Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.     

A mean field Ising model for cortical rotation in amphibian one-cell stage embryos

We propose a new physical mechanism of cortical rotation generation in one-cell embryos of amphibians based on a phase transition in the ensemble of microtubules localized to the cortical region of the cell interior. Microtubules, protein polymers formed from tubulin heterodimers, are highly negatively charged, which results in strong electrostatic interactions over tens of nanometers, even in the presence of counterions that partially screen electrostatic interactions. A simplified model that offers a plausible representation of these effects is based on the Ising Hamiltonian, which has been robustly applied to explain a wide range of order-disorder transitions in physics, chemistry and other sciences. An Ising model phase transition, especially with the supercooperative flow alignment effect of global rotation of the cortex, provides an alternative to models of cortical rotation based on microtubule polymerization or motor molecules. Insofar as there is any reality to the concept that microtubules are involved in consciousness, we propose that cortical rotation in the one-cell embryo is a better place to look for the purported microtubule entanglement or coherence properties than the adult brain.     

The Caenorhabditis elegans microtubule-severing complex MEI-1/MEI-2 katanin interacts differently with two superficially redundant beta-tubulin isotypes

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 beta-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched beta-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2-containing microtubules. Our results demonstrate that these two superficially redundant beta-tubulin isotypes have functionally distinct roles in vivo.     

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.     

Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays

The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.     

Class V β-tubulin alters dynamic instability and stimulates microtubule detachment from centrosomes

A multigene family produces tubulin isotypes that are expressed in a tissue-specific manner, but the role of these isotypes in microtubule assembly and function is unclear. Recently we showed that overexpression or depletion of β5-tubulin, a minor isotype with wide tissue distribution, inhibits cell division. We now report that elevated β5-tubulin causes uninterrupted episodes of microtubule shortening and increased shortening rates. Conversely, depletion of β5-tubulin reduces shortening rates and causes very short excursions of growth and shortening. A tubulin conformation-sensitive antibody indicated that the uninterrupted shortening can be explained by a relative absence of stabilized patches along the microtubules that contain tubulin in an assembly-competent conformation and normally act to restore microtubule growth. In addition to these changes in dynamic instability, overexpression of β5-tubulin causes fragmentation that results from microtubule detachment from centrosomes, and it is this activity that best explains the effects of β5 on cell division. Paclitaxel inhibits microtubule detachment, increases the number of assembly-competent tubulin patches, and inhibits microtubule shortening, thus providing an explanation for why the drug can counteract the phenotypic effects of β5 overexpression. On the basis of these observations, we propose that cells can use β5-tubulin expression to adjust the behavior of the microtubule cytoskeleton.     

RGS14 is a Microtubule-Associated Protein

Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster, and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin co-immunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 co-purifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD=1.3 +/- 0.3 ?M). Both RGS14 and G?i1 in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule associated proteins. However, preincubation of RGS14 with G?i1-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule associated protein that may modulate microtubule dynamics and spindle formation.     

Oxaline, a fungal alkaloid, arrests the cell cycle in M phase by inhibition of tubulin polymerization

Oxaline and neoxaline, fungal alkaloids, were found to inhibit cell proliferation and to induce cell cycle arrest at the G(2)/M phase in Jurkat cells. CBP501 (a peptide corresponding to amino acids 211-221 of Cdc25C phosphatase), which inhibits the G(2) checkpoint, did not affect the G(2)/M arrest caused by oxaline, suggesting that oxaline causes M phase arrest but not G(2) phase arrest. The Cdc2 phosphorylation level of oxaline-treated cell lysate was lower than that of the control cells, indicating that oxaline arrests the M phase. Oxaline disrupted cytoplasmic microtubule assembly in 3T3 cells. Furthermore, oxaline inhibited polymerization of microtubule protein and purified tubulin dose-dependently in vitro. In a binding competition assay, oxaline inhibited the binding of [(3)H]colchicine to tubulin, but not that of [(3)H]vinblastine. These results indicate that oxaline inhibits tubulin polymerization, resulting in cell cycle arrest at the M phase.     

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.     

Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.     

The maternal‐to‐zygotic transition: An asynchronous split

The early cell cycles of preimplantation embryo development are unique in the scheme of mitotic cell proliferation as cell division is not coupled to cell growth, leading to a halving of blastomere volume with each cleavage event. Among the early mouse embryonic divisions, the fi rst two are particularly different, lasting almost twice as long as subsequent divisions. The third cell cycle is marked by the transition of a four‐cell embryo into an eight‐cell embryo, and represents the fi rst complete cell cycle occurring after activation of the zygotic genome. The G2/M phase of the third cell cycle is highly variable, lasting between 2–5 hours, and heterogeneity between blastomeres within the same embryo may occur as a part of normal development. The embryo in this image is actively undergoing cleavage from the four‐ to the eight‐cell stage, and blastomeres are captured in multiple phases of the cell cycle, as visualized by chromatin structure (DNA, blue) and microtubule staining (α‐tubulin, green). Two blastomeres sit in interphase with decondensed chromatin masses and a mesh‐like microtubule network, while the remaining blastomeres are actively undergoing mitosis. Of the latter, one is in metaphase, one in early anaphase, and the last in late anaphase. All together, the diversity in cell cycle stages reveals the inherit asynchrony existent within individual blastomeres of a cleavage stage embryo. Mol. Reprod. Dev. 80: 1–1, 2012. © 2012 Wiley Periodicals, Inc.     

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.     

CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2.     

Copolymerization of tubulin-colchicine complex and unliganded tubulin in a nonmicrotubular polymer

We have shown previously [Saltarelli, D., & Pantaloni, D. (1982) Biochemistry 21, 2996-3006] that the tubulin-colchicine complex is able to polymerize in vitro into peculiar "curly" polymers, under the solution conditions permitting polymerization of unliganded tubulin into microtubules. Here it is further demonstrated that unliganded tubulin can be incorporated into these "curly" polymers. The partial critical concentration of tubulin-colchicine is decreased upon incorporation of unliganded tubulin into the copolymer. GTP hydrolysis occurs on unliganded tubulin upon incorporation in the copolymer. Tubulin-podophyllotoxin does not copolymerize with tubulin-colchicine to form a large polymer but interacts with it, preventing tubulin-colchicine polymerization. The data have been analyzed within a model of random copolymerization of unliganded tubulin and tubulin-colchicine into "curly" polymers. A corollary is that unliganded tubulin is virtually able to self-assemble into curly polymers with a critical concentration 10-fold higher than the critical concentration found for microtubule assembly. Consequently, these peculiar tubulin homopolymers cannot be observed except as transients at high concentrations, or when microtubule assembly is inhibited. Kinetic measurements of the T-TC copolymerization process and associated GTP hydrolysis at different T/TC ratios provide supplementary information about some privileged interactions between tubulin and tubulin-colchicine molecules. A comprehensive phase diagram of the various possible polymers formed in the presence of tubulin and tubulin-colchicine is presented.     

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.     

G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells

The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.