LIS1 is a microtubule-associated phosphoprotein.

Lissencephaly, a severe brain malformation, may be caused by mutations in the LIS1 gene. LIS1 encodes a microtubule-associated protein (MAP) that is also part of the enzyme complex, platelet-activating factor acetylhydrolase. LIS1 is also found in a complex with two protein kinases; a T-cell Tat-associated kinase, which contains casein-dependent kinase (CDK) activating kinase (CAK), as well as CAK-inducing activity, and with a spleen protein-tyrosine kinase similar to the catalytic domain of p72syk. As phosphorylation is one of the ways to control cellular localization and protein-protein interactions, we investigated whether LIS1 undergoes this post-translational modification. Our results demonstrate that LIS1 is a developmentally regulated phosphoprotein. Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis revealed that LIS1 is phosphorylated on serine residues. Alkaline phosphatase treatment reduced the number of visible LIS1 isoforms. In-gel assays demonstrate a 50-kDa LIS1 kinase that is enriched in microtubule-associated fractions. In vitro, LIS1 was phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested. We suggest that LIS1 activity may be regulated by phosphorylation.     

WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana

Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione-S-transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules.     

Splice variant-specific cellular function of the formin INF2 in maintenance of Golgi architecture

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2-non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2-non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2-non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer-sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.     

Dystrophin is a microtubule-associated protein

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K_d of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.     

INF2 Is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization

Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed "diaphanous formins" are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory domain (DID) and a C-terminal diaphanous autoregulatory domain (DAD). Here, we found several striking features for the mouse formin, INF2. First, INF2 interacted directly with actin through a region C-terminal to the FH2. This second interacting region sequesters actin monomers, an activity that is dependent on a WASP homology 2 (WH2) motif. Second, the combination of the FH2 and C-terminal regions of INF2 resulted in its curious ability to accelerate both polymerization and depolymerization of actin filaments. The mechanism of the depolymerization activity, which is novel for formin proteins, involves both the monomer binding ability of the WH2 and a potent severing activity that is dependent on covalent attachment of the FH2 to the C terminus. Phosphate inhibits both the depolymerization and severing activities of INF2, suggesting that phosphate release from actin subunits in the filament is a trigger for depolymerization. Third, INF2 contains an N-terminal DID, and the WH2 motif likely doubles as a DAD in an autoinhibitory interaction.     

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. Here, we provide evidence for a clathrin-independent endocytic pathway in yeast. In cells lacking the clathrin-binding adaptor proteins Ent1, Ent2, Yap1801, and Yap1802, we identify a second endocytic pathway that depends on the GTPase Rho1, the downstream formin Bni1, and the Bni1 cofactors Bud6 and Spa2. This second pathway does not require components of the better-studied endocytic pathway, including clathrin and Arp2/3 complex activators. Thus, our results reveal the existence of a second pathway for endocytosis in yeast, which suggests similarities with the RhoA-dependent endocytic pathways of mammalian cells.     

MAP3: characterization of a novel microtubule-associated protein

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule-associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.     

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.     

RMD-1, a novel microtubule-associated protein, functions in chromosome segregation in Caenorhabditis elegans

For proper chromosome segregation, the sister kinetochores must attach to microtubules extending from the opposite spindle poles. Any errors in microtubule attachment can induce aneuploidy. In this study, we identify a novel conserved Caenorhabditis elegans microtubule-associated protein, regulator of microtubule dynamics 1 (RMD-1), that localizes to spindle microtubules and spindle poles. Depletion of RMD-1 induces severe defects in chromosome segregation, probably through merotelic attachments between microtubules and chromosomes. Although rmd-1 embryos also have a mild defect in microtubule growth, we find that mutants of the microtubule growth regulator XMAP215/ZYG-9 show much weaker segregation defects. This suggests that the microtubule growth defect in rmd-1 embryos does not cause abnormal chromosome segregation. We also see that RMD-1 interacts with aurora B in vitro. Our results suggest that RMD-1 functions in chromosome segregation in C. elegans embryos, possibly through the aurora B–mediated pathway. Human homologues of RMD-1 could also bind microtubules, which would suggest a function for these proteins in chromosome segregation during mitosis in other organisms as well.     

FHOD1 formin is upregulated in melanomas and modifies proliferation and tumor growth

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.     

CLAMP, a novel microtubule-associated protein with EB-type calponin homology

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.     

A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin

Actin-filament bundles (or cables) have a structural role during cell division and morphogenesis, but also serve as important "tracks" for the transport of materials during cytokinesis and polarized cell growth. However, the dynamic formation of these longitudinal actin-filament higher-order structures is not understood. Recently, several lines of evidence suggest that formins provide one avenue for the initiation of actin cables in vivo. A popular model for the mechanism of polymerization of actin filaments by formin involves the processive movement of formin attached at the barbed end of an elongating filament. In the present study, we use an in vitro system to reconstitute the dynamic formation of actin-filament bundles generated by Arabidopsis FORMIN1 (AFH1). To be able to visualize individual events in such a complex system, we used real-time evanescent-wave microscopy. Surprisingly, we find that AFH1 is a nonprocessive formin that moves from the barbed end to the side of an actin filament after the nucleation event. We show why this new mechanism of nucleation by a member of the formin family is important for bundle formation. Finally, we analyze the different parameters controlling the dynamic formation of such longitudinal actin-filament bundles.     

Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity

Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is a brain specific and F-actin-binding protein. We recently demonstrated that IP(3)K-A modulates a structural reorganization of dendritic spines through F-actin remodeling, which is required for synaptic plasticity and memory formation in brain. However, detailed functions of IP(3)K-A and its regulatory mechanisms involved in the neuronal cytoskeletal dynamics still remain unknown. In the present study, we identified tubulin as a candidate of IP(3)K-A-binding protein through proteomic screening. By various in vitro and in vivo approaches, we demonstrated that IP(3)K-A was a novel microtubule-associated protein (MAP), and the N terminus of IP(3)K-A was a critical region for direct binding to tubulin in dendritic shaft of hippocampal neurons. Moreover, PKA phosphorylated Ser-119 within IP(3)K-A, leading to a significant reduction of microtubule binding affinity. These results suggest that PKA-dependent phosphorylation and microtubule binding of IP(3)K-A are involved in its regulatory mechanism for activity-dependent neuronal events such as local calcium signaling and its synaptic targeting.     

NuSAP,a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

hSnm1B is a novel telomere-associated protein

Artemis, a member of the beta-CASP family, has been implicated in the regulation of both telomere stability and length. Prompted by this, we examined whether the other two putative DNA-binding members of this family, hSnm1A and hSnm1B, may associate with telomeres. hSnm1A was found to not interact with the telomere. Conversely, hSnm1B was found to associate with telomeres in vivo by both immunofluorescence and chromatin immunoprecipitation. Furthermore, the C terminus of hSnm1B was shown to interact with the TRF homology domain of TRF2 indicating that hSnm1B is likely recruited to the telomere via interaction with the double-stranded telomere-binding protein TRF2.