Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. Here, we show that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. Our study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities.     

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.     

Orbit/CLASP Is Required for Myosin Accumulation at the Cleavage Furrow in Drosophila Male Meiosis

Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.     

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Transforming growth factor β (TGF-β)-stimulated epithelial-mesenchymal transition (EMT) is an important developmental process that has also been implicated in increased cell invasion and metastatic potential of cancer cells. Expression of the focal adhesion protein Hic-5 has been shown to be up-regulated in epithelial cells in response to TGF-β. Herein, we demonstrate that TGF-β-induced Hic-5 up-regulation or ectopic expression of Hic-5 in normal MCF10A cells promoted increased extracellular matrix degradation and invasion through the formation of invadopodia. Hic-5 was tyrosine phosphorylated in an Src-dependent manner after TGF-β stimulation, and inhibition of Src activity or overexpression of a Y38/60F nonphosphorylatable mutant of Hic-5 inhibited matrix degradation and invasion. RhoC, but not RhoA, was also required for TGF-β- and Hic-5-induced matrix degradation. Hic-5 also induced matrix degradation, cell migration, and invasion in the absence of TGF-β via Rac1 regulation of p38 MAPK. These data identify Hic-5 as a critical mediator of TGF-β-stimulated invadopodia formation, cell migration, and invasion.     

The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules

Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) was identified recently, and D-spastin RNAi-treated or genetic null flies show neurological defects, and protein overexpression decreases the density of cellular microtubules. Elucidating spastin's function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. Here, we have investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. We show that D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities.     

Orbit/Mast,the CLASP orthologue of Drosophila,is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis

Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. We show that the Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts, the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed.     

The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. We examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted us to analyze focal adhesion dynamics. We found that the rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, we found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell–matrix adhesion.     

Microtubule bundle formation and cell death induced by the human CLASP/Orbit N-terminal fragment

Previously we have identified the Drosophila orbit gene whose hypomorphic mutations cause abnormal chromosome segregation (Inoue et al., 2000). The orbit encodes Orbit/Mast, a 165-kDa microtubule-associated protein (MAP) with GTP-binding motifs. Two human homologues of the Orbit/Mast, CLASP1 (hOrbit1) and CLASP2 (hOrbit2) have been identified. Using an antibody to CLASP1/hOrbit1 polypeptide, we confirmed that the polypeptide of about 150 kDa associates with microtubule purified from the porcine brain. Thus, we conjectured that CLASP1 may be a human orthologue of the Drosophila Orbit/Mast, and therefore we named it h (human) Orbit1. We constructed the plasmid for expression of a fusion protein of the putative microtubule-binding domain (1-662 out of 1289 residues) of hOrbit1 and the green fluorescent protein (GFP), and then, transfected the plasmid into Tet off cells derived from HeLa cell. Confocal laser scanning microscopic observation revealed that the GFP-fluorescence associated with short and thin filaments in the perinuclear region during the short period after plasmid transfection, and colocalized with only part of the microtubules. GFP fluorescence was later detected on the abnormally longer and thick bundles of microtubule filaments. Finally the bundles formed networks in the perinuclear region. The results suggest that the GFP-hOrbit1 N-terminal fragment (GFP-hOrbit1 NF) binds to the newly formed microtubules rather than the pre-formed ones, and that displacement of the endogenous hOrbit by the fragment might cause abnormal bundling of microtubules. Interestingly, the expression of the GFP-hOrbit1 NF results in cell death associated with nuclear fragmentation.     

Orbit/CLASP Is Required for Germline Cyst Formation through Its Developmental Control of Fusomes and Ring Canals in Drosophila Males

Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.     

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215^msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1–Sentin duo.     

PAR-1/MARK: a kinase essential for maintaining the dynamic state of microtubules

The serine/threonine kinase, PAR-1, is an essential component of the evolutionary-conserved polarity-regulating system, PAR-aPKC system, which plays indispensable roles in establishing asymmetric protein distributions and cell polarity in various biological contexts (Suzuki, A. and Ohno, S. (2006). J. Cell Sci., 119: 979-987; Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). PAR-1 is also known as MARK, which phosphorylates classical microtubule-associated proteins (MAPs) and detaches MAPs from microtubules (Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). This MARK activity of PAR-1 suggests its role in microtubule (MT) dynamics, but surprisingly, only few studies have been carried out to address this issue. Here, we summarize our recent study on live imaging analysis of MT dynamics in PAR-1b-depleted cells, which clearly demonstrated the positive role of PAR-1b in maintaining MT dynamics (Hayashi, K., Suzuki, A., Hirai, S., Kurihara, Y., Hoogenraad, C.C., and Ohno, S. (2011). J. Neurosci., 31: 12094-12103). Importantly, our results further revealed the novel physiological function of PAR-1b in maintaining dendritic spine morphology in mature neurons.     

TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization

TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previously, we have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions. We have now investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result we found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, we observed that TDP-43 physically interacts with futsch mRNA and that its RNA binding capacity is required to prevent futsch down regulation and synaptic defects.     

The C. elegans homologue of the spastic paraplegia protein, spastin, disassembles microtubules

Mutations in human spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia. Sequence analysis revealed that spastin contains the AAA (ATPases associated with diverse cellular activities) domain in the C-terminal region. Recently, it was reported that spastin interacts dynamically with microtubules and displays microtubule-severing activity. A plausible Caenorhabditis elegans homologue of spastin (SPAS-1) has been identified by homology search and phylogenetic analyses. To understand the function of the spastin homologue, we characterized the spas-1 deletion mutant and analyzed spas-1 expression regulation in C. elegans. SPAS-1 was localized with cytoskeletons at the perinuclear region. We found that microtubules were intensely stained at the centrosomal region in the deletion mutant. Furthermore, overexpression of SPAS-1 caused disassembly of microtubule network in cultured cells, while ATPase-deficient SPAS-1 did not. These results indicate that C. elegans SPAS-1 plays an important role in microtubule dynamics. We also found that two kinds of products were generated from spas-1 by alternative splicing in a developmental stage-dependent manner.     

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.     

The state of the chromosomes in the interphase nucleus

In the living interphase nucleus no chromosomal structures are visible. Yet in the injured cell and after treatment with most histological fixatives chromatin structures become apparent. Under certain conditions this appearance of structure in the living interphase nucleus is reversible. We have found that this change in the interphase nucleus is the result of a change in the state of the chromosomes. In the living nucleus the chromosomes are in a greatly extended state, filling the entire nucleus. Upon injury the chromosomes condense and therefore become visible. At the same time the nuclear volume decreases. This behavior of the chromosomes is connected with their content of desoxyribonucleic acid (DNA). This view is based on the following observations: (a) Distribution of DNA in the Nucleus.-(1) The living interphase nucleus of uninjured cells absorbs diffusely at 2537 A. No chromosomal structures are visible in ultraviolet photographs unless they are also distinct in ordinary light. If the chromosomes are made to condense they become visible and the absorption at 2537 A is now localized in these structures. (2) After fixation with formalin and osmic acid interphase nuclei stain diffusely with Feulgen. These fixatives preserve the extended state of the chromosomes. (3) If nuclei are teased out in non-electrolytes (sucrose, glycerin) the chromosomes are extended. Such nuclei stain homogeneously with methyl green. On adding salts the chromosomes condense and the methyl green is now restricted to the visible structures. (b) Extension and Condensation of Isolated Chromosomes.-When chromosomes isolated from interphase nuclei of calf thymus are suspended in sucrose, their volume is four to five times larger than in saline, but they retain their characteristic shapes. Chromosomes from which DNA and histone have been removed do not show this reversible extension and condensation, neither do lampbrush chromosomes of frog oocytes which contain very little DNA. During mitosis a partial condensation of the DNA occurs in prophase, so that the mitotic chromosomes now occupy a much smaller volume of the nucleus. At telophase the chromosomes swell again to fill the entire nucleus.