Astral Microtubule Organization Research Articles

SUN2 regulates mitotic duration in response to extracellular matrix rigidity

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

Importin α phosphorylation promotes TPX2 activation by GM130 to control astral microtubules and spindle orientation.

Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The orientation of the spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates astral microtubules. TPX2 in the spindle pole area is activated by GM130 (GOLGA2) on Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α1 (KPNA2) binding. Mitotic phosphorylation of importin α at serine 62 (S62) by CDK1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-based activation. Importin α S62A mutation impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by CDK1-mediated phosphorylation.

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase.

Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.

Spatial cues and not spindle pole maturation drive the asymmetry of astral microtubules between new and preexisting spindle poles.

In many asymmetrically dividing cells, the microtubule-organizing centers (MTOCs; mammalian centrosome and yeast spindle pole body [SPB]) nucleate more astral microtubules on one of the two spindle poles than the other. This differential activity generally correlates with the age of MTOCs and contributes to orienting the mitotic spindle within the cell. The asymmetry might result from the two MTOCs being in distinctive maturation states. We investigated this model in budding yeast. Using fluorophores with different maturation kinetics to label the outer plaque components of the SPB, we found that the Cnm67 protein is mobile, whereas Spc72 is not. However, these two proteins were rapidly as abundant on both SPBs, indicating that SPBs mature more rapidly than anticipated. Superresolution microscopy confirmed this finding for Spc72 and for the γ-tubulin complex. Moreover, astral microtubule number and length correlated with the subcellular localization of SPBs rather than their age. Kar9-dependent orientation of the spindle drove the differential activity of the SPBs in astral microtubule organization rather than intrinsic differences between the spindle poles. Together, our data establish that Kar9 and spatial cues, rather than the kinetics of SPB maturation, control the asymmetry of astral microtubule organization between the preexisting and new SPBs.

The MgcRacGAP SxIP motif tethers Centralspindlin to microtubule plus ends in Xenopus laevis.

Centralspindlin, a complex of the kinesin-6-family member MKLP1 and MgcRacGAP (also known as Kif23 and Racgap1, respectively), is required for cytokinesis and cell-cell junctions. During anaphase, Centralspindlin accumulates at overlapping central spindle microtubules and directs contractile ring formation by recruiting the GEF Ect2 to the cell equator to activate RhoA. We found that MgcRacGAP localized to the plus ends of equatorial astral microtubules during cytokinesis in Xenopus laevis embryos. How MgcRacGAP is stabilized at microtubule plus ends is unknown. We identified an SxIP motif in X. laevis MgcRacGAP that is conserved with other proteins that bind to EB1 (also known as Mapre1), a microtubule plus-end tracking protein. Mutation of the SxIP motif in MgcRacGAP resulted in loss of MgcRacGAP tracking with EB3 (also known as Mapre3) on growing microtubule plus ends, abnormal astral microtubule organization, redistribution of MgcRacGAP from the contractile ring to the polar cell cortex, and mislocalization of RhoA and its downstream targets, which together contributed to severe cytokinesis defects. Furthermore, mutation of the MgcRacGAP SxIP motif perturbed adherens junctions. We propose that the MgcRacGAP SxIP motif is functionally important both for its role in regulating adherens junction structure during interphase and for regulating Rho GTPase activity during cytokinesis.

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we studied the effect of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from 5-100 μm, which covers the size range of typical cell bodies. We found that droplet size alone had a major organizing influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organizations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. Our results provide insights into the combined organizing influence of spatial constraints and cross-linking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid-monolayered droplet system characterized here can be expanded readily to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organization inside liposomes surrounded by a deformable lipid bilayer.

Rab11 Endosomes Contribute to Mitotic Spindle Organization and Orientation

During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin invitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Spindle Pole Body History Intrinsically Links Pole Identity with Asymmetric Fate in Budding Yeast

Budding yeast is a unique model for exploring differential fate in a cell dividing asymmetrically. In yeast, spindle orientation begins with the old spindle pole body (SPB) (from the preceding cell cycle) contacting the bud by its existing astral microtubules (aMTs) while the new pole delays astral microtubule organization. This appears to prime the inheritance of the old pole by the bud. The basis for this asymmetry and the discrimination of the poles by virtue of their history remain a mystery. Here, we report that asymmetric aMT organization stems from an outstanding structural asymmetry linked to the SPB cycle. We show that the γ-tubulin nucleation complex (γTC) favors the old spindle pole, an asymmetry inherent to the outer plaque (the cytoplasmic face of the SPB). Indeed, Spc72 (the receptor for the γTC) is acquired by the new SPB outer plaque partway through spindle assembly. The significance of this asymmetry was explored in cells expressing an Spc72(1-276)-Cnm67 fusion that forced symmetric nucleation at the SPB outer plaques. This manipulation triggered simultaneous aMT organization by both spindle poles from the outset and led to symmetric contacts between poles and the bud, effectively disrupting the program for spindle polarity. Temporally symmetric aMT organization perturbed Kar9 polarization by randomizing the choice of the pole to be guided toward the bud. Accordingly, the pattern of SPB inheritance was also randomized. Spc72 differential recruitment imparting asymmetric aMT organization represents the most upstream determinant linking SPB historical identity and fate.

TPPP acts downstream of RhoA-ROCK-LIMK2 to regulate astral microtubule organization and spindle orientation

The actin cytoskeleton in eukaryotic cells undergoes drastic rearrangement during mitosis. The changes to the actin cytoskeleton are most obvious in the adherent cells, where the actin stress fibres are disassembled, and the cortical actin network becomes more prominent with concomitant increase in cell rigidity as cells round up and enter mitosis. Although the regulatory connections between the actin cytoskeleton and the early mitotic events are apparent, the mechanisms that govern these links are not well understood. Here, we report that LIMK1 and LIMK2, the downstream effectors of RhoA and ROCK, regulate centrosome integrity and astral microtubule organization, respectively. Surprisingly, LIMK1 and cofilin are not involved downstream of RhoA and ROCK in the regulation of astral microtubule organization. Instead, we find that LIMK2 acts through TPPP in the regulation of astral microtubule organization, whereas both LIMK1 and LIMK2 affect centrosome focusing. Both phenotypes are tightly coupled to spindle orientation in the mitotic cells. Thus, our results reveal a new regulatory link between the actin cytoskeleton and the mitotic spindle during the early stages of mitosis.

Kif18B interacts with EB1 and controls astral microtubule length during mitosis

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.

Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe

Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which play essential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 and Mcp7 are required for their function as a putative recombinase cofactor complex during meiotic recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required for horsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these proteins plays an important role during the progression of cells through meiosis. However, there are still many unanswered questions on how these proteins operate. In this paper, we briefly review recent studies on the meiotic coiled-coil proteins in Sz. pombe.

Mcp6, a meiosis-specific coiled-coil protein ofSchizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination

We report here that a meiosis-specific gene of Schizosaccharomyces pombe denoted mcp6+ (meiotic coiled-coil protein) encodes a protein that is required for the horsetail movement of chromosomes at meiosis I. The mcp6+ gene is specifically transcribed during the horsetail phase. Green fluorescent protein (GFP)-tagged Mcp6 appears at the start of karyogamy, localizes to the spindle-pole body (SPB) and then disappears before chromosome segregation at meiosis I. In the mcp6Delta strain, the horsetail movement was either hampered (zygotic meiosis) or abolished (azygotic meiosis) and the pairing of homologous chromosomes was impaired. Accordingly, the allelic recombination rates of the mcp6Delta strain were only 10-40% of the wild-type rates. By contrast, the ectopic recombination rate of the mcp6Delta strain was twice the wild-type rate. This is probably caused by abnormal homologous pairing in mcp6Delta cells because of aberrant horsetail movement. Fluorescent microscopy indicates that SPB components such as Sad1, Kms1 and Spo15 localize normally in mcp6Delta cells. Because Taz1 and Swi6 also localized with Sad1 in mcp6Delta cells, Mcp6 is not required for telomere clustering. In a taz1Delta strain, which does not display telomere clustering, and the dhc1-d3 mutant, which lacks horsetail movement, Mcp6 localized with Sad1 normally. However, we observed abnormal astral microtubule organization in mcp6Delta cells. From these results, we conclude that Mcp6 is necessary for neither SPB organization nor telomere clustering, but is required for proper astral microtubule positioning to maintain horsetail movement.

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body

Background: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic.Results: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis.Conclusions: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.

Nud1p links astral microtubule organization and the control of exit from mitosis.

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the gamma-tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell-cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.