503325279802
] |
[
0.9990265369415283,
0.000275448604952544,
0.0005390199366956949,
0.00015896395780146122
] |
en
| 0.999999 |
In this report, we introduce a novel mouse model for microphthalmia, which is characterized by the targeted overexpression of the Gli3 gene in the lens. This model, as a consistent homologous mutant line, displays uniform ocular features across individuals, making it an ideal model for investigating the underlying mechanisms and potential treatments for microphthalmia.
|
39007834_p25
|
39007834
|
Discussion
| 4.06467 |
biomedical
|
Study
|
[
0.9996352195739746,
0.00024021473655011505,
0.0001244877785211429
] |
[
0.9982117414474487,
0.001057046465575695,
0.0005538508994504809,
0.00017743496573530138
] |
en
| 0.999999 |
Cyclic nucleotide-gated (CNG) channels are nonselective cation channels that transduce sensory stimuli into electrical signals in visual and olfactory receptor cells . Six different genes encode CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3) . CNG channels function as homotetramers and heterotetramers in exogenous expression systems, but appear to be exclusively heterotetramers in vivo . They share the same membrane topology as voltage-gated channels, with six transmembrane segments, intracellular NH 2 and COOH termini, and sequence similarity in the pore-lining region . However, CNG channels are activated by the direct binding of the cyclic nucleotides cAMP and cGMP to a cyclic nucleotide-binding domain in the COOH-terminal region of each subunit, and not by voltage.
|
15738051_p0
|
15738051
|
INTRODUCTION
| 4.506366 |
biomedical
|
Study
|
[
0.999437153339386,
0.00029284183983691037,
0.000270025891950354
] |
[
0.9782243967056274,
0.006190388463437557,
0.015212583355605602,
0.0003726338327396661
] |
en
| 0.999997 |
Although both the cyclic nucleotide-binding domain and the ion-conducting pore in CNG channels have been structurally defined, based on their homology to ion channels whose structural elements have been solved, the mechanism by which the energy provided by cyclic nucleotide binding is converted into opening of the pore is not understood. A region linking the last membrane-spanning region (S6) to the cyclic nucleotide binding domain in the COOH terminus (the C-linker) has been shown to play an important role in channel gating. Previous experiments suggest that the C-linker controls coupling of ligand binding to channel gating, and that the movement of this region is closely coupled to channel activation .
|
15738051_p1
|
15738051
|
INTRODUCTION
| 4.493751 |
biomedical
|
Study
|
[
0.9994574189186096,
0.0003286399005446583,
0.00021396348893176764
] |
[
0.9937742948532104,
0.0009212561999447644,
0.005121349822729826,
0.00018308137077838182
] |
en
| 0.999996 |
Divalent transitional metal (Ni 2+ ) binding to the C-linker of CNG channels has been used to demonstrate that open-state specific intersubunit interactions occur between proximal C-linker regions . Given that amino acids coordinating Ni 2+ must be within ∼4 Å of each other , these experiments strongly suggest that at least part of the proximal C-linker region must be a site for intersubunit proximity. Furthermore, at several sites, Ni 2+ coordination occurs preferentially in the open state, providing a constraint on the proximity of this region in the open state. A later study using a histidine scan of the proximal C-linker of CNGA1 reported that stripes of sites separated by 50 degrees on an α-helix produced Ni 2+ potentiation or Ni 2+ inhibition . These results suggest that the C-linker region undergoes a rotational movement during channel activation. This rotation may initiate movement of S6 and pore opening.
|
15738051_p2
|
15738051
|
INTRODUCTION
| 4.402272 |
biomedical
|
Study
|
[
0.9993351101875305,
0.00037367199547588825,
0.00029110288596712053
] |
[
0.9989930987358093,
0.00034073065035045147,
0.0005665464559569955,
0.00009964999480871484
] |
en
| 0.999996 |
Hyperpolarization-activated, cyclic nucleotide-modulated channels (HCN) contain a C-linker region that is closely related to CNGA1 based on sequence similarity. The sequences of HCN2 and CNGA1 in the C-linker are 22% identical and 45% conserved overall . Recently, a crystal structure of the COOH-terminal region of the HCN2 channel was solved to 2.0 Å resolution . This structure includes the C-linker and the cyclic nucleotide-binding domain (CNBD) bound to cAMP. In this structure, four HCN2 COOH termini are associated to form a tetramer. The C-linker region consists of six α-helices, designated A' to F', which are separated by short loops . The C-linker regions also form a large interface of subunit–subunit interaction with 2,300 Å 2 of buried solvent-accessible surface area. In contrast, the CNBD regions show very little intersubunit contact. This structure suggests that the C-linker regions mediate the primary intersubunit interactions of the COOH termini of HCN2. Based on the high sequence similarity between the COOH termini of HCN2 and CNGA1 , the CNGA1 C-linker regions may have similar structural and functional importance.
|
15738051_p3
|
15738051
|
INTRODUCTION
| 4.741302 |
biomedical
|
Study
|
[
0.9987187385559082,
0.0008335281163454056,
0.00044782794429920614
] |
[
0.9947953820228577,
0.0012288247235119343,
0.0035246210172772408,
0.00045123579911887646
] |
en
| 0.999998 |
We constructed a model of the CNGA1 C-linker by threading the sequence of CNGA1 through the structure of HCN2 . In this model, four CNGA1 C-linkers are associated to form a tetramer. Surprisingly, the structural relationships between C-linker regions in this model are not compatible with previous experiments on functional channels. For example, in this model, the distances between 420H residues in adjacent and opposite subunits are 35.12 Å and 50.18 Å, respectively. These distances are too large to allow high-affinity Ni 2+ coordination, and raise the question of whether our homology model represents a meaningful structure of the CNG channel C-linker under physiological conditions.
|
15738051_p4
|
15738051
|
INTRODUCTION
| 4.30113 |
biomedical
|
Study
|
[
0.9994596838951111,
0.0002728192484937608,
0.00026747435913421214
] |
[
0.9989688396453857,
0.00043008217471651733,
0.0005068215541541576,
0.00009427077020518482
] |
en
| 0.999996 |
In this study, we explored the intersubunit proximity between the C-linker regions of CNGA1 in functional channels using a site-specific cysteine substitution method . We found that intersubunit disulfide bonds can be formed between the A' helices of the C-linker regions during channel activation, indicating that these regions are in close proximity. Under our experimental conditions, most of the intersubunit disulfide bonds in this region potentiated channel activation, reflected as an increased apparent affinity for cGMP. We mapped the residues that formed intersubunit disulfide bonds onto a homology model of CNGA1, based on the structure of HCN2. Our data are not compatible with a model in which the C-linker conformation represents that of the open state, and suggest that the conformation seen in the structure may in fact represent the closed state.
|
15738051_p5
|
15738051
|
INTRODUCTION
| 4.26114 |
biomedical
|
Study
|
[
0.9993720650672913,
0.0004016548627987504,
0.00022630694729741663
] |
[
0.9993256330490112,
0.0002191410312661901,
0.00035823130747303367,
0.00009702231909614056
] |
en
| 0.999996 |
We introduced individual cysteines into the amino acid positions from 417 to 424 in a cysteineless background CNGA1 channel in the pGEMHE vector according to previously published methods . In brief, fragments of cDNA were amplified in PCR using oligonucleotides synthesized to contain a cysteine mutation in combination with other wild-type oligonucleotides. The cDNA fragments were then cut with two different restriction enzymes to generate a cassette containing the mutation. The cassette was then ligated into the cysteineless CNGA1 cut with the same two restriction enzymes. After transformation of bacteria with the ligation product, single positive colonies were selected, and the entire region of the amplified cassette was sequenced to check for the mutation and ensure no second-site mutations. cDNAs of the new constructs were linearized with NheI and transcribed in vitro, using the T7 mMessage mMachine kit (Ambion). For all the mutants, the native histidine at the 420 amino acid position was mutated to glutamine to eliminate the potential modification of 420H in our oxidation experiments . Throughout the text, CNGA1 cysless refers to the cysteineless background CNGA1 channel, and other cysteine mutants are named by the position of the mutated amino acid followed with “C,” for example, 417C and 418C.
|
15738051_p6
|
15738051
|
Site-directed Mutagenesis
| 4.253051 |
biomedical
|
Study
|
[
0.9995402097702026,
0.00027018869877792895,
0.00018958533473778516
] |
[
0.9987092018127441,
0.0006416509859263897,
0.0005321655771695077,
0.00011699655442498624
] |
en
| 0.999998 |
Segments of ovary were removed from anesthetized Xenopus laevis . After gross mechanical isolation, oocytes were isolated by agitating the sections of ovary in a Ca 2+ -free collagenase (2.3 mg/ml) solution for up to 1.5 h. The cells were then rinsed and stored in frog Ringers solution at 14°C. Oocytes were generally injected with 40 nl cRNA solutions (∼1 μg/μl) within 2 d of harvest. Electrophysiological recordings were performed 2–10 d after injection.
|
15738051_p7
|
15738051
|
Heterologous Expression of Channels in Xenopus Oocytes
| 4.166467 |
biomedical
|
Study
|
[
0.9994392991065979,
0.0003340349649079144,
0.00022656038345303386
] |
[
0.9969894289970398,
0.0022895452566444874,
0.000575962767470628,
0.00014508847380056977
] |
en
| 0.999996 |
Inside-out membrane patches were obtained by using fire polished borosilicate pipettes (1.5 mM OD, 1.2 mM ID; Sutter Instrument Co.). Recordings were made using symmetrical NaCl/HEPES/EDTA solutions consisting of 130 mM NaCl, 3 mM HEPES, 0.2 mM EDTA (pH 7.2) with cGMP or cAMP added to the intracellular solution only. Copper (II) phenanthroline (Cu/P) was prepared as follows: a 5 mM 1,10-phenanthroline stock in dry ethanol and a 1.5 mM cupric sulfate stock in water were diluted to final concentrations of 5 μM phenanthroline and 1.5 μM cupric sulfate in a solution containing 130 mM NaCl and 3 mM HEPES (pH 7.2); 2 mM cGMP or 128 μM cGMP (as indicated in the text) were present in this solution as well. Cu/P solutions were used for up to 2 d after preparation. Cu/P was applied to the intracellular side of patches for 3–10 min, as indicated in the text and figure legends. Dithiothereitol (DTT) was made as a 1 M stock in the NaCl/HEPES/EDTA solution and diluted to a final concentration of 5 mM in the NaCl/HEPES/EDTA solution together with 64 μM cGMP. The DTT solution was made fresh daily.
|
15738051_p8
|
15738051
|
Electrophysiology
| 4.144338 |
biomedical
|
Study
|
[
0.9995384216308594,
0.0002736365713644773,
0.00018807625747285783
] |
[
0.9971745014190674,
0.0022047637030482292,
0.0004880263877566904,
0.00013272961950860918
] |
en
| 0.999999 |
For macroscopic current measurements, pipettes were polished to a resistance between 0.3 and 1 MΩ. Currents were low-pass filtered at 2 kHz and sampled at 10 kHz with an Axopatch 200B (Axon Instruments, Inc.). All recordings were made at room temperature. Data were acquired and analyzed with PULSE data acquisition software (Heka Elektronik) and were plotted and fitted using Igor Pro (Wavemetrics Inc.). All currents shown have had currents in the absence of cyclic nucleotide subtracted. All currents were measured at +100 mV. For each experiment, currents at different cGMP concentrations were measured, and we waited until the currents had stabilized before proceeding. This delay was necessary because of the “run-up” caused by dephosphorylation of the channel by endogenous patch-associated phosphatases . Smooth curves shown in dose–response relations are fits of the data to the Hill equation: I = I max ([cGMP] n /(K 1/2 n + [cGMP] n )). Data are reported as the mean ± SEM. Statistical significance was assayed with two-tailed Student's t tests.
|
15738051_p9
|
15738051
|
Electrophysiology
| 4.131506 |
biomedical
|
Study
|
[
0.9995476603507996,
0.0002809421275742352,
0.00017129025945905596
] |
[
0.9987753033638,
0.0006019939901307225,
0.0005323215737007558,
0.0000903278196346946
] |
en
| 0.999997 |
For single channel recordings, pipettes were polished to a resistance between 8 and 12 MΩ. Currents were low-pass filtered at 5 kHz (eight-pole Bessel) and sampled at 10 kHz with an EPC10 amplifier (Heka Elektronik). Patches were held at 0 mV and stepped to +50 mV for 1 s. The presence of single channels in the patches was confirmed by application of 2 mM cGMP. Open probability (P o ) was calculated from fits to the data with two Gaussians. The area under the curve representing open channels relative to the total area was taken as P o . Cu/P was applied to the intracellular side of the patches for 10 min for all single-channel experiments. Other solutions and software for data acquisition and analysis were the same as for the macroscopic current measurements.
|
15738051_p10
|
15738051
|
Electrophysiology
| 4.135889 |
biomedical
|
Study
|
[
0.9994971752166748,
0.00033323210664093494,
0.00016960559878498316
] |
[
0.9984394907951355,
0.0010663897264748812,
0.000384204467991367,
0.00010987588757416233
] |
en
| 0.999998 |
Fig. S1 shows that Ni 2+ both potentiates and inhibits 417H. cGMP dose–response curves without Ni 2+ (open circles) and with 1 μM Ni 2+ (filled circles). The smooth curves represent fits with the Hill equation with K 1/2 = 32.3 μM, Hill slope = 1.9 initially, and K 1/2 = 22 μM, Hill slope = 1.1 with 1 μM Ni 2+ . The mean K 1/2 for activation by cGMP decreased from 32 ± 5.7 μM in the absence of Ni 2+ to 17.4 ± 3.5 μM in the presence of Ni 2+ ( n = 3). The supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200409187/DC1 .
|
15738051_p11
|
15738051
|
Online Supplemental Material
| 4.120181 |
biomedical
|
Study
|
[
0.999534010887146,
0.00018359169189352542,
0.00028237185324542224
] |
[
0.9992002844810486,
0.0005336342146620154,
0.00020481384126469493,
0.00006135421426733956
] |
en
| 0.999997 |
We used disulfide bond formation between cysteine residues as a reporter of proximity between subunits. We introduced individual cysteines into different amino acid positions in the A' helix of the C-linker region of CNGA1 in the context of a cysteineless background (CNGA1 cysless ). Copper (II) phenanthroline (Cu/P) oxidation was used to promote disulfide bond formation. Using disulfide bonds as indicators of proximity has several advantages. For our mutant channels, which contain only one cysteine in each subunit, a disulfide bond can only be formed between cysteines from two different subunits; there is no ambiguity about whether the proximity is between two subunits or within a subunit. In addition, for a disulfide bond to form, the α carbons of the two cysteines must be within ∼7 Å of each other . This distance is much shorter than the distance between 420H in our homology model , and provides an important test of this model.
|
15738051_p12
|
15738051
|
Cu/P Induced Potentiation in Three of the Seven Cysteine Mutants in the A' Helix of the C-linker Region
| 4.229136 |
biomedical
|
Study
|
[
0.9993357062339783,
0.0003614562447182834,
0.0003028546925634146
] |
[
0.9993174076080322,
0.0003151848795823753,
0.0002845617418643087,
0.00008277886809082702
] |
en
| 0.999997 |
We substituted a cysteine for each of the amino acids from 417 to 424 individually in the CNGA1 cysless background. All the mutants were functional except 421C. Mutant channels were expressed in Xenopus oocytes and examined using the inside-out configuration of the patch-clamp technique. To ensure that introduction of each cysteine did not prevent formation of functional, cyclic nucleotide-activated channels, we first measured the dose–response relation for activation of the mutant channels by cGMP and calculated the K 1/2 ( Table I , K 1/2 initial; see materials and methods ), and also measured the fractional activation by a saturating concentration of the partial agonist cAMP . We next used Cu/P to induce disulfide bond formation in each of the cysteine mutants. Two general types of outcomes are possible. If the regions containing the cysteines move during gating, then a disulfide bond that locks two of these regions together would be expected to perturb gating. If relative movement of the region containing the cysteines does not occur as part of gating, then locking the regions together through disulfide bond formation is not expected to perturb function. A third possibility, that no disulfide bond forms, cannot be functionally distinguished from a scenario in which a disulfide bond forms but does not alter channel properties.
|
15738051_p13
|
15738051
|
Cu/P Induced Potentiation in Three of the Seven Cysteine Mutants in the A' Helix of the C-linker Region
| 4.262377 |
biomedical
|
Study
|
[
0.9994136095046997,
0.0003453846147749573,
0.00024102305178530514
] |
[
0.999267041683197,
0.00026439447537995875,
0.0003815945819951594,
0.00008704846550244838
] |
en
| 0.999995 |
After incubating the patches in Cu/P with 2 mM cGMP for 3–10 min, we remeasured the cGMP dose–response curve and fractional activation by cAMP . For mutants 419C, 422C, 423C, 424C, and the cysteineless channel CNGA1 cysless , Cu/P did not cause a significant change in the cGMP dose–response curve or of the fractional activation by cAMP . In contrast, for 417C, 418C, and 420C Cu/P significantly shifted the cGMP dose–response curve to the left , causing a decrease in K 1/2 ( Table I ). For these three cysteine mutants, the fractional activation by cAMP was also significantly increased . Although decreases in K 1/2 could arise from either a potentiation of gating or an increased affinity for binding cGMP, the increase in maximal activation by the partial agonist cAMP can arise only from potentiation of gating. Thus, Cu/P produced potentiation of gating in 417C, 418C, and 420C. Interestingly, for mutant 418C, Cu/P also decreased the maximal cGMP activated current , a phenomenon we will discuss later.
|
15738051_p14
|
15738051
|
Cu/P Induced Potentiation in Three of the Seven Cysteine Mutants in the A' Helix of the C-linker Region
| 4.228129 |
biomedical
|
Study
|
[
0.9993973970413208,
0.00031546212267130613,
0.00028717127861455083
] |
[
0.9994545578956604,
0.0002076512319035828,
0.000265024253167212,
0.00007285171886906028
] |
en
| 0.999997 |
Under our oxidation conditions, Cu/P is expected to induce formation of a disulfide bond between cysteines from two subunits. However, it is possible that Cu/P would overoxidize the cysteines into sulfinic, sulfenic, or sulfonic acid . One way to differentiate between such overoxidation and disulfide bond formation is to use the reducing agent DTT, which will reduce disulfide bonds but not overoxidized cysteines . Fig. 4 shows examples of experiments with 417C, in which DTT was applied after Cu/P treatment. For 417C, the cGMP dose–response curve was measured initially, after Cu/P treatment, and after DTT treatment . Cu/P potentiated 417C function, shifting the cGMP dose–response curve to the left. Subsequent application of 5 mM DTT for 10 min to the intracellular side of the patch substantially reversed the Cu/P effect, shifting the dose–response relation for activation by cGMP back to the right.
|
15738051_p15
|
15738051
|
Cu/P Effect Can Be Reversed by DTT
| 4.16717 |
biomedical
|
Study
|
[
0.999466598033905,
0.0002619008009787649,
0.0002715658047236502
] |
[
0.9994433522224426,
0.00029816103051416576,
0.0001973509497474879,
0.0000611913637840189
] |
en
| 0.999998 |
We also measured the time course of disulfide bond reduction by DTT. An example of the time course is shown in Fig. 4 B. For these experiments, we measured a saturating concentration of cGMP (2 mM) as well as a subsaturating concentration (either 1 or 2 μM). As described above, Cu/P produced a large increase in the current activated by the subsaturating concentration of cGMP . Treatment with 5 mM DTT reversed this potentiation. For four patches examined, the mean time constant for reversal was 37.3 s (± 6.0 s), and the ratio of the current after recovery to the initial current (ratio of the current at t = 180s to the initial current) was 1.1 (±0.12). These experiments indicate that Cu/P perturbed channel function by inducing formation of disulfide bonds between cysteines from different subunits. Our experiments therefore suggest that the A' helices of the C-linker region must be in very close proximity. Furthermore, the shift in the K 1/2 toward lower cGMP concentrations and the increased fractional activation by cAMP indicate that disulfide bond formation stabilized the open state of the channels relative to the closed state. The simplest interpretation of this potentiation of gating is that the intersubunit proximity caught by disulfide bond formation reflects open state proximity.
|
15738051_p16
|
15738051
|
Cu/P Effect Can Be Reversed by DTT
| 4.35577 |
biomedical
|
Study
|
[
0.9993165731430054,
0.0004929500864818692,
0.0001905344397528097
] |
[
0.9989601373672485,
0.00039650543476454914,
0.000482365139760077,
0.00016088997654151171
] |
en
| 0.999997 |
We next asked whether the combination of potentiation and inhibition observed at position 418 was an artifact of disulfide bond formation. We wondered if a mixture of potentiation and inhibition could be observed with Ni 2+ as well. When we applied 1 μM Ni 2+ to 417H channels, we observed both a reduction in the current activated by high concentrations of cGMP and an increase in the current activated by quite low concentrations of cGMP , in contrast to previous reports . This finding suggests that the dual effect of potentiation and inhibition is not unique to Cu/P modulation, but can be observed with Ni 2+ binding as well.
|
15738051_p17
|
15738051
|
Potentiation and Inhibition by Cu/P
| 4.123359 |
biomedical
|
Study
|
[
0.99950110912323,
0.00025231766630895436,
0.00024646540987305343
] |
[
0.9994052648544312,
0.0002884817367885262,
0.0002482115232851356,
0.00005813855386804789
] |
en
| 0.999998 |
In our experiments, Cu/P treatment of 418C potentiated gating, as measured by low concentrations of cGMP and by the fractional activation by cAMP, but also decreased the maximal current activated by cGMP. Does disulfide bond formation at 418C produce potentiation or inhibition? This is an important point, because we interpret potentiation as open-state specific proximity and inhibition as closed-state specific proximity, two mutually exclusive possibilities.
|
15738051_p18
|
15738051
|
Potentiation and Inhibition by Cu/P
| 4.163808 |
biomedical
|
Study
|
[
0.9995476603507996,
0.0001877396716736257,
0.000264565838733688
] |
[
0.9983716607093811,
0.0012194670271128416,
0.0003178584447596222,
0.00009096086432691664
] |
en
| 0.999996 |
There are two general ways in which both potentiation and inhibition could be observed. Both potentiation and inhibition could be present in every channel, with each channel behaving in the same way. Alternatively, Cu/P could produce two populations of channels, each showing only potentiation or only inhibition. One simple way for each channel to show both inhibition and potentiation is if an intersubunit disulfide bond between 418C subunits causes a decrease in channel conductance (the decrease in the maximal current), but an increase in channel open probability (the potentiation at subsaturating cGMP). The proximal C-linker region is at the end of S6, a likely location of the channel gate . It is conceivable that a conformational change in this region induced by disulfide bond formation could cause changes in both ion permeation and in gating. The second general way to produce potentiation and inhibition is if two populations of channels are present, one with a decreased open probability, and the other with an increased open probability. We ought to be able to distinguish between these two models using single-channel recording.
|
15738051_p19
|
15738051
|
Potentiation and Inhibition by Cu/P
| 4.272862 |
biomedical
|
Study
|
[
0.9994376301765442,
0.0002899258688557893,
0.0002724588557612151
] |
[
0.9984592199325562,
0.0008554289233870804,
0.0005909096798859537,
0.00009444263559998944
] |
en
| 0.999998 |
We recorded the activity of single 418C and CNGA1 cysless (as a control) channels before and after treatment with Cu/P , and plotted all-points histograms to determine their open probability . In each experiment, Cu/P was applied for 10 min, and currents were recorded at +50 mV with a low cGMP concentration . The single channel conductance was 26 pS for both channels before Cu/P application, which was in the same range as that of the wild-type CNGA1 channel . Interestingly, we found that Cu/P treatment produced two populations of channels in 418C, one potentiated and the other inhibited. Among the seven patches tested, three of them showed an increase in open probability (P o ) at 4 μM cGMP after Cu/P application . For a saturating concentration of cGMP (2 mM), the P o in these patches was initially 0.91 ± 0.054 and was 0.90 ± 0.066 after Cu/P treatment (unpublished data). The single channel conductance was not altered by Cu/P treatment. This indicates Cu/P potentiated 418C through an effect on channel gating. For the other four patches, P o was 0.08 ±0.001 initially, and was reduced to essentially zero after Cu/P treatment both at 4 μM and 2 mM cGMP (not depicted). In all four of these inhibited patches, we recorded a total of <10 opening events after Cu/P treatment. This rate of opening was not sufficient to calculate P o , but is interesting because those few openings that were observed had the same unitary conductance as observed before Cu/P treatment (unpublished data). The inhibition of this population of channels correlates with the inhibition of current produced by Cu/P treatment at the macroscopic level. As a control, we also measured the single-channel effects of Cu/P treatment on CNGA1 cysless channels. We did not find a significant effect of Cu/P on the open probability or the single-channel conductance .
|
15738051_p20
|
15738051
|
Potentiation and Inhibition by Cu/P
| 4.274072 |
biomedical
|
Study
|
[
0.9992842078208923,
0.00038337099249474704,
0.0003325053257867694
] |
[
0.9994520545005798,
0.00021628283138852566,
0.0002504841540940106,
0.00008121666905935854
] |
en
| 0.999997 |
There are at least two ways in which Cu/P could produce two populations of channels. One is if a disulfide bond formed between opposite subunits produced one effect (e.g., potentiation), but disulfide bond formation between adjacent subunits produced another effect . Another possibility is that the number of disulfide bonds per channel is important. With four subunits each containing one cysteine, it is possible that one disulfide bond might form, resulting in potentiation, but that later a second forms, resulting in inhibition . To address this question, we examined the time dependence of the effects of Cu/P on 418C . In the model shown in Fig. 6 B, it might be expected that one effect (e.g., potentiation) might be observed early, with formation of one disulfide bond, followed later by the second effect (e.g., inhibition) when the second disulfide bond formed. We measured the current after various times exposed to Cu/P, and plotted it versus cumulative Cu/P treatment time at subsaturating and saturating cGMP concentrations . We found that Cu/P produced an initial increase in current activated by 2 μM cGMP, followed by a decrease in the current. In contrast, Cu/P produced only a slow decrease in the current activated by 2 mM cGMP, where the P o initially was already near 1. This experiment suggests that the two populations of channels we observed in the single channel studies reflected channels with either one (potentiated) or two (inhibited) disulfide bonds present per channel. The distribution of potentiated versus inhibited channels observed would depend on the arbitrary time chosen for the Cu/P treatment. Thus, the potentiation we observed in Figs. 2 and 3 indeed represents open-state specific proximity, but is complicated at later times by a formation of a second disulfide bond, giving channel inhibition.
|
15738051_p21
|
15738051
|
Potentiation and Inhibition by Cu/P
| 4.353453 |
biomedical
|
Study
|
[
0.9993718266487122,
0.0003502757754176855,
0.0002779301139526069
] |
[
0.9991494417190552,
0.00036030932096764445,
0.00039403661503456533,
0.00009625463280826807
] |
en
| 0.999998 |
Is the crystal structure of the HCN2 C-linker a reasonable model for that of CNG channels? The sequences of HCN2 and CNGA1 in this region are 22% identical and 45% conserved , suggesting that they likely have a similar structure. Equally important, they perform similar roles in the two channel types. We and others have shown that the C-linker of CNG channels energetically couples cyclic nucleotide binding to opening of the pore . In HCN channels, it has been shown that the C-linker inhibits opening of the pore, and that this inhibition can be relieved by cAMP binding to the CNBD, by deleting the C-linker and CNBD, or by deleting the CNBD . Thus, the C-linker regions of the two channel types share sequence similarity and are functionally homologous; it seems likely that they are structurally homologous as well.
|
15738051_p22
|
15738051
|
DISCUSSION
| 4.442207 |
biomedical
|
Study
|
[
0.9995012283325195,
0.0002583115710876882,
0.00024048186605796218
] |
[
0.9893335700035095,
0.0014788471162319183,
0.008976447395980358,
0.0002110420900862664
] |
en
| 0.999998 |
In the homology model of the C-linker of CNGA1 shown in Fig. 1 B, the residues at the 420 position of both adjacent and opposite subunits are much too far apart to either form a Ni 2+ coordination site (420H) or to form a disulfide bond (420C). Our data now show that the functional proximity between subunits is observed not only at the 420 position, but at 417 and 418 as well. Furthermore, in all cases, formation of a disulfide bond between cysteines at a given site produced potentiation. Potentiation of gating indicates an energetic stabilization of the open conformation of the channel relative to the closed conformation. Another way to state this is that the channels require less energy to open when the disulfide bond is formed compared with when the cysteines are reduced and the A' helices are free to move. We infer from this potentiation that the conformation of the channel trapped by the disulfide bond most closely resembles the open conformation.
|
15738051_p23
|
15738051
|
DISCUSSION
| 4.372691 |
biomedical
|
Study
|
[
0.9993420243263245,
0.0004092226445209235,
0.0002487932506483048
] |
[
0.9990812540054321,
0.0003524230851326138,
0.00045583696919493377,
0.0001105391638702713
] |
en
| 0.999997 |
Our data are incompatible with a model in which 417, 418, and 420 in different subunits are tens of Angstroms apart, as depicted in Fig. 1 B. We are led, then, to propose the following model for intersubunit interactions in the C-linkers of both CNG and HCN channels. Our hypothesis is depicted in Fig. 7 . This cartoon shows the C-linker region represented in both the closed and open state. The closed state is identical to that of the HCN2 crystal structure. Although cAMP is bound to the CNBD in the HCN2 crystal structure, and the CNBD structure likely represents that of the open configuration, we believe that the C-linker in the structure is in the closed conformation. For the open state , we have rotated the A' and B' helices as a unit, by introducing a rotation around the B'-C' loop. This rotation brings the A' helices nearly perpendicular to the membrane, as in the Johnson and Zagotta model . We moved both the A' and B' helices as a unit because movement around the A'-B' loop alone could not bring the 417–420 positions close enough to form a disulfide bond. However, the required proximity between the A' helices does not uniquely constrain the B' helices or other regions of the model. In functional channels, binding of cyclic nucleotide to the CNBD (not depicted) would cause a conformational change from the closed state, with A' helices at a distance, to the open state, with A' helices in proximity. This conformational change would be directly coupled to opening of the ion-conducting pore.
|
15738051_p24
|
15738051
|
DISCUSSION
| 4.538757 |
biomedical
|
Study
|
[
0.9990129470825195,
0.0006668948917649686,
0.00032009714050218463
] |
[
0.9977977275848389,
0.0009778081439435482,
0.0009942445904016495,
0.0002301840140717104
] |
en
| 0.999998 |
Why does locking subunits together through disulfide bond formation potentiate gating, but not lock the channels open? A given energetic stabilization of the open state relative to the closed state could produce either a locked open channel or a potentiated channel, depending on the relative stability of the two states in the absence of disulfide bonds. Furthermore, formation of a disulfide bond between two subunits traps them only along one axis of symmetry. The two pairs of subunits are still free to move relative to each other; perhaps this movement facilitates closing. Finally, the disulfide bonds formed here trap the subunits at only one point. It is likely that the opening conformational change involves a concerted movement throughout all the subunits. Restricting the movement of the protein at just one point does not appear to be sufficient to prevent closing. Of course, we must exercise caution when drawing conclusions about the structure of the channel in the absence of disulfide bonds from the perturbed structure when disulfide bonds are present. Closed channels whose subunits are locked together by disulfide bonds are very likely to have a different structure than closed channels without disulfide bonds, at least in the arrangement of the A' helices.
|
15738051_p25
|
15738051
|
DISCUSSION
| 4.621551 |
biomedical
|
Study
|
[
0.9983262419700623,
0.000804112758487463,
0.0008695751312188804
] |
[
0.9295005798339844,
0.014708761125802994,
0.054966460913419724,
0.0008242208859883249
] |
en
| 0.999997 |
What is the source of the two types of intersubunit interactions observed in 418C? Could one population arise from disulfide bond formation between subunits of different channels? Our observation of two populations at the single-channel level indicates this is not the case. We present two different ways different populations could arise in Fig. 6 . In one model, disulfide bonds could form between either adjacent or opposite subunits, each with a different functional effect . In the second model, formation of one disulfide bond would produce one effect, and formation of the second would produce the opposite functional effect . We observed that the current activated by 2 μM cGMP was first potentiated by Cu/P, and was then inhibited after longer cumulative exposure to Cu/P. Although this time dependence does not rule out the model in which the two functional populations are based on disulfide bonds with two different orientations, it would seem that time dependence is more compatible with the model in which the two functional populations are based on having either one or two disulfide bonds. The potentiated population, observed with short time Cu/P treatments, would arise from formation of a single disulfide bond. The inhibited population would then arise later, as the second disulfide bond formed.
|
15738051_p26
|
15738051
|
DISCUSSION
| 4.287623 |
biomedical
|
Study
|
[
0.9994211196899414,
0.00029225932667031884,
0.00028665244462899864
] |
[
0.998878538608551,
0.0005085879238322377,
0.000532979320269078,
0.00007983082468854263
] |
en
| 0.999996 |
Inhibition of CNGA2 channels with Ni 2+ has also been reported . Wild-type CNGA2 channels have a histidine at the position corresponding to 417, and a glutamine at the position corresponding to 420. Treatment of wild-type CNGA2 channels with Ni 2+ shifts the dose–response relation for cGMP to the right, increasing the K 1/2 from 2.3 to 6 μM . The inhibition of CNGA2 channels by Ni 2+ is unambiguous. We propose that this inhibition arises from the same mechanism that produced inhibition in CNGA1-417H. However, subtle differences in structure between CNGA1 and CNGA2 might prevent the relatively small potentiation produced by Ni 2+ in CNGA1-417H from occurring in CNGA2. This is most likely to occur if 417 is at a position that undergoes less movement during channel gating, relative to 420. Our model in Fig. 7 depicts 417 as near the fulcrum for movement, with proximity between subunits in both open and closed channels.
|
15738051_p27
|
15738051
|
DISCUSSION
| 4.360552 |
biomedical
|
Study
|
[
0.9993736147880554,
0.0003342500713188201,
0.00029209634521976113
] |
[
0.9990866184234619,
0.00046192333684302866,
0.0003485974157229066,
0.00010290722275385633
] |
en
| 0.999997 |
In summary, we have used trapping of the A' helices through disulfide bond formation to ask whether the crystal structure of the C-linker of HCN2 represents an open or closed conformation in CNG channel. Because each subunit contains only a single cysteine, any disulfide bond formation must occur between the same cysteine on different subunits. Three positions, 417, 418, and 420, formed disulfide bonds that potentiated activation of the channels. We interpret this potentiation to mean that the disulfide bond trapped the channels in a conformation more similar to the open state than to the closed state. These data are not compatible with a model in which the C-linker of the HCN2 structure represents the open channel conformation. Although cAMP is bound to the CNBD in the crystal structure and the CNBD observed in the structure almost certainly represents the open-channel conformation, our results indicate that the C-linker may be in the closed-channel conformation. Furthermore, the open-state specific proximity between subunits in the 417–420 region indicates that the A' helices form tilted bundles perpendicular to the axis of the membrane in open channels. Channel closure may then involve a relaxation of the A' helices to the position parallel to the axis of the membrane, as observed in the crystal structure of HCN2.
|
15738051_p28
|
15738051
|
DISCUSSION
| 4.430012 |
biomedical
|
Study
|
[
0.99920254945755,
0.0005399842048063874,
0.0002575752150733024
] |
[
0.9988691210746765,
0.0004315996775403619,
0.0005423533730208874,
0.00015691503358539194
] |
en
| 0.999995 |
The properties of hydrophobic substances, such as self-aggregation, interference with cellular membrane structure and stability, and binding to hydrophobic proteins, impede normal cellular processes and need to be counteracted effectively. Therefore, cells across phyla simply store several of these substances in storage depots termed lipid droplets, storage droplets, lipid bodies, lipid particles, or adiposomes. In the retinal pigment epithelium (RPE) of the eye these retinyl ester storage particles are termed retinosomes.
|
15314061_p0
|
15314061
|
Lipid bodies are autonomous intracellular structures with metabolic functions
| 4.015672 |
biomedical
|
Study
|
[
0.9991911053657532,
0.0002703548816498369,
0.0005384848918765783
] |
[
0.5681118965148926,
0.4072510004043579,
0.023639367893338203,
0.0009977398440241814
] |
en
| 0.999996 |
Typically, lipid bodies contain triacylglycerides, cholesteryl esters, and/or retinyl esters in their hydrophobic core, which is most likely surrounded by a hemi-membrane of unique fatty acid composition . Although they are clearly identified as morphologically distinct and autonomous entities found in close proximity to the plasma membrane or intracellular organelle membranes , their formation is not fully understood. Most likely, the lipid bodies are born at or in the ER, which is where acyltransferases are catalyzing the last steps in the synthesis of triacylglycerides and where cholesteryl ester and all-trans-retinyl ester syntheses take place. An attractive model proposes that the triacylglycerides and sterol esters accumulate between the two leaflets of the ER bilayer and, after reaching a certain critical concentration, bud off into the cytoplasm. Thereby the highly hydrophobic esters would form the core of a particle surrounded by a monolayer of ER membrane lipids oriented with their acyl chains toward the particle core and with their polar headgroups toward the cytoplasm. Because the limiting ER monolayer corresponds to the cytoplasmic leaflet of the ER membrane, proteins residing in this membrane or those peripherally bound to it can be found on the surface of the lipid bodies .
|
15314061_p1
|
15314061
|
Lipid bodies are autonomous intracellular structures with metabolic functions
| 4.693863 |
biomedical
|
Study
|
[
0.9990978240966797,
0.0005195913254283369,
0.00038254447281360626
] |
[
0.9901391267776489,
0.003590676002204418,
0.005867660976946354,
0.0004025727102998644
] |
en
| 0.999998 |
Lipid droplets have traditionally been regarded as inert inclusions used as storage vessels for hydrophobic intermediates such as triacylglycerides and all-trans-retinyl esters. However, the recent identification of proteins involved in lipid metabolism, signaling, and membrane trafficking in fractions of purified lipid droplets is suggestive of a more active role . Among others, key enzymes of sterol metabolism, some of which are controlled in their expression by the sterol regulatory element-binding protein, are found in lipid droplet fractions. Likewise, lipid droplet fractions of yeast cells contain enzymes involved in sterol and triacylglyceride metabolism. Important metabolic enzymes found in lipid particles of the yeast Saccharomyces cerevisiae are Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p . Thus, anabolic as well as catabolic steps in lipid metabolism might occur in or at lipid bodies. In most cases the exact location of metabolic enzymes in the lipid bodies remains to be determined, along with how and when they gain access to their substrates.
|
15314061_p2
|
15314061
|
Lipid bodies are autonomous intracellular structures with metabolic functions
| 4.507073 |
biomedical
|
Study
|
[
0.999427318572998,
0.0003158535691909492,
0.0002569274802226573
] |
[
0.9829750657081604,
0.0008918720996007323,
0.015926070511341095,
0.0002070734917651862
] |
en
| 0.999996 |
Several small GTPases of the Rab family, which are known to participate in membrane trafficking events, have also been identified in lipid droplet fractions . These GTPases include Rab1 and Rab2, which have been shown to regulate ER-Golgi transport, and Rab5, Rab7, and Rab11, which regulate different steps in early and late endocytosis. Another protein possibly involved in signaling and trafficking steps, which is targeted to lipid bodies under certain conditions, is the cholesterol-binding protein caveolin . Most caveolin isoforms are found in the cytosolic leaflets of cellular membranes, e.g., ER, Golgi membranes, and the plasma membrane (caveolae). However, caveolin-1 was recently also identified in lipid droplets . In this study, immunolabeling of freeze-fracture replicas revealed the presence of caveolin-1 in the exoplasmic leaflets of ER membranes and in lipid droplet cores. This localization is not easily compatible with previous findings identifying caveolin at the surface of lipid droplets . Thus, it remains to be seen whether and how caveolin-1 transits the lipid bilayer at some point, how it gains access to the hydrophobic core of lipid droplets, and how hydrophilic regions of the protein are organized in the hydrophobic interior of the droplet. Regardless of these controversial issues, it is evident that several caveolin isoforms can associate with lipid bodies. Ectopic expression studies reveal that an NH 2 -terminally truncated caveolin-2 isoform, caveolin-2β, is also present in lipid bodies . Moreover, NH 2 -terminal truncation mutants of other caveolins as well as full-length caveolins tagged with an ER retention signal are directed to lipid bodies . Interestingly, one of the mutants accumulating on lipid bodies also blocks microtubule-dependent lipid droplet motility and the redistribution and/or catabolism of lipids out of the lipid bodies . Together with the finding that endogenous caveolins can redistribute to lipid bodies in lipid-loaded cells , these data suggest a direct or indirect role for caveolin in the transport of lipid from lipid bodies to other destinations within the cell. This process could include the transport of cholesterol, although the major biosynthetic and endocytic transport routes of cholesterol, requiring either vesicular carriers or carrier protein complexes , appear not to include lipid bodies.
|
15314061_p3
|
15314061
|
Lipid bodies are autonomous intracellular structures with metabolic functions
| 4.720037 |
biomedical
|
Study
|
[
0.9988906979560852,
0.0007261299178935587,
0.00038322623004205525
] |
[
0.9977782368659973,
0.0006487835198640823,
0.0012757432414218783,
0.0002972093061544001
] |
en
| 0.999997 |
In addition to the enzymes and putative transport proteins mentioned in the previous section, at least one of a set of four related proteins (ADRP, perilipin, S3-12, and TIP47) has been found to associate with all lipid bodies analyzed thus far. The role of these proteins is not fully understood, but they may stabilize the droplet structure, control the lipolysis of core lipids, and/or provide an anchor for specific subcellular locations within the cell. Structurally, these proteins share a 100-residue-long region of limited homology at the NH 2 -terminal part called the PAT (perilipin, ADRP or adipophilin, and TIP47) domain.
|
15314061_p4
|
15314061
|
Adipose differentiation-related protein (ADRP) and related proteins are present on lipid bodies
| 4.26717 |
biomedical
|
Study
|
[
0.999554455280304,
0.0001677207910688594,
0.0002777970221359283
] |
[
0.9937798380851746,
0.0015408998588100076,
0.004554199054837227,
0.00012494139082264155
] |
en
| 0.999997 |
Perilipin is the predominant PAT protein in mature adipocytes. It coats the hydrophobic particles, limiting the access of lipases to their substrates in the particle core and thereby restraining lipolysis in resting cells . Upon β-adrenergic receptor stimulation and subsequent PKA activation, perilipin becomes phosphorylated, allowing lipases to gain access to the lipid bodies . Consistently, perilipin null mice have a reduced fat cell mass and a resistance to obesity combined with an elevation in basal lipolysis .
|
15314061_p5
|
15314061
|
Adipose differentiation-related protein (ADRP) and related proteins are present on lipid bodies
| 4.312664 |
biomedical
|
Study
|
[
0.9996597766876221,
0.00018416863167658448,
0.00015610450645908713
] |
[
0.9860966801643372,
0.0070560164749622345,
0.006521359086036682,
0.00032600003760308027
] |
en
| 0.999996 |
The amino acid sequence of ADRP (∼50 kD) is not suggestive of transmembrane domains, but the protein appears to be acylated . Fluorescent protein-tagged ADRP is directed to the surface of lipid bodies in human hepatocyte HuH7 and Chinese hamster ovary K2 cells . FRAP experiments using GFP-tagged ADRP protein demonstrate that photobleached lipid bodies do not recover fluorescence, suggesting that ADRP is not recruited rapidly to the surface of lipid bodies . ADRP and perilipin are important in serving as a nucleation center for the assembly of lipids to form nascent lipid bodies , and overexpression experiments in COS-7 cells suggest that ADRP can enhance the uptake of long-chain fatty acids by increasing their influx velocity . PAT proteins are not found in yeast ( S. cerevisiae ) lipid bodies. This finding raises the question of possible substitution or different mechanisms of lipid droplet generation and/or physiology in yeast as compared with animal cells.
|
15314061_p6
|
15314061
|
Adipose differentiation-related protein (ADRP) and related proteins are present on lipid bodies
| 4.457549 |
biomedical
|
Study
|
[
0.9994775652885437,
0.00030626740772277117,
0.00021612216369248927
] |
[
0.9984028935432434,
0.0005923359422013164,
0.0008486383594572544,
0.00015621363127138466
] |
en
| 0.999996 |
Studies on lipid bodies were advanced recently when a novel method of imaging these structures was introduced . This method took advantage of the intrinsic fluorescent properties of vitamin A combined with noninvasive infrared two-photon microscopy , which allowed for deep tissue penetration in live animals to monitor regeneration of rhodopsin without introduction of artificial fluorophores. Using this approach, we were able to identify in RPE cells previously uncharacterized structures, which were termed retinosomes (or REST particles, for retinyl ester-storage particles). Retinosomes have been shown to participate in 11-cis-retinal recycling in vivo, a process that is necessary for production of rhodopsin and maintenance of the light sensitivity of the photoreceptors . The retinosomes were characterized as specific sites of all-trans-retinyl ester accumulation because of genetic evidence and because spectrally sensitive detectors revealed that the λ max of retinosome fluorescence (488–499 nm) corresponds to that of model all-trans-retinyl esters . They appear to exist as stable organelles because the number of retinosomes does not change after all-trans-retinol is mobilized from photoreceptor cells, whereas their all-trans-retinyl ester content significantly increases . However, they appear to participate actively in 11-cis-retinoid production because the amount of all-trans-retinyl esters in retinosomes increases after bleaching of visual pigments and subsequently decreases during the process of rhodopsin regeneration. Interestingly, the density of retinosomes seems to vary between species. Mouse RPE appears to have fewer retinosomes as compared with monkey RPE within the macular region . These differences loosely correlate with the much faster regeneration of visual pigments, particularly cones, in primates as compared with the slow regeneration process in rodents .
|
15314061_p7
|
15314061
|
Retinosomes are lipid bodies allowing retinoid compartmentalization during the production of visual pigment chromophore in the eye
| 4.481527 |
biomedical
|
Study
|
[
0.9993494153022766,
0.00043072100379504263,
0.00021985109196975827
] |
[
0.9982095956802368,
0.00047396228183060884,
0.001151946489699185,
0.00016442866763100028
] |
en
| 0.999998 |
In mice, retinosomes are uniquely elongated structures of lipid particles oriented perpendicularly to the cell layer with a length of 6.9 ± 1.1 μm, a diameter of 0.8 ± 0.2 μm, and a density of 36.2 ± 2.2 per double-nuclei cell . They are clearly distinct from Golgi membranes, mitochondria, the majority of lysosomes, the plasma membrane, and the endoplasmic reticulum. Retinosomes participate in 11-cis-retinal recycling, as demonstrated by in vivo experiments using wild-type and certain knockout mice .
|
15314061_p8
|
15314061
|
Retinosomes are lipid bodies allowing retinoid compartmentalization during the production of visual pigment chromophore in the eye
| 4.300282 |
biomedical
|
Study
|
[
0.9995429515838623,
0.00023882892855908722,
0.00021834541985299438
] |
[
0.9986054301261902,
0.000813253631349653,
0.00048111710930243134,
0.00010017931344918907
] |
en
| 0.999999 |
Genetically engineered mice lacking key components of the retinoid cycle have been generated to define the role, formation, and utilization of retinoid intermediates in the live RPE. In the retinal photoreceptor cells, photoisomerization of the chromophore 11-cis-retinal that is coupled to the visual pigment leads to the production and release of all-trans-retinal. The photoisomerized chromophore must then be reverted back to 11-cis-retinal to maintain the light sensitivity of the photoreceptors . Specific metabolic transformations of retinoids (i.e., the retinoid cycle) are responsible for this conversion . In the photoreceptor cells, the majority of all-trans-retinal is released from the photoactivated pigment and accessible to a set of short-chain alcohol dehydrogenases on the surface of the disk membrane. However, a fraction of all-trans-retinal escapes into the intracellular disk space and needs to be transported across the disk membrane. This process requires the action of the ATP-binding cassette transporter (ABCR), which can transport all-trans-retinal or flip all-trans-retinylidene-phosphatidylethanolamine across the membrane, thereby allowing efficient reduction of the aldehyde to all-trans-retinol . The significance of transport across the disk membrane is underscored by the finding that mutations in the ABCR gene in humans or ABCR gene ablation in mice cause a buildup of bis-retinoids in photoreceptor cells . Two nucleotide-binding domains of ABCR have interdependent functions . One domain appears to play a noncatalytic, regulatory role, whereas the second binds and hydrolyzes ATP in a process that produces energy needed for the transport of hydrophobic substrates across photoreceptor disc membranes. The newly formed all-trans-retinol then diffuses to RPE, where the exclusive enzymatic activity of lecithin retinol acyltransferase (LRAT) catalyses the esterification of all-trans-retinol (vitamin A) with fatty acids. Coenzyme A-retinol transferase cannot substitute for LRAT in this reaction . Importantly, LRAT is present in the ER, indicating that this organelle is the site of esterification and genesis of retinosomes .
|
15314061_p9
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.742523 |
biomedical
|
Study
|
[
0.9986051917076111,
0.0010414052521809936,
0.0003534485585987568
] |
[
0.9955897331237793,
0.001601325231604278,
0.0022948889527469873,
0.0005140625289641321
] |
en
| 0.999998 |
In the next step of the cycle, all-trans-retinyl esters, which have the propensity to self-aggregate, bud off from the ER as independent structures, forming the retinosomes. This process is followed by trans-cis isomerization of the retinoid 11–12 double bond. In the dissected eye, this isomerization does not occur, and the retinoid cycle stops at either all-trans-retinol at lower temperatures or all-trans-retinyl esters at body temperatures. It is possible that exhaustion of ATP and GTP blocks isomerization under those conditions. However, such an explanation is not compatible with the conventional view of isomerization by an isomerohydrolase, where hydrolysis of the all-trans-retinyl esters is coupled to the isomerization reaction . Such reactions would not require energy input because high-energy esters are formed in the dissected eye. Thus, small G-proteins or other energy-using enzymes could be required, not for the actual isomerization reaction but to achieve a specific cellular distribution of all-trans-retinyl esters.
|
15314061_p10
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.450696 |
biomedical
|
Study
|
[
0.9994704127311707,
0.0003032631357200444,
0.0002264194772578776
] |
[
0.9969313144683838,
0.0018279313808307052,
0.0010618772357702255,
0.00017886859131976962
] |
en
| 0.999997 |
One possible site of isomerization could be the ER. However, in this case all-trans-retinyl ester would need to be transported from retinosomes back to the ER. RPE65 (an RPE-specific 65-kD protein), which binds retinyl esters , could mobilize them and function as a retinosome-ER shuttle protein. This shuttle would be unidirectional, as retinosomes probably form by budding off from the ER and the lack of RPE65 in knockout mice does not affect the formation of retinosomes. However, Rpe65 − / − mice accumulate all-trans-retinyl esters in overgrown structures. Why are these overgrown structures formed? In wild-type mice, all-trans-retinol enters the RPE efficiently from the bloodstream, but also exchanges rapidly with a pool of all-trans-retinol present in the blood . RPE65 could be a key protein that works together with a hydrolase (REH) to produce all-trans-retinol, an alternative substrate for a putative isomerase . The absence of RPE65 prevents all-trans-retinol's exchange with the blood circulation. Thus, once all-trans-retinol enters the eye and is esterified, it remains in the retinosomes and cannot be liberated to diffuse to blood circulation . Two types of RPE65 were characterized, a membrane-associated and a soluble form . The molecular switch between the two forms was proposed to involve triple palmitoylation of specific Cys residues catalyzed by LRAT. However, two out of three Cys residues are not conserved in vertebrate RPE65s, raising the questions of whether or not triple palmitoylation is a general feature of the isomerization process and whether the proposed mechanism is relevant.
|
15314061_p11
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.676261 |
biomedical
|
Study
|
[
0.9991686344146729,
0.0005440216045826674,
0.0002873564080800861
] |
[
0.9963666200637817,
0.0014196463162079453,
0.0019056000746786594,
0.0003081224858760834
] |
en
| 0.999996 |
The retinoid isomerization reaction could take place in retinosomes. Consistent with this concept is the phenotype of CRALBP (soluble 11-cis-retinol/11-cis-retinal binding protein) knockout mice. CRALBP appears to be crucial for an efficient and stereospecific isomerization reaction , and CRALBP-deficient mice accumulate all-trans-retinyl esters, most likely in the retinosomes . Because the isomerization to 11-cis-retinol is drastically attenuated in these mice, retinosomes are a likely place for the actual isomerization reaction. However, it must be noted that sluggish isomerization does occur in the absence of CRALBP due to the presence of other retinoid-binding proteins, eventually restoring the visual pigment.
|
15314061_p12
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.369803 |
biomedical
|
Study
|
[
0.9995263814926147,
0.00026564986910670996,
0.00020806898828595877
] |
[
0.9978020787239075,
0.0012838701950386167,
0.0007498989580199122,
0.00016422569751739502
] |
en
| 0.999997 |
Once produced, 11-cis-retinol is oxidized to 11-cis-retinal and exported to the photoreceptors , where it recombines with opsins to form 11-cis-retinylidene-opsins . Thus, retinosomes appear to be essential structures for retaining esterified vitamin A in the eye to support its utilization in forming the chromophore for visual pigments.
|
15314061_p13
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.096266 |
biomedical
|
Other
|
[
0.9986345171928406,
0.0007002613274380565,
0.0006651917356066406
] |
[
0.23484669625759125,
0.757293701171875,
0.006266060285270214,
0.0015936039853841066
] |
en
| 0.999998 |
On average, between 20–40 rod outer segments project toward one RPE cell . For efficient transfer of retinoids between the RPE and the photoreceptor cells, the retinoid processing enzymes should be widely distributed throughout the cell. This prediction has been confirmed by revealing the ER distribution of LRAT poised to trap all-trans-retinol to form all-trans-retinyl esters . Self-associating complexes of all-trans-retinyl esters would facilitate retinosome formation, and the resulting clustering of all-trans-retinyl esters in retinosomes may prevent diffusion of retinoids throughout the retina. However, the symmetric nature of these structures and their intracellular distribution suggest that additional proteins are involved in forming or maintaining the particles. ADRP, which is present on retinosomes as well as other lipid bodies, could play such a role, either alone or in conjunction with other yet to be identified proteins. Interestingly, neither retinosomes nor ADRP are found in mouse or bovine retinal Müller cells, which are implicated in the cone-dominant retina (e.g., chicken) as cells functioning alternatively to RPE in 11-cis-retinal production in the course of cone photoreceptor regeneration .
|
15314061_p14
|
15314061
|
Formation and utilization of retinosomes in transgenic mice
| 4.530106 |
biomedical
|
Study
|
[
0.9994217157363892,
0.0003069113299716264,
0.00027142156613990664
] |
[
0.9979665279388428,
0.000756608962547034,
0.0011490067699924111,
0.00012795007205568254
] |
en
| 0.999997 |
Several conditions or genetic alterations can result in an aberrant intracellular accumulation of lipids, either in the endosomal system when internalized lipids are not properly degraded or transported or in overgrown lipid bodies. Altered lipid storage is clearly evident in several cells, e.g., in adipocytes and hepatocytes after excess food intake. Modified retinosomes are also observed in the retina, where they are the result of blinding disorders affecting the enzymes of the retinoid cycle . As discussed, retinosomes accumulate in mice incapable of carrying out the enzymatic isomerization process (RPE65 knockout) and are absent in mice deficient in vitamin A in the eye , which are animal models of inherited early onset dystrophies that are a subset of Leber congenital amaurosis. Thus, although many retinoid processing enzymes are thought to localize to the ER and cytoplasm, retinosomes that are found close to the plasma membrane of RPE cells could not only be a storage place for retinoids, but could also play a direct, metabolic role in the regeneration of visual pigment chromophores . Therefore, retinosomes are novel components of the retinoid cycle critical to the formation of 11-cis-retinal, and their structure is aberrant in certain disease stages.
|
15314061_p15
|
15314061
|
Increased accumulation or lack of lipid bodies causes diseases
| 4.692469 |
biomedical
|
Study
|
[
0.9992600083351135,
0.00045459819375537336,
0.0002854420163203031
] |
[
0.9675244092941284,
0.0019283117726445198,
0.030080944299697876,
0.0004663607105612755
] |
en
| 0.999996 |
Spectrally sensitive noninvasive two-photon microscopy in conjunction with genetically engineered mice lacking key components of the retinoid cycle can be used to follow the production of the visual chromophore 11-cis-retinal in intact eyes. Two-photon microscopy permits deep tissue penetration of infrared excitation light in anesthetized mice and allows monitoring of the regeneration processes of rhodopsin without introducing external fluorophores. This novel approach has revealed the existence of novel compartments, retinosomes, which are critical to the formation of 11-cis-retinal. It also provides an opportunity to study the formation of the lipid bodies in vivo with spatiotemporal resolution, taking advantage of the accessibility of the visual system to physiological testing. Thus, the eye is one of the most accessible systems for studying the formation, utilization, and disease-causing abnormalities of lipid bodies.
|
15314061_p16
|
15314061
|
Conclusions: the eye as an experimental model system to study lipid bodies
| 4.23686 |
biomedical
|
Study
|
[
0.9996316432952881,
0.00021040832507424057,
0.00015793144120834768
] |
[
0.9948118925094604,
0.0018626991659402847,
0.0031786849722266197,
0.00014671242388430983
] |
en
| 0.999997 |
During mitosis, a bipolar spindle is assembled to accurately segregate the replicated DNA into two daughter cells. The shape and size of the spindle determine the distance over which DNA is segregated and impact the forces acting on chromosomes. Spindle organization depends on microtubules, polymers of tubulin. Spindle microtubules have been shown to undergo a process referred to as polewards flux . At metaphase, this involves the continuous addition of tubulin subunits to microtubule plus-ends, polewards translocation of microtubules, and microtubule disassembly at spindle poles. Surprisingly, all of these events are coordinated so that spindle length and average spindle microtubule length remain unchanged whereas microtubules flux polewards. Recent studies have shown that microtubule polymerization and transport can each be regulated by microtubule-associated proteins . These include motor proteins, such as Eg5 and dynein, that can slide microtubules, as well as kinesins in the KinI family and nonmotor proteins that can regulate microtubule polymerization. It is not known how activities of these proteins are regulated at different sites within spindles to maintain steady-state length.
|
15314063_p0
|
15314063
|
Introduction
| 4.555806 |
biomedical
|
Study
|
[
0.9991635084152222,
0.0005040731048211455,
0.00033248672843910754
] |
[
0.975987434387207,
0.001716898288577795,
0.02196686528623104,
0.0003288831503596157
] |
en
| 0.999995 |
During polewards flux, microtubule disassembly occurs at spindle poles, whereas microtubule ends remain focused. Dynein and dynactin are large (>1 MD) multi-subunit complexes that localize to spindle poles . Recent studies suggest that a complex of dynein and dynactin plays an important role in transporting and targeting a number of proteins to spindle poles . NuMA, a microtubule cross-linking protein that plays a key role in spindle pole formation, is transported to spindle poles, most likely through interactions with dynein/dynactin . Additionally, dynein contributes to the polewards transport of short stabilized exogenous microtubules added to spindles . These data suggest that dynein and dynactin may also drive the translocation of spindle microtubules during polewards flux. However, this possibility has not been directly tested. Here, we show that dynein, dynactin, and their binding partner NuMA, can control spindle microtubule length by contributing to the targeting of the depolymerizing activities of a KinI kinesin to spindle poles without directly affecting the translocation of microtubules associated with polewards flux.
|
15314063_p1
|
15314063
|
Introduction
| 4.385046 |
biomedical
|
Study
|
[
0.9993081092834473,
0.000450548657681793,
0.00024140544701367617
] |
[
0.9990529417991638,
0.00041941326344385743,
0.0004014645819552243,
0.0001261647412320599
] |
en
| 0.999997 |
To examine the role of dynactin in polewards flux in bipolar spindles, we used an inhibitor named p150-CC1. This recombinant protein fragment consists of amino acids 217–548 of the p150 Glued subunit of dynactin and has been shown to bind dynein in vitro and inhibit dynactin function . For these experiments we used spindles assembled in Xenopus egg extracts . An advantage of this cell-free system is that spindles are not constrained in fixed volumes and cell cortices are absent, allowing mechanisms intrinsic to the spindle to be examined.
|
15314063_p2
|
15314063
|
Inhibition of dynein and dynactin results in the elongation of spindle microtubules
| 4.175572 |
biomedical
|
Study
|
[
0.9994832277297974,
0.00022693643404636532,
0.00028974670567549765
] |
[
0.9988645315170288,
0.0006960391183383763,
0.00037022167816758156,
0.00006925519846845418
] |
en
| 0.999997 |
p150-CC1 was added to spindles assembled in egg extracts cycled through interphase to replicate their DNA and centrosomes. Individual spindles were monitored by time-lapse microscopy. Within ∼7 min of p150-CC1 (2 μM) addition, spindle length doubled while bipolar organization was maintained . Measurements revealed that the distance between opposite poles increased at 4.5 ± 0.9 μm/min (12 live recordings, two independent experiments) after p150-CC1 treatment, whereas control spindles did not change length . Microtubule focusing at poles was not significantly perturbed in p150-CC1–treated spindles that were twice as long as untreated spindles . At >25 min after p150-CC1 addition, structures were often longer than 3.5 times the length of control spindles (∼140 μm; unpublished data). Analysis of fixed samples revealed that the effect of p150-CC1 on spindle length increase was dose dependent, and the effect saturated by 2 μM . These data demonstrate that dynactin is required for maintaining constant spindle length.
|
15314063_p3
|
15314063
|
Inhibition of dynein and dynactin results in the elongation of spindle microtubules
| 4.337038 |
biomedical
|
Study
|
[
0.9994186162948608,
0.0003833117661997676,
0.00019813430844806135
] |
[
0.9990261793136597,
0.0004518081550486386,
0.0003998622123617679,
0.00012207157851662487
] |
en
| 0.999995 |
To examine whether the effect of p150-CC1 on spindle length was due to inhibition of the activity of the dynein/dynactin complex, we tested the effect of available dynein inhibitors, the antibody 70.1 and vanadate. Spindles treated with 70.1 (1 mg/ml; note: ∼800 nM dynein in egg extracts), an antibody to dynein intermediate chain, increased in length at 3.7 ± 0.9 μm/min . Similar effects were observed for vanadate-treated (100 μM) spindles . These data are consistent with both dynein motor activity and dynactin regulating spindle length.
|
15314063_p4
|
15314063
|
Inhibition of dynein and dynactin results in the elongation of spindle microtubules
| 4.16645 |
biomedical
|
Study
|
[
0.9995119571685791,
0.0002690920664463192,
0.00021903585002291948
] |
[
0.9993000030517578,
0.0003379601694177836,
0.00029514089692384005,
0.0000668767825118266
] |
en
| 0.999996 |
We find p150-CC1 to be significantly more potent than the commonly used dynactin inhibitor p50 dynamitin . No effect on assembled spindles was observed at 18 μM p50 dynamitin, the maximum concentration that we could use without perturbing extracts by dilution alone. However, as previously reported, p50 dynamitin (18 μM) added at the start of spindle assembly resulted in structures with unfocused poles and lengths within 20% of that of untreated spindles . Addition of 2 μM p150-CC1 at the start of spindle assembly resulted in very long spindles similar to that shown in Fig. 1 G. An effect similar to that of p50 dynamitin was observed with low concentrations p150-CC1 (56 nM), if added at the start of spindle assembly. It is possible that differences in p50 dynamitin and p150-CC1 potencies reflect their different mechanisms of inhibiting dynactin function. It noteworthy that addition of p150-CC1 (to 2 μM) to spindles with unfocused poles, that were assembled in the presence of high concentration of p50 dynamitin or low concentrations of p150-CC1, resulted in spindle elongation at the same rate as spindles treated with p150-CC1 alone . These data indicate that the role of dynactin in spindle microtubule length regulation is independent of its function in pole focusing.
|
15314063_p5
|
15314063
|
Inhibition of dynein and dynactin results in the elongation of spindle microtubules
| 4.234229 |
biomedical
|
Study
|
[
0.9994958639144897,
0.00028180607478134334,
0.00022233523486647755
] |
[
0.9992825388908386,
0.0002672102418728173,
0.0003822010476142168,
0.00006812207720940933
] |
en
| 0.999998 |
Dynein and dynactin associate with centrosomes and play a role in tethering centrosomes to spindle microtubules . To examine whether the spindle elongation observed upon dynein or dynactin inhibition depended on centrosome function, we tested the effect of p150-CC1 addition to spindles assembled in the absence of centrosomes. DNA-coated beads, when added to Xenopus egg extracts, induce the assembly of centrosome-free spindles morphologically indistinguishable from spindles assembled around sperm chromatin . Analysis of fixed samples , as well as real-time analysis (not depicted), revealed that inhibition of dynactin with p150-CC1 resulted in the elongation of centrosome-free spindles. In the presence of p150-CC1 (2 μM), spindles elongated at 3.1 ± 0.2 μm/min (average of 26 spindles, two independent experiments). Similar results were obtained when antibody 70.1 was added to DNA-bead spindles (unpublished data). These data are consistent with a role for dynein and dynactin in maintaining the length of spindle microtubules independent of their function at centrosomes.
|
15314063_p6
|
15314063
|
Inhibition of dynein and dynactin results in the elongation of spindle microtubules
| 4.206679 |
biomedical
|
Study
|
[
0.9995088577270508,
0.000313952739816159,
0.00017721303447615355
] |
[
0.9992311000823975,
0.00037949412944726646,
0.0003041288291569799,
0.0000852795346872881
] |
en
| 0.999997 |
Dynein/dynactin inhibition could, in principle, increase spindle microtubule length by affecting the polewards transport of microtubules or by suppressing disassembly at spindle poles. To distinguish between these possibilities, we used fluorescent speckle microscopy to analyze microtubule dynamics in spindles treated with p150-CC1, the dynactin inhibitor for which we had determined dose-dependence and saturating concentrations. To track microtubule organization in time-evolving structures we used multi-wavelength fluorescence microscopy, recording fluorescent speckles, overall microtubule organization, and as needed, the position of DNA . Rates of tubulin translocation were measured by kymography and confirmed by manual tracking of individual speckles .
|
15314063_p7
|
15314063
|
Inhibition of dynactin in bipolar spindles suppresses disassembly of microtubules at spindle poles
| 4.196982 |
biomedical
|
Study
|
[
0.9994456171989441,
0.0003286733408458531,
0.00022564474784303457
] |
[
0.9992640614509583,
0.000317125377478078,
0.0003430547076277435,
0.00007572874892503023
] |
en
| 0.999996 |
Fig. 2 (A and F) show polewards flux in an untreated spindle . Analysis revealed that tubulin speckles moved toward the spindle pole at 2.4 ± 0.7 μm/min . In p150-CC1 treated spindles the rate of polewards microtubule sliding, was 2.1 ± 0.4 μm/min, indistinguishable from that in untreated spindles . The speckles did not move relative to the spindle pole, but moved along with the pole at half the speed the poles moved apart . Analysis of fluorescent speckle microscopy data also revealed that there was not a significant difference in the size of the region of bi-directional speckle movement in untreated and p150-CC1–treated spindles, suggesting that dynactin inhibition did not affect the extent of antiparallel microtubule overlap in the spindle .
|
15314063_p8
|
15314063
|
Inhibition of dynactin in bipolar spindles suppresses disassembly of microtubules at spindle poles
| 4.188485 |
biomedical
|
Study
|
[
0.9994031190872192,
0.0003808991750702262,
0.00021605579240713269
] |
[
0.9992955923080444,
0.00030349765438586473,
0.00031794366077519953,
0.00008298936882056296
] |
en
| 0.999995 |
These data indicate that the spindle elongation observed upon inhibition of dynactin with p150-CC1 is due to a lack of microtubule minus-end disassembly at spindle poles, and that this occurs without any significant perturbation of polewards microtubule sliding in the spindle.
|
15314063_p9
|
15314063
|
Inhibition of dynactin in bipolar spindles suppresses disassembly of microtubules at spindle poles
| 4.219462 |
biomedical
|
Study
|
[
0.9993928670883179,
0.00041863511432893574,
0.0001884857047116384
] |
[
0.9976316690444946,
0.0017027714056894183,
0.0004958296776749194,
0.00016973423771560192
] |
en
| 0.999996 |
NuMA has been shown to interact with dynein/dynactin, and NuMA targeting to spindle poles depends on dynactin function . Consistent with these data, we found that p150-CC1 treatment disrupted NuMA targeting to spindle poles . To examine whether NuMA cooperates with dynein and dynactin in regulating the length of spindle microtubules, we perturbed NuMA function in spindles using the NuMA-binding domain of LGN, a mammalian Pins homologue . This recombinant protein fragment, consisting of amino acids 1–373 of LGN (hereafter named LGN-N), inhibits NuMA interactions with microtubules both in vitro and in vivo .
|
15314063_p10
|
15314063
|
NuMA inhibition results in elongation of spindle microtubules
| 4.217877 |
biomedical
|
Study
|
[
0.9995079040527344,
0.00025595768238417804,
0.00023611947835888714
] |
[
0.9993815422058105,
0.00028823729371652007,
0.00025598000502213836,
0.00007420145266223699
] |
en
| 0.999995 |
Addition of LGN-N to spindles assembled in Xenopus extracts had an effect similar to that of perturbing dynein or dynactin . Real-time analysis of individual spindles and measurements of fixed samples revealed that spindles treated with LGN-N increased in length while maintaining bipolar organization. At 4 μM LGN-N spindle length increased at 4.3 ± 0.6 μm/min ( n = 26 spindles, two independent experiments). At these concentrations of LGN-N , NuMA was displaced from spindle poles . Fluorescent speckle microscopy revealed that the poleward sliding rate of microtubules in the spindle was not perturbed by LGN-N treatment (2.3± 0.7 μm/min; unpublished data) relative to control spindles . Recombinant LGN, fused to either GST, or to a polyhistidine tag, gave similar results in these experiments. As with dynein or dynactin inhibition, the length of centrosome-free spindles assembled around DNA beads also increased upon LGN-N treatment .
|
15314063_p11
|
15314063
|
NuMA inhibition results in elongation of spindle microtubules
| 4.245324 |
biomedical
|
Study
|
[
0.9994974136352539,
0.00030746604898013175,
0.0001950533624039963
] |
[
0.9992602467536926,
0.00031163764651864767,
0.0003390554338693619,
0.0000890902301762253
] |
en
| 0.999997 |
These data demonstrate that NuMA cooperates with dynein/dynactin to regulate microtubule minus-end dynamics at spindle poles without directly contributing to the sliding component of polewards flux.
|
15314063_p12
|
15314063
|
NuMA inhibition results in elongation of spindle microtubules
| 4.190784 |
biomedical
|
Study
|
[
0.9993973970413208,
0.00036176841240376234,
0.00024074599787127227
] |
[
0.9968920350074768,
0.0023839608766138554,
0.0005224415799602866,
0.00020164270245004445
] |
en
| 0.999996 |
We considered the possibility that dynein, dynactin, and NuMA may regulate spindle microtubule length by targeting microtubule-depolymerizing activities to spindle poles. Two vertebrate kinesins in the KinI family, Kif2a and MCAK/XKCM1/Kif2c (hereafter referred to as MCAK), have been shown to have microtubule depolymerizing activities in vitro . The function of MCAK during spindle assembly in the Xenopus extract system has been extensively studied . Although a functional role for Kif2a has been described in neurons , a role in spindle assembly has not been shown.
|
15314063_p13
|
15314063
|
Kif2a plays a key role in spindle assembly and maintenance
| 4.228024 |
biomedical
|
Study
|
[
0.9995527863502502,
0.00021873855439480394,
0.000228417688049376
] |
[
0.9985612034797668,
0.0008363373926840723,
0.0005091498023830354,
0.00009331165347248316
] |
en
| 0.999998 |
To examine Kif2a function in spindles, we used a polyclonal antibody raised against the NH 2 terminus of Kif2a to block its function. Similar antibodies, raised against the NH 2 terminus of the closely related KinI MCAK have been shown to block MCAK microtubule destabilizing activity . In Western blots, the Kif2a antibody was found to be mono-specific, recognizing a ∼100-kD band, which is consistent with the predicted size of Xenopus Kif2a . Addition of Kif2a antibody (0.7 mg/ml) at the start of spindle assembly, after DNA and centrosome replication, resulted in the formation of large monopolar structures with unusually long extended microtubule bundles . These results are similar to those observed in mammalian cells after suppression of Kif2a protein levels using siRNA , which are consistent with the antibody inhibiting Kif2a function in Xenopus egg extracts.
|
15314063_p14
|
15314063
|
Kif2a plays a key role in spindle assembly and maintenance
| 4.200766 |
biomedical
|
Study
|
[
0.9994754195213318,
0.00027551795938052237,
0.00024910399224609137
] |
[
0.9993245601654053,
0.0003432821831665933,
0.00025964219821617007,
0.00007246780296554789
] |
en
| 0.999995 |
Addition of Kif2a antibody (0.7 mg/ml) to assembled bipolar spindles resulted in an increase in spindle microtubule length, evident from buckled and wavy microtubule bundles in spindles , and also from long microtubules extending out from spindles . However, the pole to pole distance did not increase to accommodate these increases in microtubule length. Real-time analysis also revealed increases in microtubule length that did not result in corresponding increases in spindle length . At ∼20 min after antibody addition, spindles often became highly disorganized and lost bipolar organization. Due to continuous changes in microtubule curvature, and complex trajectories of spindle pole movements (in 3-dimensions), we were unable to quantitatively analyze microtubule dynamics upon Kif2a inhibition. However, similar to the consequences of inhibiting dynein, dynactin, or NuMA, Kif2a inhibition increased the lengths of spindle microtubules.
|
15314063_p15
|
15314063
|
Kif2a plays a key role in spindle assembly and maintenance
| 4.252992 |
biomedical
|
Study
|
[
0.9994314312934875,
0.00036061988794244826,
0.00020785605011042207
] |
[
0.9991360306739807,
0.0003632573352660984,
0.0003971662372350693,
0.00010350049706175923
] |
en
| 0.999999 |
To test whether increases in spindle microtubule length upon dynein, dynactin, or NuMA inhibition were due to perturbation of KinI activities at spindle poles, we examined the localization of Kif2a and MCAK in spindles treated with p150-CC1 or antibody 70.1. In control samples, we found that MCAK targeted to spindle poles, which is consistent with previous reports . Under these fixation conditions we found that Kif2a also localized to spindle poles . In elongating spindles treated with p150-CC1 or 70.1, MCAK targeted to spindle poles, and its distribution, relative to microtubules, was not significantly perturbed . The accumulation of Kif2a at spindle poles in the presence of p150-CC1 or 70.1 was diminished . LGN-N treated spindles also revealed that Kif2a, but not MCAK, targeting was perturbed (not depicted). Spindles fixed using different conditions (methanol instead of formaldehyde) revealed that Kif2a, like MCAK, also targeted to sites of chromosome-microtubules attachment, which is consistent with these proteins targeting to kinetochores . Treatment with p150-CC1 did not disrupt the targeting of Kif2a to kinetochores. As another measure of the dependence on dynactin of KinI targeting to spindles, we partially purified spindles by pelleting through a glycerol cushion, and determined the amounts of spindle-associated MCAK and Kif2a by immunoblotting. Using this assay, we also found that dynactin inhibition reduced the amount of spindle-associated Kif2a, but not MCAK, relative to tubulin . Although we are unable to detect a direct interaction between NuMA or dynein/dynactin and Kif2a using available reagents, our data are consistent with dynactin and NuMA targeting, or maintaining, Kif2a at spindle poles.
|
15314063_p16
|
15314063
|
Dynein/dynactin and NuMA are required for the targeting of Kif2a to spindle poles
| 4.338988 |
biomedical
|
Study
|
[
0.9993170499801636,
0.00040895750862546265,
0.0002739500778261572
] |
[
0.999218225479126,
0.00031816225964576006,
0.000357503246050328,
0.00010611959442030638
] |
en
| 0.999996 |
It is possible that Kif2a is the functional homologue of Klp10, the Drosophila KinI motor , and its effect on microtubule dynamics may be specific to spindle poles . Our works demonstrate that Kif2a plays an important role in regulating the length of spindle microtubules, but do not confirm its site-specific activity. We propose that dynein, dynactin, and NuMA regulate spindle microtubule length by localizing the depolymerizing activity of Kif2a to spindles poles. Our work also reveals that NuMA and dynein/dynactin play a role in maintaining a mechanical constraint that keeps spindle poles from separating, even when spindle microtubules get unusually long after Kif2a inhibition. Our data also provide insight into mechanisms of polewards flux. The fact that microtubule assembly/disassembly can do mechanical work, has been demonstrated in vitro and in the context of spindles . This raises the possibility that the force driving flux could come from microtubule disassembly at poles. Microtubules could be “reeled in” from poles without a separate force-generating component driving microtubule sliding. Our data show that microtubule disassembly can be suppressed without affecting the rates of microtubule translocation in spindles. Therefore, the disassembly of microtubules is not required for the force generating mechanism driving polewards flux and this force is more likely to be produced by the sliding mechanism. Furthermore, this sliding mechanism does not involve dynein/dynactin or NuMA.
|
15314063_p17
|
15314063
|
Dynein/dynactin and NuMA are required for the targeting of Kif2a to spindle poles
| 4.533691 |
biomedical
|
Study
|
[
0.9992097616195679,
0.0004972272436134517,
0.0002930394548457116
] |
[
0.9986166954040527,
0.000605246692430228,
0.0005910311592742801,
0.00018694247410167009
] |
en
| 0.999996 |
Spindles were assembled in cytostatic factor–arrested Xenopus laevis extracts cycled once though interphase to replicate DNA and centrosomes as described previously . Tubulins labeled with fluorophores , and p50 dynamitin were prepared as described previously. p150-CC1 was expressed and purified as described previously , and dialyzed into buffer XB1 (10 mM Hepes, 150 mM sucrose, 250 mM KCl, 1 mM MgCl 2 , 1 mM DTT plus protease inhibitors). Expression construct was a gift from T. Schroer (Johns Hopkins University, Baltimore, MD). LGN-N fused to GST was expressed in E. coli BL21(DE3) from the pGEX2T vector (Amersham Biosciences), purified on a glutathione-agarose column (Sigma-Aldrich), and dialyzed into buffer XB1. Expression construct was a gift from I. Macara (University of Virginia, Charlottesville, VA). Dynein intermediate chain antibody 70.1 (Sigma-Aldrich) was prepared as described previously , and used at 1:10 dilution. Polyclonal rabbit anti-Kif2a was a gift from D.A. Compton (Dartmouth Medical School, Hanover, NH).
|
15314063_p18
|
15314063
|
Reagents
| 4.152565 |
biomedical
|
Study
|
[
0.9996150732040405,
0.00014543702127411962,
0.00023949235037434846
] |
[
0.9982643723487854,
0.0010482118232175708,
0.000605047564022243,
0.00008240021270466968
] |
en
| 0.999998 |
Samples for immunofluorescence were fixed in 2% formaldehyde processed for immunofluorescence as described previously . Anti-Kif2a was used at 1 μg/ml and anti–α-tubulin (clone DM1a; Sigma-Aldrich) was used at 1:3,000 dilution. Images were acquired as 3-D volumes using a DeltaVision system (Applied Precision Instruments) and processed using iterative constrained deconvolution.
|
15314063_p19
|
15314063
|
Immunofluorescence and microscopy
| 4.044586 |
biomedical
|
Study
|
[
0.999528169631958,
0.00022190899471752346,
0.00024994483101181686
] |
[
0.9874511957168579,
0.011717933230102062,
0.0006119081517681479,
0.00021896800899412483
] |
en
| 0.999997 |
Fluorescence imaging of spindles in extract was done using an Axiovert 200M (Carl Zeiss MicroImaging, Inc.) with either a 40× (Plan Neo, NA 0.75, for tracking spindle length) or 63× (Plan Apo, NA 1.4, for fluorescence speckle microscopy and imaging microtubule organization) objective. An Orca ER CCD camera (Hamamatsu) was used. Fluorophore-conjugated tubulins were used for fluorescent speckle microscopy at ∼11 nM (X-rhodamine) and for visualizing microtubule organization at ∼300 nM (X-rhodamine or Alexa-488). For fluorescent speckle microscopy, 350–500-msec exposures were acquired at 2–5-s intervals. All real-time imaging experiments were performed at 18°C.
|
15314063_p20
|
15314063
|
Immunofluorescence and microscopy
| 4.142427 |
biomedical
|
Study
|
[
0.9994683861732483,
0.0003187844413332641,
0.00021281054068822414
] |
[
0.9941402077674866,
0.00497009651735425,
0.0006787031306885183,
0.00021097793069202453
] |
en
| 0.857141 |
Extract samples were diluted 1:10 in BRB80, 30% glycerol, 0.5% Triton X-100, overlaid on 1-ml cushion (BRB80, 40% glycerol), and centrifuged for 15 min at 6,000 g . Spindle pellets were resuspended in 1-ml cushion and recentrifuged for 15 min at 6,000 g . The final spindle pellet was analyzed by immunoblotting. KinI and tubulin band intensities were recorded by scanning , and quantified using Metamorph software.
|
15314063_p21
|
15314063
|
Spindle pelleting
| 4.152503 |
biomedical
|
Study
|
[
0.9994171857833862,
0.0003076965222135186,
0.000275088706985116
] |
[
0.9921990036964417,
0.007131528574973345,
0.0004684950108639896,
0.00020097442029509693
] |
en
| 0.999998 |
Analysis was performed using Metamorph software (Universal Imaging Corp.). The length of a line drawn from one spindle pole to the other was used to measure spindle length. Kymographs were prepared from unprocessed images by selecting the maximum intensity across a 7-pixel-wide (x dimension) region aligned with the spindle axis (y dimension). Slopes of fluorescent speckle streaks in the kymographs were measured to calculate speckle velocity. Line scans were generated from the average intensity across a 10-pixel-wide line across the image.
|
15314063_p22
|
15314063
|
Data analysis
| 4.087074 |
biomedical
|
Study
|
[
0.9995450377464294,
0.00027782487450167537,
0.00017711688997223973
] |
[
0.9986186027526855,
0.0008930645999498665,
0.0004000201588496566,
0.00008831774903228506
] |
en
| 0.999998 |
Online supplemental material includes: (a) methods for immunofluorescence to examine kinetochore localization of Kif2a; (b) Fig. S1 showing the effect of vanadate on spindle length and comparing the effects of p50 dynamitin and p150-CC1; (c) Fig. S2 showing NuMA localization in spindles treated with p150-CC1 or LGN-N; (d) Fig. S3 showing Kif2a localization at kinetochores; and (e) five videos, corresponding to data in Figs. 1 – 4 . Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404015/DC1 .
|
15314063_p23
|
15314063
|
Online supplemental material
| 3.388041 |
biomedical
|
Study
|
[
0.9989755153656006,
0.00032816926250234246,
0.000696274102665484
] |
[
0.8388875722885132,
0.15740986168384552,
0.002215250162407756,
0.001487292698584497
] |
en
| 0.999996 |
Eukaryotic ribosome biogenesis is a highly regulated, evolutionary conserved process in the nucleolus. A large precursor ribosomal RNA (pre-rRNA) is transcribed by Pol I and rapidly packaged into the 90S ribonucleoprotein particle (90S pre-RNPs) containing ribosomal proteins, non-ribosomal proteins and snoRNA-containing ribonucleoprotein particles (snoRNPs). The 90S pre-RNPs are processed into intermediates, which finally give rise to mature 40S and 60S ribosomal subunits ( 1 ).
|
16738141_p0
|
16738141
|
INTRODUCTION
| 4.307831 |
biomedical
|
Study
|
[
0.99933260679245,
0.0003600791096687317,
0.00030718615744262934
] |
[
0.9528560042381287,
0.03506505861878395,
0.011591248214244843,
0.0004876819148194045
] |
en
| 0.999997 |
Ribosome biogenesis is the major metabolic challenge of rapidly proliferating cells, particular in tumour cells, as it consumes up to 80% of the total energy. However, little is known about the molecular mechanism that ensure the equilibrium between cell division and ribosome biogenesis required for balanced cell proliferation ( 1 ). Recently it has become evident that ribosome synthesis is cell cycle controlled and sensitive to growth factor and nutrient signalling, and likewise inhibited upon stress signals. Interestingly, several pivotal regulators of cell cycle progression and senescence, such as p19ARF reside within the nucleolus and are also involved in the control of ribosome biogenesis. Moreover, nucleolar proteins like nucleophosmin not only function in the maturation of ribosomes, but are also implicated in the control of the tumour suppressors p53 and p19ARF ( 2 , 3 ). Noteworthy, dysfunction of nucleophosmin is frequently associated with acute myeloid leukaemia and heterozygous mice develop myelodysplastic syndromes ( 4 , 5 ). In conclusion, the recent years have unravelled remarkable links between the nucleolus and cell cycle regulation, thus underlining the importance of the nucleolus far beyond the production of ribosomes.
|
16738141_p1
|
16738141
|
INTRODUCTION
| 4.751624 |
biomedical
|
Review
|
[
0.996336817741394,
0.001829382381401956,
0.00183389603625983
] |
[
0.26490676403045654,
0.002233818406239152,
0.7318065762519836,
0.0010527924168854952
] |
en
| 0.999998 |
The nucleolus owns a particular ability in sensitizing cellular stress after ultraviolet (UV) radiation of cells. Using micropore irradiation, Rubbi and Milner ( 6 ) demonstrated that large amounts of nuclear DNA damage failed to stabilize p53 unless the nucleolus was affected. In addition, forcing nucleolar disruption by anti-upstream binding factor (UBF) antibody microinjection (in the absence of DNA damage), different chemotherapeutic drugs, or cre-mediated deletion of the Pol I specific transcription factor TIF-IA also caused p53 stabilization and a p53-dependent cell cycle arrest. This suggests that the nucleolus is a stress sensor responsible for the maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress ( 6 , 7 ).
|
16738141_p2
|
16738141
|
INTRODUCTION
| 4.248592 |
biomedical
|
Study
|
[
0.9995817542076111,
0.00021555945568252355,
0.00020271808898542076
] |
[
0.998848557472229,
0.0002834133920259774,
0.0007985847187228501,
0.00006935528654139489
] |
en
| 0.999996 |
How does nucleolar stress result in the activation of cell cycle check points? Several mechanisms have been proposed that link ribosome biogenesis to the cell cycle machinery in mammalian cells. A central player in all models is Mdm2, a p53-specific ubiquitin ligase. Disturbance of ribosome biogenesis may decrease the demand for ribosomal proteins and thus lead to an excess of free ribosomal proteins as L5, L11 and L23, which directly bind and inactivate Mdm2 resulting in the accumulation of p53 ( 8 – 12 ). Alternatively, export of ribosomal subunits to the cytoplasm may be a critical step for p53 degradation, which does not take place, if rRNA processing is inhibited ( 13 ). However, other models cannot be excluded.
|
16738141_p3
|
16738141
|
INTRODUCTION
| 4.401766 |
biomedical
|
Study
|
[
0.9994990825653076,
0.0002495136286597699,
0.00025140130310319364
] |
[
0.9825950860977173,
0.002753651700913906,
0.014429870992898941,
0.0002212855324614793
] |
en
| 0.999996 |
We have recently described the nucleolar complex PeBoW, consisting of Pes1 (Pescadillo), Bop1 (block of proliferation) and WDR12 (WD-repeat protein) in mammalian cells. Knockdown of WDR12 by siRNA technology or expression of a dominant-negative WDR12 mutant blocked processing of the 32S pre-rRNA, evoked stabilization of p53 and induced a strong cell cycle arrest ( 14 ). Likewise, expression of dominant-negative mutants of other members of the complex, Pes1 and Bop1, inhibit rRNA processing and cell cycle progression ( 15 , 16 ).
|
16738141_p4
|
16738141
|
INTRODUCTION
| 4.273264 |
biomedical
|
Study
|
[
0.9995805621147156,
0.00020062668772879988,
0.00021882746659684926
] |
[
0.9990828037261963,
0.00032705749617889524,
0.000519944413099438,
0.00007019215991022065
] |
en
| 0.999997 |
The structure and function of the PeBoW-complex appears to be highly conserved throughout evolution. A homolog complex consisting of Nop7 (Yph1p), Erb1 and Ytm1p has been identified in yeast. As in mammals, mutants of Nop7 and Ytm1 inhibit rRNA processing and cell cycle progression ( 17 ). Mutations in Ytm1 disrupt interactions between Ytm1 and Erb1, destabilize the heterotrimer, and significantly reduce association of all three proteins with 66S pre-ribosomes ( 18 ).
|
16738141_p5
|
16738141
|
INTRODUCTION
| 4.354407 |
biomedical
|
Study
|
[
0.9995972514152527,
0.0001681046123849228,
0.0002346215769648552
] |
[
0.9982739686965942,
0.0006376306992024183,
0.0010007726959884167,
0.0000875820915098302
] |
en
| 0.999994 |
Even though a function of the PeBoW-complex in processing of the 32S rRNA precursor is established, the structure of the complex and the role of its components Pes1, Bop1 and WDR12 in other cellular processes is largely unresolved e.g. mouse embryos lacking Pes1 arrest at morula stages of development, their nucleoli fail to differentiate and accumulation of ribosomes is inhibited, suggesting an essential role of Pes1 for ribosome biogenesis and nucleologenesis ( 19 ). Overexpression of Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar growth but not in inducing cell immortalization ( 20 ). Transient depletion of Pes1 resulted in an increase of abnormal mitoses with appearance of binucleate or hyperploid cells, of cells with multipolar spindles and of aberrant metaphase plates ( 21 ). Thus, Pes1 appears to be involved in multiple cellular processes, yet it is unclear, whether all these processes require the PeBoW-complex.
|
16738141_p6
|
16738141
|
INTRODUCTION
| 4.353517 |
biomedical
|
Study
|
[
0.999528169631958,
0.00021873755031265318,
0.0002530326892156154
] |
[
0.9935958981513977,
0.00038172490894794464,
0.005913543049246073,
0.00010888500401051715
] |
en
| 0.999997 |
Recently, the interaction of Pes1 transposon insertion mutants with Bop1 protein has been studied. Several of the mutants displayed a dominant-negative phenotype for rRNA processing. Interestingly, the dominant-negative phenotype required the interaction of the mutant Pes1 with Bop1, while Pes1 mutants, which did not interact with Bop1, failed to induce a dominant-negative phenotype ( 15 ), suggesting that Pes1 mutants might act only in the context of the PeBoW-complex.
|
16738141_p7
|
16738141
|
INTRODUCTION
| 4.163058 |
biomedical
|
Study
|
[
0.9993553757667542,
0.00021369308524299413,
0.00043088317033834755
] |
[
0.9993610978126526,
0.0002279604523209855,
0.00036255206214264035,
0.0000483798939967528
] |
en
| 0.999996 |
Here we report the generation of a set of Pes1 deletion mutants and their interaction with the PeBoW-complex. Two mutants with a N- and C-terminal deletion displayed a dominant-negative effect on rRNA processing and cell growth. Both mutants were incorporated into the PeBoW-complex and induced accumulation of p53. Our data suggests that essential cellular functions of Pes1 are mediated by its incorporation into the PeBoW-complex.
|
16738141_p8
|
16738141
|
INTRODUCTION
| 4.1732 |
biomedical
|
Study
|
[
0.9994282126426697,
0.00032790121622383595,
0.00024387749726884067
] |
[
0.9994043111801147,
0.0002346362452954054,
0.000280549778835848,
0.00008046276343520731
] |
en
| 0.999996 |
Pes1 was cloned from human cDNA using the following primers: HsPescadillo fwd: 5′-GCCACCATGGGAGGCCTTGAGAAGAAG-3′; HsPescadillo bwd: 5′-CTCCGGCCTTGCCTTCTTGGCCTTC-3′. Cloning into the modified pUC18 vector resulted in the addition of a C-terminal HA-tag to the Pes1 open reading frame. The mutant Pes1-HA alleles were created by standard techniques of molecular biology using restriction enzymes and site directed mutagenesis. Pes1-HA wild-type and mutants were cloned into the vector pRTS-1 using the SfiI restriction site ( 22 ).
|
16738141_p9
|
16738141
|
Cloning/plasmids
| 4.183811 |
biomedical
|
Study
|
[
0.9995545744895935,
0.0002089824847644195,
0.00023645862529519945
] |
[
0.9987450838088989,
0.0008824963006190956,
0.00028396869311109185,
0.00008841659291647375
] |
en
| 0.999997 |
TGR-1 rat fibroblasts (provided by J. Sedivy, Brown University, Providence, RI) and U2OS osteosarcoma cells were cultured in DMEM with 10% FBS at 8% CO 2 . For generation of polyclonal cell lines, 6 × 10 5 cells were transfected with 7.5 µg pRTS-1 plasmids using Polyfect (QIAGEN) and selected in the presence of 200 µg/ml hygromycin B for 10 to 14 days. Conditional gene expression was induced with 1 µg/ml doxycycline. The percentage of induced cells was monitored by FACS analysis for EGFP expression.
|
16738141_p10
|
16738141
|
Tissue culture
| 4.129915 |
biomedical
|
Study
|
[
0.9995337724685669,
0.0002765831886790693,
0.00018967254436574876
] |
[
0.9972856044769287,
0.002231455408036709,
0.00036709432606585324,
0.00011579564306885004
] |
en
| 0.999998 |
BrdU light assays were performed essentially as described previously ( 23 ). Briefly, stable polylclonal TGR-1 cells were seeded in the presence of 1 µg/ml doxycycline at a density of 10 5 cells per 100 mm well. After 30 h seeding, the cells were incubated with 100 µM BrdU and doxycycline for 72 h. Culture medium was then removed and replaced by medium containing doxycycline and Hoechst 33 258 at 2 µg/ml for 3 h. Finally, cells were placed on sheet of glass 11 cm above a 30 W fluorescent daylight bulb and irradiated from beneath for 15 min. Cells were washed in phosphate-buffered saline (PBS) two times and regular culture medium without doxycycline was added.
|
16738141_p11
|
16738141
|
BrdU light assay
| 4.121247 |
biomedical
|
Study
|
[
0.9995020627975464,
0.0002616794081404805,
0.0002362796221859753
] |
[
0.998019814491272,
0.0014898157678544521,
0.00040470826206728816,
0.00008566689939470962
] |
en
| 0.999998 |
Total RNA was isolated from TGR-1 cells using Trifast (PeqLab). A total of 2 µg of RNA were separated on a 1% agarose-formaldehyde gel and blotted on Hybond N + membranes (GE Healthcare). The following 32 P-labelled oligonucleotides were used to visualize rRNA precursors: ITS-1, 5′-CCGGAGAGATCACGTACCACCCCCGGTGCACACGAGATCACGGAGCCG-3′; ITS-2, 5′-GGAGCGGTCGGCCCCGGTAGAGGGAGCGGGGGAGGAGAGGGACGCGAG-3′; 18S, 5′-CACCCGTGGTCACCATGGTAGGCACGGCGACTACCATCGAAAGTTGATAG-3′.
|
16738141_p12
|
16738141
|
RNA analysis and 32 P in vivo labelling
| 4.090641 |
biomedical
|
Study
|
[
0.9996150732040405,
0.00018213849398307502,
0.00020287398365326226
] |
[
0.9973222613334656,
0.002184443874284625,
0.00039712805300951004,
0.00009623239020584151
] |
en
| 0.999996 |
For metabolic labelling of rRNA, TGR-1 cells were induced with doxycycline for 24 h, followed by pre-incubation in phosphate-free DMEM (Gibco) with dialyzed FBS (Gibco) for 30 min. The medium was then replaced by phosphate-free DMEM/10% dialyzed FBS containing 15 µCi/ml 32 P-orthophosphate (Amersham). After 60 min the radioactive medium was removed and cells were incubated in regular DMEM/10% FBS for indicated times. A total of 2 µg of RNA were separated on 1% agarose-formaldehyde gels. After electrophoresis, gels were placed on whatman-paper and dried at 80°C under vacuum suction. Dried agarose gels were exposed to regular X-ray films (Kodak) and rRNA was visualized by autoradiography. A PhosphoImager (Fuji) was used for the quantification of signal intensities.
|
16738141_p13
|
16738141
|
RNA analysis and 32 P in vivo labelling
| 4.131366 |
biomedical
|
Study
|
[
0.9995204210281372,
0.0002786177210509777,
0.00020097345986869186
] |
[
0.9973573088645935,
0.0020618790294975042,
0.00047154189087450504,
0.00010930700955213979
] |
en
| 0.999997 |
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