PRC 1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase
2016; Springer Nature; Volume: 18; Issue: 2 Linguagem: Inglês
10.15252/embr.201642650
ISSN1469-3178
AutoresBruno Polak, Patrik Risteski, Sonja Lesjak, Iva M. Tolić,
Tópico(s)Cellular transport and secretion
ResumoScientific Report27 December 2016Open Access Source DataTransparent process PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase Bruno Polak Bruno Polak Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Patrik Risteski Patrik Risteski Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Sonja Lesjak Sonja Lesjak Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Iva M Tolić Corresponding Author Iva M Tolić [email protected] orcid.org/0000-0003-1305-7922 Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Bruno Polak Bruno Polak Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Patrik Risteski Patrik Risteski Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Sonja Lesjak Sonja Lesjak Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Iva M Tolić Corresponding Author Iva M Tolić [email protected] orcid.org/0000-0003-1305-7922 Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Author Information Bruno Polak1,‡, Patrik Risteski1,‡, Sonja Lesjak1 and Iva M Tolić *,1 1Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia ‡These authors contributed equally to this work *Corresponding author. Tel: +385 1 4571 370; E-mail: [email protected] EMBO Reports (2017)18:217-230https://doi.org/10.15252/embr.201642650 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In the mitotic spindle, kinetochore microtubules form k-fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross-linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k-fibers. However, the relationship between overlap bundles and k-fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k-fibers. We found that the number of PRC1-GFP-labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live-cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1-GFP-labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1-GFP. PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle. Synopsis In the metaphase spindle, most PRC1-decorated overlap microtubule bundles are linked with a pair of sister kinetochores, acting as a bridge between k-fibers. The number of PRC1-decorated bundles per spindle correlates with the variable chromosome numbers in HeLa cells, indicating a one-to-one relationship. PRC1 is bound to spindle microtubules during metaphase. Kinetochore movement is specifically correlated with the movement of the coupled PRC1-labeled fiber. PRC1 knockdown reduces bridging fiber thickness and interkinetochore distance throughout the spindle. Introduction The mitotic spindle is a highly dynamic and complex machinery that orchestrates progression through mitosis and cytokinesis 1. Kinetochores are protein complexes assembled on two sides of the chromosome's centromeric region, which are necessary for interaction of spindle microtubules with chromosomes and proper chromosome segregation 2. Microtubules that bind to kinetochores with their plus ends become k-fibers that can exert forces on chromosomes 3. Meanwhile, non-kinetochore microtubules interact in an antiparallel fashion in the central part of the spindle, thus forming overlap regions. In addition to k-fibers, it is thought that non-kinetochore microtubules comprise the majority of microtubules in mammalian spindles 4. During metaphase, non-kinetochore microtubules bundle together 30–50 nm apart in groups of 2–6, with antiparallel interactions apparently preferred 4. Their antiparallel region contains motor and non-motor cross-linking proteins. Motors contribute to antiparallel microtubule sliding, whereas passive cross-linkers take part in maintenance of the overlap integrity 56. PRC1 is a conserved non-motor cross-linking protein localized in the antiparallel overlaps of microtubules in vitro 789 and of the spindle midzone 1011121314 where it plays an essential role in regulating its formation and cytokinesis 15. Orthologs of PRC1 with conserved function include Ase1 (anaphase spindle elongation 1) in yeasts 1416, SPD-1 (spindle defective 1) in Caenorhabditis elegans 17, Feo (Fascetto) in Drosophila melanogaster 18, and MAP65 (microtubule-associated protein 65) in plants 19, all of which fall in a conserved family of non-motor microtubule-associated proteins (MAPs). Even though it is widely accepted that during metaphase-to-anaphase transition a conspicuous network of antiparallel non-kinetochore interdigitating microtubules assembles between separating chromosomes 41115, very little is known of PRC1-containing overlap fibers in metaphase. The affinity of PRC1 to bind to microtubules is regulated by phosphorylation and dephosphorylation events precisely timed throughout mitosis 1120. Due to dephosphorylation, PRC1 forms dimers and binds to microtubules to form cross-linkages between neighboring interdigitating fibers 1121. It was shown that only in anaphase an organized central spindle midzone forms between separating chromosomes and consists of a dense network of overlapping antiparallel microtubules cross-linked by PRC1 4111220. By combining structural flexibility and rigidity, PRC1 stabilizes antiparallel overlaps while not impeding sliding between them 22. We have recently shown that a bundle of overlap microtubules which contains PRC1 links outermost sister k-fibers 232425. This fiber, termed "bridging fiber", balances the tension between sister kinetochores and helps the spindle to obtain a rounded shape. However, the fraction of overlap bundles that function as bridging fibers, as well as the fraction of kinetochore pairs that have a microtubule bridge between them, remained unexplored. Here, we show that the number of PRC1-labeled overlap fibers is nearly the same as the number of kinetochore pairs in a spindle during metaphase. Dynamic properties of PRC1-labeled bundles and sister kinetochores revealed that the majority of overlap bundles are associated with a pair of sister kinetochores. The endogenous PRC1 visualized by immunofluorescence was predominantly localized in the central part of metaphase spindles and co-localized with bridging fibers throughout the spindle. PRC1 knockdown by siRNA generated thinner bridging fibers, as well as a reduced interkinetochore distance. Taken together, our results indicate that in metaphase nearly all overlap fibers exist as bridging fibers between pairs of sister kinetochores and that PRC1 plays a key role in linking antiparallel microtubules in the bridging fibers. Results and Discussion The number of PRC1-labeled bundles in a metaphase spindle is correlated with the number of chromosomes per cell To study the relationship between overlap bundles and kinetochores during metaphase, we used HeLa cells stably expressing PRC1-GFP from a bacterial artificial chromosome (BAC) 26, which were transiently transfected with mRFP-CENP-B to visualize kinetochores. We acquired z-stacks of images that cover a whole metaphase spindle in fixed cells. Metaphase was identified by the alignment of sister kinetochores on the metaphase plate, and kinetochores were defined as sisters if the bi-orientation was estimated within individual planes. We measured the number of PRC1-labeled overlap bundles and kinetochore pairs by using two approaches: spindles with their long axis oriented roughly parallel to the imaging plane (horizontal spindles) and spindles with their long axis oriented roughly perpendicular to the imaging plane (vertical spindles). In the first approach, we analyzed horizontal spindles, which was the most frequent spindle orientation (Figs 1A and EV1A, Video EV1). Individual PRC1-labeled overlaps appeared as slightly curved lines with a broader central part, gradually narrowing in both directions toward the spindle poles (Figs 1A and EV1A). The number of PRC1-labeled bundles per spindle was 63 ± 2 and the number of kinetochore pairs 59 ± 2 (all results are mean ± s.e.m. unless otherwise indicated, n = 29 spindles). These numbers may be somewhat underestimated due to occasional overlaying of neighboring sister kinetochores or neighboring PRC1-labeled bundles in the images. We conclude that the mean number of PRC1-labeled bundles is roughly the same as the mean number of chromosomes. Figure 1. Most PRC1-labeled overlap bundles are coupled with sister kinetochores in metaphase A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z = 0, two images below, z = −4 μm and z = −2 μm, and above, z = +3 μm and z = +5 μm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A. B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in (A). Additional z-slices of this spindle are shown in Fig EV1B. C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits. D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labeled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1,786 kinetochore-PRC1 pairs (n = 29 cells) and vertical spindles 1,133 kinetochore-PRC1 pairs (n = 16 cells). E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles. G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle. Data information: Scale bars, 2 μm; n, number of cells; R2, coefficient of determination; P, P-value from a t-test. Source data are available online for this figure. Source Data for Figure 1 [embr201642650-sup-0005-SDataFig1.xls] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Additional characterization of kinetochore-PRC1 pairs A–C. Individual z-stack images acquired in fixed HeLa cells stably expressing PRC1-GFP (green) and transiently mRFP-CENP-B (magenta). Z-stack images of (A) horizontal spindle (same spindle shown in Fig 1A), (B) vertical spindle (same spindle shown in Fig 1B), and (C) spindle imaged with different settings (line averaged 16 times with 200 nm spacing between z-slices). Regardless of depicted approaches, images reveal majority of sister kinetochores positioned in close proximity of PRC1-labeled fibers. D. Graph shows correlation between the number of sister kinetochores and PRC1 signals counted in the central z-plane of fixed HeLa cells in metaphase. E. Pie chart shows what fraction of PRC1-labeled fibers and sister kinetochores are linked (blue) in the central z-plane of fixed HeLa cells in metaphase. Percentage of single kinetochores and single PRC1 signals are shown in magenta and green, respectively. F. A cross section of vertical spindles with 81 (left) and 67 (right) chromosomes. Data information: Scale bars, 2 μm; n, number of cells; R2, coefficient of determination; P, P-value from a t-test. Download figure Download PowerPoint In the second approach, we analyzed vertical spindles, which were found occasionally in the field of view (Figs 1B and EV1B, Video EV2). The average number of PRC1-labeled bundles per spindle was 75 ± 3 and the number of kinetochore pairs 72 ± 3 (n = 16 spindles). Images of vertically oriented spindles confirmed our observation in horizontally oriented spindles that the mean number of PRC1-labeled bundles is nearly the same as the number of kinetochore pairs. In this approach, the number of sister kinetochores and PRC1-labeled fibers was larger due to less frequent overlaying of neighboring bundles. Notably, the majority of kinetochores were positioned right next to PRC1-labeled bundles. In HeLa cells, both the number of chromosomes and their structure show abnormalities 272829. The number of chromosomes has been shown to vary between 56 and 179 30313233. We found the number of kinetochore pairs per cell to be in the range of 38–96 (Fig 1C), in agreement with previous studies. We used this inherently unstable HeLa karyotype to determine whether the number of PRC1-labeled bundles is correlated with the number of chromosomes (kinetochore pairs). Indeed, we found the number of PRC1-labeled bundles to be roughly equal to the number of kinetochore pairs in individual spindles, both horizontal and vertical ones (Fig 1C), which further prompted us to investigate their association. Nearly all overlap bundles are associated with sister kinetochores, acting as a bridge between them Next, we explored how many PRC1-labeled overlap bundles are associated with kinetochore pairs and vice versa. A PRC1-labeled bundle and a kinetochore pair were defined as associated if the distance between them was smaller than 0.3 μm (see Materials and Methods), based on a previous measurement of this distance for outermost kinetochores 23. We found that > 90% of PRC1-labeled fibers were associated with a kinetochore pair, both in horizontal and vertical spindles (n > 1,000 PRC1-labeled bundles in each approach, Fig 1D). Conversely, our analysis revealed only a small fraction of PRC1-labeled bundles and kinetochore pairs that were not mutually linked (Fig 1D). To test whether the association between PRC1-labeled bundles and kinetochore pairs depends on the number of chromosomes in a cell, we grouped the cells into two groups: those with ≤ 70 and those with > 70 kinetochore pairs. In each group, we found that > 90% of PRC1-labeled fibers were associated with a kinetochore pair (Fig 1D), indicating that the association between PRC1-labeled bundles and kinetochores does not depend on the number of chromosomes in the spindle. Moreover, by using different imaging settings (higher signal-to-noise ratio, 200 nm spacing between z-slices, see Fig EV1C) to image only the central planes of horizontal spindles, we obtained similar results as above (n = 13 spindles, Fig EV1C–E). We conclude that nearly all PRC1-labeled overlap bundles are associated with pairs of sister kinetochores, acting as bridges that link sister k-fibers. Spindles with more chromosomes have a larger length and width Our finding that spindles with more chromosomes contain more overlap bundles prompted us to ask how spindle length and width vary to accommodate these differences. To answer this question, we used only horizontal spindles because spindle length and width could not have been precisely measured in vertically oriented spindles due to the variable tilt of the spindle long axis. We measured the average spindle length to be 10.65 ± 0.16 μm, and the width 11.21 ± 0.25 μm (n = 29), consistent with previous measurements 2334. Both spindle length (Fig 1E) and width (Fig 1F) increased with the number of coupled kinetochore pairs and PRC1-labeled fibers. The increase in width was similar to the increase in length, which indicates that spindles accommodate a larger number of chromosomes by increasing their length and width to a similar extent. To examine the spatial distribution of bridging fibers in spindles with different numbers of chromosomes, we measured the density of PRC1-labeled fibers coupled with kinetochores, that is, their number per unit area in the equatorial plane of horizontal and vertical spindles. We found that the density does not depend significantly on the number of coupled pairs (Figs 1G and EV1F). Thus, by accommodating its width the spindle maintains the neighboring bridging fibers at similar distances regardless of the total number of chromosomes in the spindle. Dynamic properties of PRC1-labeled fibers and kinetochores in vertical spindles confirm their association In order to understand the dynamic interplay between neighboring PRC1-labeled fibers and kinetochores, we acquired time series of vertical spindles and tracked individual bundles and kinetochores in the spindle (Fig 2A and Video EV3). We analyzed the dynamics in the transversal cross section and found that in the majority of the associated pairs, the PRC1-labeled bundle and the kinetochores moved along identical trajectories or moved in the same direction and passed similar distances, whereas some pairs showed movements in mutually independent directions (Fig 2B). Figure 2. Dynamic properties of PRC1-labeled bundles and kinetochores in vertical spindles confirm their association Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane. Examples of trajectories, with respect to the spindle's center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in (A) that moved together for at least 200 s. Dots represent starting points of trajectories, t = 0 s. Trajectories finish at t = 200 s. Gray circle represents the center of mass of the spindle. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n = 226 pairs in five cells). A small fraction of PRC1-labeled fibers (green, n = 32 in five cells) and kinetochores (magenta, n = 16 in five cells) did not move together. Correlation coefficients between trajectories (with respect to the spindle's center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled with the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (gray); n, the number of trajectories from three cells. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in (A). Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle. Data information: Scale bars, 2 μm. Source data are available online for this figure. Source Data for Figure 2 [embr201642650-sup-0006-SDataFig2.xlsx] Download figure Download PowerPoint In order to distinguish the fraction of overlap bundles and kinetochores that moved together, we tracked all PRC1-labeled fibers and kinetochores within the spindle. We observed a dynamic interaction between PRC1-labeled fibers and kinetochores in their vicinity. A PRC1-labeled fiber and a kinetochore were termed associated if they moved together for at least five time frames (~1 min) (n = 274 associated and individual PRC1-labeled fibers and kinetochores from five cells). We found 82.5 ± 2.7% (n = 226) of mutually associated fibers and kinetochores, whereas 11.7 ± 2.3% (n = 32) of PRC1-labeled fibers did not have a coupled kinetochore with which they moved along the same or similar trajectories, and 5.8 ± 0.8% (n = 16) of kinetochores were observed as free of any PRC1-labeled fiber (Figs 2C and EV2A and B). Click here to expand this figure. Figure EV2. Dynamics of PRC1-labeled bundles and kinetochores in the spindle cross section Dynamics of PRC1-labeled bundles in live HeLa cells expressing PRC1-GFP and mRFP-CENP-B. The distance between PRC1-labeled bundles and center of the mass of the spindle (CM) is plotted over t = 150 s. n, number of pairs. Dynamics between PRC1-labeled bundles and corresponding kinetochores in live HeLa cells expressing PRC1-GFP and mRFP-CENP-B. The distance between them is plotted over t = 150 s. n, number of pairs. Examples of trajectories of a kinetochore pair (magenta), its coupled PRC1-labeled bundle (green), nearest PRC1 neighbors (blue), next nearest PRC1 neighbors (black), and randomly chosen PRC1 bundles (gray) with respect to the spindle's center of the mass (CM) over t = 200 s in three cells. n, number of PRC1-labeled bundles. Download figure Download PowerPoint To quantify to which extent the kinetochores move in a correlated manner with different PRC1-labeled fibers in the spindle, we performed cross-correlation analysis 35 on the acquired trajectories. Our analysis revealed high correlation of movement between a kinetochore pair and the coupled PRC1-labeled fiber, with a median correlation coefficient of 0.93 (n = 12). The correlation coefficient decreased with an increasing distance between the PRC1-labeled fiber and the kinetochore pair: The median correlation coefficient was 0.61 for the nearest neighbor fiber that was not coupled with the kinetochore pair, 0.35 for the next nearest neighbor, and 0.02 for a randomly chosen fiber (n = 14–18 fibers in each group, Fig 2D; examples of trajectories are shown in Fig EV1C). These results indicate that kinetochores typically move together with their coupled PRC1-labeled fiber, whereas the correlation of movement with neighboring fibers decreases in a distance-dependent manner and vanishes for remote fibers. To determine the dynamic events between PRC1-labeled bundles and kinetochores in more detail, we analyzed only the outermost PRC1-labeled fibers and kinetochores, because those were most easily distinguished from their neighbors. Within this group, we observed several types of behavior of neighboring PRC1-labeled fibers and kinetochores. Due to the better clarity of events in this region, we used a more strict criterion for determining the interaction of PRC1-labeled fibers and kinetochores: PRC1-labeled fiber and kinetochore were termed associated if they were moving along the same or similar trajectories during the entire acquired video (~5 min). We found that 65.7 ± 4.1% of PRC1-labeled fibers and kinetochores were mutually associated. Within this group, we included the following occasional events as well: one PRC1-labeled fiber moved together with two kinetochores which do not seem to be sisters; PRC1-labeled fiber moved together with sister kinetochores until they both disappeared from the imaged planes in the z-direction at the same time. Other scenarios in which we term a kinetochore and PRC1-labeled fiber uncoupled (34.3 ± 4.1%) were as follows: first, the kinetochore seemed to be free of any PRC1-labeled fiber, and after a certain time, a PRC1 signal appeared and they started moving together; a kinetochore and PRC1-labeled fiber moved together and eventually separated to a distance greater than 0.3 μm; kinetochore first moved alone and at a certain time point moved to the vicinity of a neighboring PRC1 and they started moving together; a PRC1-labeled fiber merged with the neighboring bundle and they appeared as a single PRC1-labeled fiber; kinetochore moved with the PRC1 until PRC1 disappeared; kinetochore moved with the PRC1, at one point separated from it, and subsequently associated with the same bundle to continue moving together. For events described above, see Fig 2E and Video EV3. The observed events reveal the dynamic nature of the interactions between PRC1-labeled fibers and kinetochores. However, most of the time PRC1-labeled bridging fibers and kinetochores are in close proximity and move along the same or similar trajectories. Endogenous PRC1 localizes to the central part of the bridging fibers in metaphase The results described above were obtained on a cell line that expresses PRC1-GFP in addition to the endogenous PRC1. Western blot analysis showed 1.64 ± 0.10 times higher expression of PRC1 in this cell line compared with unlabeled HeLa cells (n = 6 independent experiments, P = 0.0004) while tubulin-GFP cell line showed the same expression level of PRC1 as determined in unlabeled cells (Fig EV3A and B, and Table 1). Previous studies have shown that PRC1 exhibits a slightly different localization when overexpressed, for example, a substantial fraction of the protein is cytosolic and localizes to brightly stained ring-shaped arrays around the interphase nucleus 11. Thus, we set out to define the localization and distribution of endogenous PRC1 in metaphase spindles and compare it with the localization of PRC1-GFP. Click here to expand this figure. Figure EV3. Additional characterization of PRC1-labeled bundles A, B. Western blot for PRC1 in various HeLa cell lines and conditions, as denoted in the figure. PRC1 expression in PRC1-GFP HeLa cells was 60.97 ± 10.01% higher than in unlabeled cells. Synchronized cells were lysed in lysis buffer 29 h after transfection. Cell lysates were subjected to SDS-PAGE (12% polyacrylamide), transferred on to a nitrocellulose membrane and immunoblotted with anti-PRC1 and anti-GAPDH antibodies used as a loading control. Intensities of the Western blot bands of endogenous PRC1 isoforms (61–71 kDa) and PRC1-GFP (98 kDa) were quantified as described in Materials and Methods. P-value was 0.0004. C. From left to right: image of the immunostained PRC1 in the spindle of a HeLa cell expressing tubulin-GFP and immunostained for PRC1; dependence of the length of immunostained PRC1 signal in HeLa cells expressing tubulin-GFP and immunostained for PRC1 on the distance from the spindle long axis; dependence of the signal intensity I of immunostained PRC1 signal in HeLa cells expressing tubulin-GFP and immunostained for PRC1 on the distance from the spindle long axis; dependence of Icross of immunostained PRC1 signal in HeLa cells expressing tubulin-GFP and immunostained for PRC1 on the distance from the spindle long axis. D. From left to right: image of PRC1-GFP in the spindle of a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B; dependence of the length of PRC1-GFP signal in fixed HeLa cells expressing PRC1-GFP on the distance from the spindle long axis; dependence of the signal intensity I of PRC1-GFP signal in fixed HeLa cells expressing PRC1-GFP on the distance from the spindle long axis; dependence of Icross of PRC1-GFP signal in fixed HeLa cells expressing PRC1-GFP and on the distance from the spindle long axis. E. From left to right: image of PRC1-GFP in the spindle of a live HeLa cell expressing PRC1-GFP and mRFP-CENP-B; dependence of the length of PRC1-GFP signal in live HeLa cells expressing PRC1-GFP on the distance from the spindle long axis; dependence of the signal intensity I of PRC1-GFP signal in live HeLa cells expressing PRC1-GFP on the distance from the spindle long axis; dependence of Icross of PRC1-GFP signal in live HeLa cells expressing PRC1-GFP and on the distance from the spindle long axis. Data information: Scale bars, 2 μm; R2, coefficient of determination; P, P-value from a t-test; n, number of bridging fibers; error bars, s.e.m. Source data are available online for this figure. Download figure Download PowerPoint Table 1. Properties of the PRC1-labeled overlap measured by different approaches Tubulin-GFP cells immunostained for PRC1 PRC1-GFP cells fixed PRC1-GFP cells live I/au 1778.78 ± 101.37 311.34 ± 16.49 343.69 ± 30.23 Icross/au 53.03 ± 4.85 9.56 ± 0.44 15.01 ± 1.51 LPRC1/μm 4.95 ± 0.18 5.49 ± 0.17 4.64 ± 0.08 Spindle length/μm ND 10.65 ± 0.16 ND Spindle width/μm ND 11.21 ± 0.25 ND Au, arbitrary units; ND, not determined. All values are given as mean ± s.e.m. and measured as in Materials and Methods. We used HeLa cells stably expressing tubulin-GFP, which allowed us to identify the k-fibers and bridging fibers, and immunostained them for PRC1 (see Materials and Methods). We were interested in the distribution of PRC1 i
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