Time-sequence observations of microtubule dynamics throughout mitosis in living cell suspensions of stable transgenic Arabidopsis--direct evidence for the origin of cortical microtubules at M/G1 interface

Transgenic Arabidopsis thaliana, stably expressing a GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA. In the cell suspensions, the microtubular changes in the mitotic cells could be monitored by time-sequence observations using a time-lapse system of fluorescence microscopy. We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G2 phase to early G1 phase, and found that, at the M/G1 interface, the cortical MTs were firstly reorganized in the perinuclear regions and then in the cortex, as we had previously suggested (Hasezawa and Nagata 1991, Nagata et al. 1994). The significance of this observation on the origin of cortical MTs is discussed.     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.     

Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.     

Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.     

Dynamic Organization of Microtubules and Microfilaments during Cell Cycle Progression in Higher Plant Cells

Abstract: The cytoskeleton, which mainly consists of microtubules (MTs) and actin microfilaments (MFs), plays various significant roles that are indispensable for eukaryotic viability, including determination of cell shape, cell movement, nuclear division, and cytokinesis. In animal cells, MFs appear to be of more importance than MTs, except for spindle formation in nuclear division. In contrast, higher plants have a rigid cell wall around their cells, and have thus evolved elegant systems of MTs to control the direction of cellulose microfibrils (CMFs) deposited in the cell wall, and to divide centrifugally in a physically limited space. Dynamic changes in MTs during cell cycle progression in higher plant cells have been observed over several decades, including cortical MTs (CMTs) during interphase, preprophase bands (PPBs) from late G₂ phase to prophase, spindles from prometaphase to anaphase, and phragmoplasts at telophase. The MFs also show some changes not as obvious as MT dynamics. However, questions regarding the process of formation of these arrays, and the precise mechanisms by which they fulfill their roles, remain unsolved. In this article, we present an outline of the changes in the cytoskeleton based on our studies with highly-synchronized tobacco BY-2 cells. Some candidate molecules that could play roles in cytoskeletal dynamics are discussed. We also hope to draw attention to recent attempts at visualization of cytoskeletons with molecular techniques, and to some examples of genetic approaches in this field.     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Regulation of microtubule organization during interphase and M phase

Microtubule (MT) dynamics and organization change markedly during interphase-M phase transition of the cell cycle. This mini review focuses first on p220, a ubiquitous MT-associated protein of Xenopus. p220 is phosphorylated by p34cdc2 kinase and MAP kinase in M phase, and concomitantly loses its MT-binding and MT-stabilizing activities. A cDNA encoding p220 was cloned, which identified p220 as a Xenopus homolog of MAP4, and p220 was therefore termed XMAP4. To examine the physiological relevance of XMAP4 phosphorylation during mitosis, Xenopus A6 cells were transfected with cDNA encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein (GFP). Mutations of serine and threonine within potential phosphorylation sites for p34cdc2 kinase to nonphosphorylatable alanine interfered with mitosis-associated reduction in MT-affinity of XMAP4 and their overexpression affected chromosome movement during anaphase A. These results indicated that phosphorylation of XMAP4 by p34cdc2 kinase is responsible for the decrease in its MT-binding and MT-stabilizing activities during mitosis which are important for chromosome movement during anaphase A. The second focus is on a novel monoclonal antibody W8C3, which recognizes alpha-tubulin. W8C3 stained spindle MTs but not interphase MTs of Xenopus A6 cells, although tubulin dimers in M phase and interphase were equally recognized by this antibody. The difference in MT staining pattern may be because the W8C3-recognition site on alpha-tubulin is sterically hidden in interphase MTs but not in spindle MTs.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

The Involvement of Protein Synthesis Elongation Factor 1α in the Organization of Microtubules on the Perinuclear Region during the Cell Cycle Transition from M Phase to G1 Phase in Tobacco BY-2 Cells

Association of the organization of microtubules (MTs) in the perinuclear region with a 49-kDa protein, that is immunologically cross-reactive to a 51-kDa protein isolated from sea urchin centrosomes and has been shown to play some roles in the organization of MTs in animal cells (Toriyama et al.: Cell Motil. Cytoskeleton 9, 117–128, 1988), was examined during the cell cycle transition from M phase to G₁ phase using the highly synchronized tobacco BY-2 cells under confocal laser scanning microscopy (CLSM). After double staining with an antibody against the 51-kDa protein and with an antibody against tubulin, it was revealed that the 49-kDa protein was closely associated with the organization of MTs on the perinuclear regions during this stage under the CLSM. Notably, microfilaments (MFs) were not associated with the organization of MTs in the perinuclear region. This observation suggests that the 49-kDa protein plays a specific role in the organization of MTs on the perinuclear regions during the cell cycle transition from M phase to G₁ phase. To understand the molecular characteristics of the 49-kDa protein further, the search for cDNA encoding the 49-kDa protein was conducted in a cDNA expression library prepared from rapidly growing tobacco BY-2 cells using monoclonal antibodies against the 51-kDa protein. Determination of the base sequence of the isolated clone revealed that it encodes protein synthesis elongation factor (EF)-1α. Thus the significance of the involvement of the 49-kDa protein as EF-1α in the organization of MTs on the perinuclear regions is discussed in relation to other cellular functions.     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.     

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.     

Tobacco microtubule-associated protein,MAP65-1c,bundles and stabilizes microtubules

Three genes that encode MAP65-1 family proteins have been identified in the Nicotiana tabacum genome. In this study, NtMAP65-1c fusion protein was shown to bind and bundle microtubules (MTs). Further in vitro investigations demonstrated that NtMAP65-1c not only alters MT assembly and nucleation, but also exhibits high MT stabilizing activity against cold or katanin-induced destabilization. Analysis of NtMAP65-1c-GFP expressing BY-2 cells clearly demonstrated that NtMAP65-1c was able to bind to MTs during specific stages of the cell cycle. Furthermore, in vivo, NtMAP65-1c-GFP-bound cortical MTs displayed an increase in resistance against the MT-disrupting drug, propyzamide, as well as against cold temperatures. Taken together, these results strongly suggest that NtMAP65-1c stabilizes MTs and is involved in the regulation of MT organization and cellular dynamics.     

Cell cycle-regulated proteolysis of mitotic target proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission

During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.     

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.     

A Highlights from MBoC Selection: The kinesin-14 Klp2 is negatively regulated by the SIN for proper spindle elongation and telophase nuclear positioning

In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end–directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized.     

The role of microfilaments in the organization and orientation of microtubules during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells

Summary During cell cycle transition from M to G₁ phase, micro-tubules (MTs), organized on the perinuclear region, reached the cell cortex. Microfilaments (MFs) were not involved in this process, however, MFs accumulated to form a ring-like structure in the division plane and from there they elongated toward the distal end in the cell cortex. Subsequently, when MTs elongated along the long axis of the cells, towards the distal end, the MTs ran into and then associated with the predeveloped MFs in the cell cortex, suggesting the involvement of MFs in organizing the parallel oriented MTs in the cell cortex. When cortical MTs were formed in the direction transverse to the long axis of cells, the two structures were again closely associated. Therefore, with regards to the determination of the direction of organizing MTs, predeveloped MFs may have guided the orientation of MTs at the initial stage. Disorganization of MFs in this period, by cytochalasins, prevented the organization of cortical MTs, and resulted in the appearance of abnormal MT configurations. We thus demonstrate the involvement of MFs in determining the orientation and organization of cortical MTs, and discuss the possible role of MFs during this process.Abbreviations CB cytochalasin B - CD cytochalasin D - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - EF-1 elongation factor 1 - MF microfilament - MT microtubule