Polarity of spindle microtubules in Haemanthus endosperm

Structural polarities of mitotic spindle microtubules in the plant Haemanthus katherinae have been studied by lysing endosperm cells in solutions of neurotubulin under conditions that will decorate cellular microtubules with curved sheets of tubulin protofilaments. Microtubule polarity was observed at several positions in each cell by cutting serial thin sections perpendicular to the spindle axis. The majority of the microtubules present in a metaphase or anaphase half-spindle are oriented with their fast-growing or "plus" ends distal to the polar area. Near the polar ends of the spindle and up to about halfway between the kinetichores and the poles, the number of microtubules with opposite polarity is low: 8-20% in metaphase and 2-15% in anaphase cells. Direct examination of 10 kinetochore fibers shows that the majority of these microtubules, too, are oriented with their plus ends distal to the poles, as had been previously shown in animal cells. Sections from the region near the spindle equator reveal an increased fraction of microtubules with opposite polarity. Graphs of polarity vs. position along the spindle axis display a smooth transition from microtubules of one orientation near the first pole, through a region containing equal numbers of the two orientations, to a zone near the second pole where the opposite polarity predominates. We conclude that the spindle of endosperm cells is constructed from two sets of microtubules with opposite polarity that interdigitate near the spindle equator. The length of the zone of interdigitation shortens from metaphase through telophase, consistent with a model that states that during anaphase spindle elongation in Haemanthus, the interdigitating sets of microtubules are moved apart. We found no major changes in the distribution of microtubule polarity in the spindle interzone from anaphase to telophase when cells are engaged in phragmoplast formation. Therefore, the initiation and organization of new microtubules, thought to take place during phragmoplast assembly, must occur without significant alteration of the microtubule polarity distribution.     

ASPM and Citron Kinase Co-Localize to the Midbody Ring during Cytokinesis

Mutations in ASPM (abnormal spindle-like microcephaly associated) and Citron Kinase (CITK) cause primary microcephaly in humans and rodents, respectively. Both proteins are expressed during neurogenesis and play important roles in neuronal progenitor cell division. ASPM is localized to the spindle pole, and is essential for maintaining proliferative cell division. CITK is present at the cytokinesis furrow and midbody ring, and it is essential for cellular abscission. We report here that ASPM also localizes to the midbody ring in mammalian cells. ASPM co-localizes with CITK at the midbody ring and co-immunoprecipitates with CITK in lysates prepared from HeLa cells and embryonic neuroepithelium. Furthermore, a GFP-tagged fragment of the N-terminus of ASPM localizes to centrosomes and spindle poles, while a GFP-tagged fragment of the C-terminus localizes to midbodies. All reported ASPM mutations that cause microcephaly involve a truncation or mutation of the C-terminus. In addition, at least two other microcephaly-related proteins, CENPJ and CDK5RAP2, previously localized to spindle poles, also localize to midbodies. Together our observations support a model of neurogenesis in which spindle dynamics and cellular abscission are coordinated.     

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.     

Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold- treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold- treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.     

Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells

Monoclonal antibodies were raised against isolated spindles of CHO (Chinese hamster ovary) cells to probe for molecular components specific to the mitotic apparatus. One of the antibodies, CHO1, recognized an antigen localized to the midbody during mitosis. Immunofluorescence staining of metaphase cells showed that although the total spindle area was labeled faintly, the antigen corresponding to CHO1 was preferentially localized in the equatorial region of the spindle. With the progression of mitosis, the antigen was further organized into discrete short lines along the spindle axis, and eventually condensed into a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells. Parallel immunostaining of tubulin showed that the CHO1-stained area corresponded to the dark region where microtubules are entrapped by the amorphous dense matrix components and possibly blocked from binding to tubulin antibody. Immunoblot analysis indicated that CHO1 recognized two polypeptides of mol wt 95,000 and 105,000. The immunoreaction was always stronger in preparations of isolated midbodies than in mitotic spindle fractions. The protein doublet was retained in the particulate matrix fraction after Sarkosyl extraction (Mullins, J. M., and J. R. McIntosh. 1982. J. Cell Biol. 94:654-661), suggesting that CHO1 antigen is indeed a component of the dense matrix. In addition to the equatorial region of spindles and midbodies, CHO1 also stained interphase centrosomes, and nuclei in a speckled pattern that was cell cycle-dependent. Thus, the midbody appears to share either common molecular component(s) or a similar epitope with interphase centrosomes and nuclei.     

Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.     

LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation

LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), which is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, we now show that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. We therefore propose that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization.     

Midbodies and beaded threads.

When the dorsal surface of the young chick embryo is examined by SEM, long threads are visible, each of which appears to connect pairs of cells; these cells may be separated from each other by several intervening cells. Many of the threads possess a bead-like structure about half way along their length. When sections of the beads are examined by TEM they are found to resemble midbodies. Furthermore, the threads possess longitudinally arranged structures within them, which are probably the remnants of the microtubules which were part of a mitotic spindle. It is concluded that each bead is a midbody and that each beaded thread is the remains of a telophase bridge connecting two daughter cells which were incompletely separated after mitosis had taken place. The possible function of the beaded threads is considered.     

Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle

The structural polarity of cellular microtubules can be visualized in situ by lysing cells in special buffers containing tubulin. Under these conditions, the tubulin polymerizes to form curved sheets which attach to the walls of the endogenous microtubules. When such decorated microtubules are cut in cross section and viewed in the electron microscope, they appear to bear hooks curving clockwise or counter- clockwise. The direction of hook curvature is defined by the orientation of the decorated microtubule and thus serves as a probe for microtubule polarity. In this paper we describe a way to analyze the relative frequencies of hooks of different curvatures so as to measure the fidelity of the relation between hook curvature and microtubule polarity. The assumptions of the method are tested and found to be valid to a reasonable accuracy. The correlation between hook curvature and microtubule orientation is shown to be at least 0.98 for the spindles of PtK cells and Haemanthus endosperm at all stages of division and at all places in the spindle. The correlation is shown to be valid for each hook that forms, so the polarity of those microtubules that bear multiple hooks is specified with even better certainty than 0.98. This property of hook decoration is used to reinvestigate the possibility that some of the microtubules of the kinetochore fiber might be oriented with their plus ends distal to the kinetochore (opposite to the direction previously shown to predominate). Close analysis fails to identify such oppositely oriented microtubules. The scoring of tubules bearing multiple hooks also shows that individual interzone fibers at anaphase are constructed from clusters of antiparallel microtubules. The method for estimating the correlation between hook decoration and microtubule polarity is shown to be applicable to many structures and circumstances, but we find that the hook decoration assay for microtubule polarity is not uniformly accurate. We suggest that future studies using hook decorations should employ the method of data analysis presented here to assess the accuracy of the results obtained.     

Analysis of microtubule arms and bridges in mitotic and interphase amebae of Dictyostelium discoideum

We have analyzed transparencies of electron micrographs from ultrathin longitudinal sections through mitotic spindles of undifferentiated amebae of Dictyostelium discoideum for the presence of arms on microtubules (MTs) and bridges between them. We used the technique of microdensitometer scanning and computer-based model matching by cross-correlation and autocorrelation. We also determined that spindle MTs are composed of 13 protofilaments. Although regularly arranged lateral appendages are not a universal feature of MTs in these cells, both cross-correlation and autocorrelation analysis revealed that bridges between a kinetochore MT and its neighbor, and between MTs in the zone of overlap of the central spindle were significantly arranged on a 12-dimer superlattice. In addition, the autocorrelation analysis indicated a slight match with the 12-dimer model for neighboring non-kinetochore MTs. Although electron micrographs revealed putative arms on cytoplasmic and astral MTs, as well as bridges between central spindle MTs outside the zone of overlap, their arrangement did not match any of the models tested. Bridges between non-kinetochore MTs in the half-spindles possibly serve to reinforce the spindle scaffold. Bridges between kinetochore MTs and their neighbors may contribute to the mechanical stability of kinetochore fibers or they may be involved in poleward movements of the chromosomes. In the zone of overlap of the central spindle, the occurrence of frequent and regularly spaced bridges is consistent with models predicting that a sliding mechanism operates between MTs of opposite polarity in this region of the spindle to produce its elongation.     

Talin concentrates to the midbody region during mammalian cell cytokinesis.

In this study we investigated the cellular distribution of talin, a cytoskeletal protein, during mammalian cell cytokinesis. Immunohistochemical experiments on various carcinoma cell lines and mesenchyme-derived cells reveal that talin displays a cell cycle-dependent cellular localization. During metaphase, talin is located in the centromeric region of the chromosome, like the TD-60 protein and intrinsic centromere components detected by a CREST serum. From anaphase to telophase, talin is present in the cleavage furrow. As the cells progress to cytokinesis, when the furrow is complete, talin is concentrated in the midbody structures, as assessed by immunofluorescence and confirmed by Western blot experiments on purified midbodies. Double staining experiments reveal that alpha-tubulin, TD-60 protein, and talin co-localize in the midbodies. These results suggest that talin, in addition to its implication in focal adhesion organization and signaling, may play a critical role in cytokinesis. (J Histochem Cytochem 47:1357-1367, 1999)     

Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules

During the transition from prometaphase to metaphase, the cross- sectional area of the central spindle of Diatoma decreases by a factor of nearly two, both at the poles and at the region of overlapping microtubules (MTs) near the spindle equator. The density of spindle MT packing stays approximately constant throughout mitosis. Optical diffraction analysis of electron micrographs shows that the packing of the MTs at the poles at all stages of mitosis is similar to that expected for a two-dimensional liquid. Analysis of the region of overlap reveals more packing regularity: during prometaphase, a square packing emerges that displays sufficient organization by late metaphase to generate five orders of diffraction; during anaphase the packing in the overlap region shifts to hexagonal; at telophase, it returns to square. From the data provided by serial section reconstructions of the central spindle, it is possible to identify the polarity of almost every spindle MT, that is, to identify one pole with which the MT is associated. Near neighbor analyses of MTs in cross sections of the overlap region show that MTs prefer antiparallel near neighbors. These near neighbors are most often found at a spacing of approximately 40 nm center-to-center, while parallel near neighbors in the zone of overlap are spaced essentially at random. These results are evidence for a specific interaction between antiparallel MTs. In some sections definite bridges between MTs can be seen. Our findings show that certain necessary conditions for a sliding filament model of anaphase spindle elongation are met.     

Microtubule flux and sliding in mitotic spindles of Drosophila embryos

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 microm/s, too slow to drive chromatid-to-pole motion at 0.11 microm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.     

Isolation and initial characterization of the mammalian midbody

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

Contribution of midbody channels to embryogenesis in the mouse

Summary We have examined the persistence of midbody channels during the second, third, and fourth cleavage cycles of the mouse using immunofluorescence to map the distribution of midbody microtubule bundles in intact embryos. Electron microscopy showed these bundles to be a characteristic feature of midbodies throughout the interphase period. In recently-divided embryos at each cleavage stage the number of midbodies was half the number of blastomeres, and declined towards zero as the next cleavage approached. This indicated to us that the only midbodies present in each stage were those which had arisen in the immediately-preceding division. Of those blastomeres which were in mitosis at the time of fixation, less than 4% were connected via a midbody to another blastomere, demonstrating that persistence of midbodies beyond a single cleavage cycle is a rare event. We conclude that midbody channels in our embryos are likely to connect only pairs of sister blastomeres because midbodies do not persist through multiple cleavage cycles. Midbody channels cannot, therefore, be regarded as providing extensive cell coupling in advance of the onset of gap junctional communication.     

Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skp1 at the midbody and participates in cytokinesis. CENP-E interacts with Skp1 in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955-1571 aa) and the C-terminal 33 amino acids of Skp1, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skp1 and the midbody localization of CENP-E becomes diminished as Skp1 arrives at the midbody. Suppression of Skp1 in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2-3 times longer than that of normal cells. These Skp1-eliminated or -suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skp1 lacking the CENP-E-binding domain confirmed that Skp1-CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skp1 link.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

FAM29A promotes microtubule amplification via recruitment of the NEDD1–γ-tubulin complex to the mitotic spindle

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a γ-tubulin–dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1–γ-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.