The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B

INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Stabilization of anaphase midzone microtubules is regulated by Rho during cytokinesis in human fibrosarcoma cells

The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but more severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was sufficient to maintain the midzone structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, P. DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529–9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during mitosis and cytokinesis.     

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position‐fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against­ the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti‐centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti‐centering pulling forces localize the mitotic spindles within dividing C. elegans cells.     

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.     

MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150^glued, a subunit of the dynein–dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.     

Vesicle-like biomechanics governs important aspects of nuclear geometry in fission yeast

It has long been known that during the closed mitosis of many unicellular eukaryotes, including the fission yeast (Schizosaccharomyces pombe), the nuclear envelope remains intact while the nucleus undergoes a remarkable sequence of shape transformations driven by elongation of an intranuclear mitotic spindle whose ends are capped by spindle pole bodies embedded in the nuclear envelope. However, the mechanical basis of these normal cell cycle transformations, and abnormal nuclear shapes caused by intranuclear elongation of microtubules lacking spindle pole bodies, remain unknown. Although there are models describing the shapes of lipid vesicles deformed by elongation of microtubule bundles, there are no models describing normal or abnormal shape changes in the nucleus. We describe here a novel biophysical model of interphase nuclear geometry in fission yeast that accounts for critical aspects of the mechanics of the fission yeast nucleus, including the biophysical properties of lipid bilayers, forces exerted on the nuclear envelope by elongating microtubules, and access to a lipid reservoir, essential for the large increase in nuclear surface area during the cell cycle. We present experimental confirmation of the novel and non-trivial geometries predicted by our model, which has no free parameters. We also use the model to provide insight into the mechanical basis of previously described defects in nuclear division, including abnormal nuclear shapes and loss of nuclear envelope integrity. The model predicts that (i) despite differences in structure and composition, fission yeast nuclei and vesicles with fluid lipid bilayers have common mechanical properties; (ii) the S. pombe nucleus is not lined with any structure with shear resistance, comparable to the nuclear lamina of higher eukaryotes. We validate the model and its predictions by analyzing wild type cells in which ned1 gene overexpression causes elongation of an intranuclear microtubule bundle that deforms the nucleus of interphase cells.     

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.     

Astral microtubules control redistribution of dynein at the cell cortex to facilitate spindle positioning

Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis.     

Slk19p is a centromere protein that functions to stabilize mitotic spindles.

We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

The Spindle Midzone Microtubule-Associated Proteins Ase1p and Cin8p Affect the Number and Orientation of Astral Microtubules in Saccharomyces cerevisiae

The nucleus of the budding yeast S. cerevisiae has to move to the bud neck during mitosis in order for proper DNA segregation to take place. This movement is mediated by spindle and astral microtubules, and it relies on forces generated by microtubule-associated motor proteins. When budding yeast cells express the non-cleavable cohesin subunit, Scc1-RRDD, sister chromatid separation is blocked, preventing the spindle from elongating. Thus, in the presence of Scc1-RRDD nuclear positioning is mediated solely by forces acting through astral microtubules. We have previously shown that under these conditions cells exit mitosis with the nucleus in the mother cells, and that the position of the nucleus is determined, at least in part, by the FEAR pathway, which regulates various aspects of mitotic exit. When the FEAR pathway is inactivated, cells expressing Scc1-RRDD exit mitosis with the nucleus in the daughter cells (referred to as a “daughterly phenotype”). In order to find additional proteins that participate in nuclear positioning, we screened a series of mutant strains for those that displayed a daughterly phenotype when Scc1-RRDD was expressed. The most prominent defects were seen in ase1Δ and cin8Δ mutant cells. Both Ase1p and Cin8p were previously shown to be nuclear and to be involved in spindle function. We show here that deletion of ASE1 or CIN8 causes a defect in SPB separation and leads to an abnormal number of astral microtubules and a change in their orientation within the cell. Taken together, these results suggest that in budding yeast Ase1p and Cin8p affect nuclear positioning through astral microtubule-dependent mechanisms.     

Three-dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe

Mitotic spindles of Schizosaccharomyces pombe have been studied by EM, using serial cross sections to reconstruct 12 spindles from cells that were ultrarapidly frozen and fixed by freeze substitution. The resulting distributions of microtubules (MTs) have been analyzed by computer. Short spindles contain two kinds of MTs: continuous ones that run from pole to pole and MTs that originate at one pole and end in the body of the spindle. Among the latter there are three pairs of MT bundles that end on fibrous, darkly staining structures that we interpret as kinetochores. The number of MTs ending at each putative kinetochore ranges from two to four; all kinetochore-associated MTs disappear as the spindle elongates from 3-6 microns. At this and greater spindle lengths, there are no continuous MTs, only polar MTs that interdigitate at the spindle midzone, but the spindle continues to elongate. An analysis of the density of neighboring MTs at the midzone of long spindles shows that their most common spacing is approximately 40 nm, center to center, and that there is a preferred angular separation of 90 degrees. Only hints of such square-packing are found at the midzone of short spindles, and near the poles there is no apparent order at any mitotic stage. Our data suggest that the kinetochore MTs (KMTs) do not interact directly with nonkinetochore MTs, but that interdigitating MTs from the two spindle poles do interact to form a mechanically stable bundle that connects the poles. As the spindle elongates, the number of MTs decreases while the mean length of the MTs that remain increases. We conclude that the chromosomes of S. pombe become attached to the spindle by kinetochore MTs, that these MTs disappear as the chromosomes segregate, that increased separation of daughter nuclei is accompanied by a sliding apart of anti-parallel MTs, and that the mitotic processes of S. pombe are much like those in other eukaryotic cells.     

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.     

Spindle positioning in fibroblasts supports an astral microtubule length dependent force generation at the basal membrane.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.     

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.     

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.     

Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.