Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends
2018; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês
10.15252/embr.201846196
ISSN1469-3178
AutoresJohn C. Meadows, Liam J. Messin, Anton Kamnev, Theresa C. Lancaster, Mohan K. Balasubramanian, Robert A. Cross, Jonathan Millar,
Tópico(s)Ubiquitin and proteasome pathways
ResumoScientific Report11 September 2018Open Access Source DataTransparent process Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends John C Meadows Corresponding Author John C Meadows [email protected] orcid.org/0000-0001-7024-3014 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Liam J Messin Liam J Messin Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Anton Kamnev Anton Kamnev Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Theresa C Lancaster Theresa C Lancaster Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Mohan K Balasubramanian Mohan K Balasubramanian Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Robert A Cross Robert A Cross orcid.org/0000-0002-0004-7832 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Jonathan BA Millar Corresponding Author Jonathan BA Millar [email protected] orcid.org/0000-0002-9930-3989 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author John C Meadows Corresponding Author John C Meadows [email protected] orcid.org/0000-0001-7024-3014 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Liam J Messin Liam J Messin Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Anton Kamnev Anton Kamnev Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Theresa C Lancaster Theresa C Lancaster Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Mohan K Balasubramanian Mohan K Balasubramanian Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Robert A Cross Robert A Cross orcid.org/0000-0002-0004-7832 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Jonathan BA Millar Corresponding Author Jonathan BA Millar [email protected] orcid.org/0000-0002-9930-3989 Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Author Information John C Meadows *,1,‡, Liam J Messin1,‡, Anton Kamnev1, Theresa C Lancaster1, Mohan K Balasubramanian1, Robert A Cross1 and Jonathan BA Millar *,1 1Division of Biomedical Sciences, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 24 76150414; E-mail: [email protected] *Corresponding author. Tel: +44 24 76150414; E-mail: [email protected] EMBO Reports (2018)19:e46196https://doi.org/10.15252/embr.201846196 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 fission yeast, the lengths of interphase microtubule (iMT) arrays are adapted to cell length to maintain cell polarity and to help centre the nucleus and cell division ring. Here, we show that length regulation of iMTs is dictated by spatially regulated competition between MT-stabilising Tea2/Tip1/Mal3 (Kinesin-7) and MT-destabilising Klp5/Klp6/Mcp1 (Kinesin-8) complexes at iMT plus ends. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus ends of iMT bundles, thereby restricting access to the plus ends by Klp5/Klp6/Mcp1, which accumulate behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus ends and MT catastrophe. These data show that in vivo, whilst an iMT length-dependent model for catastrophe factor accumulation has validity, length control of iMTs is an emergent property reflecting spatially regulated competition between distinct kinesin complexes at the MT plus tip. Synopsis During interphase, the de-stabilising Klp5/Klp6/Mcp1 (Kinesin-8) complex accumulates behind the stabilising Tea2/Tea1/Tip1 (Kinesin-7) complex at microtubule plus tips. When microtubules encounter the cell end, Kinesin-8 invades the space occupied by Kinesin-7 to trigger microtubule catastrophe. Mcp1 is an interphase specific component of the fission yeast Klp5/Klp6 (Kinesin-8) complex. Mcp1 is required for Kinesin-8-mediated microtubule de-stabilisation but not the processivity of the motor complex. Klp5/Klp6/Mcp1 accumulates behind Tea2/Tea1/Tip1 (Kinesin-7) complex, which blocks Kinesin-8 access to microtubule plus tips until the microtubule reaches the cell end. Competition between Kinesin-7 and Kinesin-8 ensures microtubule catastrophe occurs at cell ends. Introduction Microtubule (MT) length control is important for multiple cellular processes including vesicle transport, mitotic spindle size, chromosome bi-orientation and ciliary function 1-4. In the fission yeast, Schizosaccharomyces pombe, arrays of interphase microtubules (iMT) grow along the long axis of the cell and undergo catastrophe at cell ends. Interaction of iMTs with the cell end cortex is required to maintain cell polarity and correctly position the nucleus and cell division ring 5-7. The maintenance of cell polarity and positioning of the division ring require transport of the Kelch-repeat protein Tea1 and SH3-domain protein Tea4 to the plus ends of iMTs by association with Tea2 (Kinesin-7) and their deposition at cell ends following interaction of iMTs with the cell end cortex 6, 8-12. Reconstitution and live-cell imaging experiments reveal that association of Tea2 (Kinesin-7) with the growing plus ends of MTs requires two other components, Mal3 (EB1 homologue) and Tip1 (Clip170 homologue) 13-19. The presence of the Tea2/Tip1/Mal3 complex, but not its cargo (Tea1, Tea3 and Tea4), at the MT plus end also prevents premature MT catastrophe in the cytoplasm 20, at least in part through a mechanism whereby Mal3 alters the structural architecture of MTs 21-23. Members of the Kinesin-8 family have attracted particular attention as regulators of MT length because they are both highly processive motors and undergo a conformational switch at the MT plus end that promotes MT disassembly 24-30. These features have given rise to the “antenna model” for MT length control whereby more Kinesin-8 accumulates at the plus ends of longer MTs, thus increasing the likelihood of catastrophe and MT shrinkage 27-29, 31. Fission yeast contains two Kinesin-8 motors, Klp5 and Klp6, that operate as a functional heterocomplex in interphase. Deletion of either gene causes numerous mitotic defects and overgrowth of interphase MTs, which result in defective nuclear positioning and loss of cell polarity, particularly in longer cells 32-36. Timely shrinkage of interphase MTs also requires Mcp1, a +TIP that is distantly related to the Ase1/PRC1/MAP65 family of anti-parallel MT binding proteins 37-39. Although association of Mcp1 with MT plus ends requires Klp6, it remains unclear as to whether Mcp1 functions co-ordinately or in parallel with Klp5/Klp6 to control iMT length 31. In this study, we employ quantitative fluorescence imaging to propose a novel mechanism of MT length control that is based on spatially regulated competition between opposing kinesin motor complexes for binding sites at the MT plus end. Results and Discussion Mcp1 is an interphase-specific component of the Klp5/Klp6 complex We first set out to establish the relation of Mcp1 to the Klp5/Klp6 complex. We find that, like Klp5 and Klp6, the intensity of Mcp1 increases at iMTs plus ends as they grow and dwell at cell ends and decreases as iMTs undergo shrinkage (Figs 1A and EV1A). Binding of Mcp1 to iMT plus ends requires the motor activity of Klp5/Klp6, indicating that Mcp1 is indeed a cargo of the Klp5/Klp6 complex (Fig EV1B; 38). Consistently, Mcp1 binds weakly to Klp5 in co-immunoprecipitates from cell extracts (Fig EV1C). Importantly, by monitoring MT dynamics using GFP-atb2 (α2-tubulin), we find that the dwell time of iMTs at the cell end is extended in the absence of both Klp5 and Klp6 to the same extent as in the absence of Mcp1 and this effect is not additive, indicating that Mcp1 controls destabilisation of iMTs via its association with the Klp5/Klp6 complex (Fig 1B). It should be noted that, as with previous studies, it is not possible to determine whether these fluorescent signals represent individual MTs or bundles of a small number of MTs. Notably though, unlike deletion of either Klp5 or Klp6, loss of Mcp1 does not cause cell polarity defects in elongated cdc25-22 cells (Fig EV1D: 36) and does not influence mitotic timing or accuracy of chromosome segregation (Fig EV2A–E). These functions may instead be due to association of Klp5/Klp6 with PP1, a type-1-phosphatase (Dis2) 40, 41. Consistently, Mcp1 is not required for Klp5 and Klp6 to bind the mitotic spindle or kinetochores during mitosis and is not present in the nucleus during mitosis (Fig EV2F and G). These results indicate that Mcp1 is an interphase-specific regulator of Kinesin-8-mediated interphase MT length control in fission yeast, confirming and extending previous observations 31. Figure 1. Mcp1 is required for control of interphase microtubule stability by Klp5/Klp6 but not for its motility Interphase microtubules (iMTs) (magenta) in fission yeast grow towards the cell end (i), dwell (ii) then shrink (iii). Cells expressing fluorescently tagged α2-tubulin (atb2) were imaged every 5 s and the dwell time of ˜100 individual iMTs recorded within the final 1.1 μm of the cell for each strain. Red bars signify the mean. Kymographs showing fluorescently tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end. MT growth speed was calculated from kymographs from control (n = 16) and ∆mcp1 cells (n = 11), and Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n = 44) and ∆mcp1 cells (n = 32). Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n = 19) or ∆mcp1 cells (n = 14). Mixing experiment to compare fluorescently tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5 μm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n = 44) and ∆mcp1 cells (n = 45) and at the MT plus end in control (n = 64) and ∆mcp1 cells (n = 35) prior to shrinkage. Data information: In (E), data are presented as mean ± s.d. *P < 0.001, n.s. (non-significant) P > 0.05 (Kolmogorov–Smirnov test). In (D) and (F), boxes show the interquartile range with the median represented between the lower and upper quartiles, and whiskers show the highest and lowest values. Source data are available online for this figure. Source Data for Figure 1 [embr201846196-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Mcp1 interacts with Klp5/Klp6 and requires its motor activity to accumulate at microtubule plus ends but is dispensable for cellular polarity control Upper panels showing fluorescently tagged microtubules (MT) and either Klp5-GFP (left panel) or Mcp1-GFP (right panel) imaged every 4.8 s. Dashed yellow line indicates the cell end. Scale bar, 5 μm. Lower panels show the mean intensity ± s.d. of either Klp5-GFP (left panel, n = 20) or Mcp1-GFP (right panel, n = 20) at the plus ends of iMTs. Plots show the mean distance moved over time of GFP puncta associated with growing iMTs from each of the indicated backgrounds. Error bars show standard deviation from five replicates. Log phase cultures of GFP-mcp1 klp5-13Myc cells were harvested and lysed. Proteins were immunoprecipitated from 2 mg of whole cell extract (WCE) using rabbit α-GFP antibodies (I) or pre-immune control (PI), migrated by SDS–PAGE and probed with either sheep α-GFP or mouse α-Myc antibodies. 50 μg of WCE was run and immunoblotted for comparison. Images show cdc25-22 cells (left panel) or cdc25-22 Δklp5 Δklp6 cells (right panel) arrested at the restrictive temperature (35.5°C) for 6 h. Scale bar, 5 μm. Cellular curvature was quantitated, as in the schematic, by measuring both the cell length (length, L) and the distance between cell ends (Euclidean distance, E) and then calculating the ratio (L:E). These ratios, converted to percentages, are displayed on the plot, with red lines showing the mean value. ˜850 cells were measured for each strain. Log phase cultures of cells expressing klp5-GFP (left panels) or klp6-GFP (right panel) in control or Δmcp1 cells were lysed and proteins extracted. 50 μg of each was then migrated by SDS–PAGE, transferred to nitrocellulose membrane and probed with both α-GFP to determine protein level and α-Tat1 to use tubulin as a loading control. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Mcp1 does not control the mitotic functions of Klp5/Klp6 Log phase cultures of control, Δklp5 or Δmcp1 cells expressing fluorescently tagged kinetochore (Fta3) and spindle pole body (Sid4) proteins were imaged. The proportion of pre-anaphase mitotic cells with unseparated kinetochore pairs between poles was determined (PM & M). Log phase cultures of control, Δklp5 or Δmcp1 cells expressing fluorescently tagged cyclin B (Cdc13) and Sid4 were imaged. The proportion of cells with Cdc13-GFP on separated poles and spindles was determined. Log phase cultures of control, Δklp5 or Δmcp1 cells expressing ade6-M210 and carrying the Ch16(ade6-M216) mini-chromosome were grown in media lacking adenine and then individual cells plated onto media with minimal adenine. After 3 days, the proportion of cells that had formed colonies that were ≥ 50% red sectored, having lost the mini-chromosome in the first mitotic division, was determined. Log phase cultures of control, Δklp5, Δdam1 or Δmcp1 cells were serially diluted onto plates containing the indicated concentrations of the MT poison thiabendazole (TBZ) and grown for 3 days. Cells lacking Klp5 have elevated resistance to TBZ, whereas cells lacking Dam1, a component of the DASH complex, display enhanced sensitivity to TBZ. Viability of strains lacking Dam1, Dis2 or Bub3 without either Klp5 or Mcp1. Mitotic cells expressing fluorescently tagged MTs and Klp5/Klp6 in the presence (left panels) or absence (right panels) of Mcp1. Scale bar, 2 μm. Mitotic cell expressing fluorescently tagged MTs and Mcp1. Scale bar, 2 μm. Data information: In (A–C), the mean from three independent experiments is plotted + s.d. Source data are available online for this figure. Download figure Download PowerPoint Mcp1 is required for the destabilisation activity of Klp5/Klp6 but not the motility To further understand the role of Mcp1 in Kinesin-8 function, we examined Klp5/Klp6 motility and accumulation at iMT plus ends in control and Δmcp1 cells by dual camera live-cell imaging (Fig 1C). We find that Klp5/Klp6 walks along the lattice of iMTs at 134 ± 28 nm/s, compared to average iMT growth speed of 69 ± 20 nm/s, and accumulates at iMT plus ends, particularly whilst iMTs dwell at the cell end, and then dissociates following MT catastrophe (Fig 1B–E). In fact, Klp5/Klp6 translocates faster (168 ± 37 nm/s) along the lattice of iMTs in the absence of Mcp1, although the growth speed of iMTs is unaffected. These data indicate that Mcp1 is required for the iMT-destabilising function of Klp5/Klp6, perhaps by promoting its association with curved tubulin, but not for its processive motility. We suggest it is likely that, like KIP3 and KIF18A, the highly processive motility of Klp5/Klp6 depends on MT binding site(s) in the C-terminal tail of those proteins 26, 42-44. To quantify the intensity of Klp5/Klp6 at plus ends, mixed populations of cells lacking Mcp1 and control cells were imaged in the same field of view. Although the absence of Mcp1 does not influence Klp5 or Klp6 stability, as judged by Western blot of whole cell extracts (Fig EV1E), nor its intensity in the nucleus, Klp5/Klp6 accumulated to approximately half the level at iMT plus ends in the absence of Mcp1, even though iMTs dwell for longer at cell ends (Fig 1F). This may reflect a role for Mcp1 in enhancing the affinity of Klp5/Klp6 for the iMT lattice, resulting in additional runs to plus ends, or a role for Mcp1 in either retaining Klp5/Klp6 at plus ends or controlling the oligomeric status of the Klp5/Klp6 complex. Reconstitution and biochemical analysis of the Klp5/Klp6/Mcp1 complex will be needed to distinguish between these possibilities. Kinesin-7 and Kinesin-8 complexes antagonistically control microtubule length We next examined the functional relationship between the Klp5/Klp6/Mcp1 and Tea2/Tip1/Mal3 kinesin complexes. In contrast to Klp5/Klp6/Mcp1, which accumulates in both a MT length- and a dwell time-dependent manner, binding of Tea2 kinesin to plus ends is independent of MT length (Fig 2A) but, like Klp5/Klp6/Mcp1, Tea2 dissociates from plus ends following MT catastrophe. To our surprise, we find that Tea2 remains bound, at the same average intensity, to dwelling MT plus ends for longer in the absence of Klp6 or Mcp1 (Figs 2B and C, and EV3A), indicating that the Klp5/Klp6/Mcp1 complex is required for timely dissociation of the Tea2 complex from plus ends. In the absence of Tea2, iMTs dwell at cell ends for less time than in control cells and undergo frequent MT catastrophe in the cytoplasm before iMTs reach the cell end, as previously observed (Fig 2D–F: 20). Importantly, we find that deletion of klp5 and klp6 or mcp1 in Δtea2 cells largely restores MT catastrophe near the cell end and increases MT dwell time, although not quite to that observed in control cells (Fig 2E and F). A similar effect is seen in the absence of Tip1 (Fig EV3B). By contrast, MTs dwell only marginally longer at cell ends in Δtea1 cells than the control, and this is exacerbated in Δtea1 Δmcp1 double mutants (Fig EV3B). This suggests that, in the absence of the Tea2/Tip1/Mal3 complex, but not its cargo, Klp5/Klp6/Mcp1 induces premature MT catastrophe in the cytoplasm. Consistently, Klp5/Klp6 accumulates at iMT plus ends in the absence of Tea2, albeit to a lower intensity than in control cells, as iMTs rarely last long enough to reach the cell end (Fig EV3C and D). In the absence of both Tea2 and Mcp1, Klp5/Klp6 accumulates at the plus ends of iMTs that reach the cell end, but at greatly reduced levels compared to control cells (Fig EV3E). In this situation, iMT catastrophe may purely be reliant on the force exerted by interaction of the MT plus end with the cortex at the cell end 27. Figure 2. Tea2 and Klp5/Klp6/Mcp1 antagonistically control microtubule stability Log phase cells expressing fluorescently tagged MTs were imaged, and the levels of either fluorescently tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs were determined. Measurements (klp5-mNG klp6-mNG, n = 305; tea2-GFP, n = 359) were plotted against microtubule (MT) length and third-order polynomial curves fitted to the data. Kymographs showing fluorescently tagged MTs co-imaged with fluorescently tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end. Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n = 16), ∆klp6 (n = 13) or ∆mcp1 (n = 14) cells. Kymographs showing fluorescently tagged MTs co-imaged with fluorescently tagged Klp5/Klp6 in control (top panels, repeated from 1C) or Δtea2 cells (bottom panels). Dashed yellow line indicates the cell end. Cells expressing fluorescently tagged tubulin were imaged every 5 s and the dwell time of ˜100 individual iMTs for each condition recorded within the final 1.1 μm of the cell. Red bars signify the mean. The strains in (E) were analysed to determine the position of 20 MT shrinkage events relative to the cell end, which are represented both graphically and as a box plot. Data information: In (C), data are presented as mean ± s.d. *P < 0.001, **P < 0.05, n.s. (non-significant) P > 0.05 (Kolmogorov–Smirnov test). In (F), boxes show the interquartile range with the median represented between the lower and upper quartiles, and whiskers show the highest and lowest values. Source data are available online for this figure. Source Data for Figure 2 [embr201846196-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Effect of mutants on kinesin accumulation at plus ends and microtubule dwell time Box plots show data from mixing experiments comparing the intensity of fluorescently tagged Tea2 on MT plus ends between either ∆klp6 (n = 50) and control cells (n = 49) (left panel) or between ∆mcp1 (n = 50) and control cells (n = 50; right panel). Cells expressing fluorescently tagged tubulin were imaged every 5 s and the dwell time of ˜100 individual iMTs for each condition recorded within the final 1.1 μm of the cell. Red bars signify the mean. Intensity of Klp5/Klp6 at MT plus ends relative to the time of MT shrinkage was quantified from multiple kymographs (n = 16) of Δtea2 cells (top panel). Box plots (bottom panel) from mixing experiments comparing the amount of fluorescently tagged Klp5/Klp6 localised either in the nucleus or on MT plus ends before shrinkage between control (n = 37) and ∆tea2 (n = 37) cells. Log phase cells expressing fluorescently tagged MTs were imaged, and the levels of fluorescently tagged Klp5/Klp6 at the plus ends of growing MTs were determined. Measurements (control, n = 100; ∆tea2, n = 100) were plotted against microtubule (MT) length and second-order polynomial curves fitted to the data. Dashed line indicates the estimated division between MT growth, where MT lifetime directly correlates to MT length, and MT dwell, where MT lifetime no longer directly correlates to MT length due to MT pausing at cell ends. Experiments as for (C) but with kymographs of Δmcp1 Δtea2 cells (n = 24) (top panel) and for box plots (bottom panel) between control (n = 50) and ∆mcp1 ∆tea2 (n = 50) cells. Data information: In (C and E, upper panels), data are presented as mean ± s.d. *P < 0.001, **P < 0.01, n.s. (non-significant) P > 0.05 (Kolmogorov–Smirnov test). In (A, C and E), boxes show the interquartile range with the median represented between the lower and upper quartiles, and whiskers show the highest and lowest values. Source data are available online for this figure. Download figure Download PowerPoint Kinesin-7 restricts access of Kinesin-8 to the microtubule plus end We next considered how the antagonistic activities of the MT-stabilising Tea2/Tip1/Mal3 and MT-destabilising Klp5/Klp6/Mcp1 complexes are coordinated in space and time. One possibility is that they compete for the same physical space at the iMT plus end. As Klp5/Klp6/Mcp1 concentration increases, it might physically displace the Tea2/Tip1/Mal3 complex from the plus end and induce MT catastrophe. An alternative, but not necessarily exclusive, possibility is that the Tea2/Tip1/Mal3 complex might occlude access by the Klp5/Klp6/Mcp1 complex to the iMT plus end until the iMT encounters the cell end. To test these models, we monitored the relative positions and levels of components of the Tea2/Tip1/Mal3 and Klp5/Klp6/Mcp1 complexes at the iMT plus end. Importantly, dual imaging of tea2-GFP tip1-tdTomato cells shows that Tea2 and Tip1 occupy the same region of the MT plus end and remain at the same intensity after MT contact with the cell end cortex, consistent with the fact that these proteins are components of the same complex (Fig 3A and B). By contrast, dual imaging of klp5-mNG klp6-mNG tip1-tdTomato cells reveals that Klp5/Klp6 accumulates behind Tip1 as the iMT grows and dwells at the cell end, as judged by maximal intensity measurements of the fluorescence (Figs 4A and B, and EV4, and Movie EV1). Importantly, the Tea2/Tip1/Mal3 and Klp5/Klp6/Mcp1 complexes converge at the plus end approximately 10 s before MT shrinkage is detected. This behaviour is also observed in the absence of Mcp1, although in this case Klp5/Klp6 remains behind the Tea2/Tip1/Mal3 complex at the MT plus end for longer as it dwells at the cell end (Figs 4C and EV5). Figure 3. Tea2 and Tip1 co-localise at the microtubule plus end Kymograph (top left panels) showing fluorescently tagged Tea2 (Kinesin) co-imaged with fluorescently tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2 μm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right). Data quantitated from multiple kymographs (n = 5) of the type illustrated in (A) to display Tea2 and Tip1 intensity at the MT plus end (left plot) and the position of Tea2 relative to Tip1 (right panel) and relative to initial MT contact with the cell end. Error bars show standard deviation. Download figure Download PowerPoint Figure 4. Klp5/Klp6 accumulates behind Tea2 at the plus end until just prior to microtubule catastrophe Kymograph (top left panels) showing fluorescently tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2 μm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph. Data quantitated from multiple kymographs (n = 7) of the type illustrated in (A) and Fig EV4 to display Klp5/Klp6 and Tip1 levels at the MT plus end (top plot) and the position of Klp5/Klp6 relative to Tip1 (bottom plot) before MT shrinkage. Data quantitated from multiple kymographs (n = 8) of the type illustrated in Fig EV5 where fluorescently tagged Klp5/Klp6 is co-imaged with fluorescently tagged Tip1 in the absence of Mcp1 and presented as in (B). Data information: In (B, C), data are presented as mean ± s.d. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Location of Klp5/Klp6 and Tea2/Tip1 at the plus end during microtubule growth, dwell and shrinkageFour additional kymographs (i–iv) from cells expressing fluorescently tagged Klp5/Klp6 (green) co-imaged with fluorescently tagged Tip1 (magenta). Dashed yellow lines indicate cell ends. Associated plots show both the relative fluorescence intensity and position for Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymographs. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Klp5/Klp6 queues behind Tea2 for longer in the absence of Mcp1Kymograph (top panels) showing fluorescently tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently tagged Tip1 (Tip1-Tdtom) in the absence of Mcp1. Dashed yellow line indicates the cell end. Plots (second top panel) showing both the relative fluorescence intensity and position for Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2 μm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end over time for both Klp5/K
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