Myelin proteolipid protein expressed in COS-1 cells is targeted to actin-associated surfaces

The compact myelin sheath represents one of the largest expanses of membrane-membrane contact in the body and, in the central nervous system, requires the myelin proteolipid protein (PLP) for assembly, To determine whether the molecular properties of PLP promote membrane adhesion and direct its subcellular localization in the absence of oligodendrocyte-specific targeting mechanisms, PLP was expressed in COS-I fibroblasts, Immunofluorescence staining indicated that PUP was translated effectively, transited the rough endoplasmic reticulum and Golgi apparatus, was delivered to the cell surface, and was endocytosed, In the plasma membrane, the PLP distribution was patchy and only sporadically coincided with sites of membrane-membrane contact between PLP-expressing cells, PLP was not randomly distributed, however, but correlated closely with microfilament locations in leading edge membranes and microvilli, as demonstrated by phalloidin double labeling, Our results indicate that even in non-myelinating cells, PLP can be concentrated in membranes associated with movement and growth, and suggest possible roles for the actin cytoskeleton in PLP localization, As PLP, DM20, and the DM20-like M6 protein all associate with actin-enriched membranes, this may be a common feature of PLP/DM20 gene family members. (C) 1997 Wiley-Liss, Inc.

CCTα and CCTδ chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena

Background - The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTalpha to CCTzeta). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (alpha, beta and gamma) and actin, as quantitatively major substrates. Methodology/Principal findings - We disrupted the genes encoding CCTalpha and CCTdelta subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTalpha or CCTdelta showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTalpha rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTalpha, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTalpha localization in cilia. Conclusions/Significance - Our results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.

Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

Dissertation presented to obtain the Ph.D degree in Molecular Medicine

The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles.

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

Pob1 participates in the Cdc42 regulation of fission yeast actin cytoskeleton.

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.

Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in traf- ficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

Actin cytoskeleton regulates pollen tube growth and tropism

La fertilisation chez les plantes dépend de la livraison des cellules spermatiques contenues dans le pollen à l’ovule. Au contact du stigmate, le grain de pollen s’hydrate et forme une protubérance, le tube pollinique, chargé de livrer les noyaux spermatiques à l’ovule. Le tube pollinique est une cellule à croissance rapide, anisotrope et non autotrophe; ainsi tout au long de sa croissance à travers l’apoplaste du tissu pistillaire, le tube pollinique puise ses sources de carbohydrates et de minéraux du pistil. Ces éléments servent à la synthèse des constituants de la paroi qui seront acheminés par des vésicules de sécrétion jusqu’à l’apex du tube. Ce dernier doit aussi résister à des pressions mécaniques pour maintenir sa forme cylindrique et doit répondre à différents signaux directionnels pour pouvoir atteindre l’ovule. Mon projet de doctorat était de comprendre le rôle du cytosquelette dans la croissance anisotrope du tube pollinique et d’identifier les éléments responsables de sa croissance et de son guidage. Le cytosquelette du tube pollinique est composé des microfilaments d’actine et des microtubules. Pour assurer une bonne croissance des tubes polliniques in vitro, les carbohydrates et les éléments de croissance doivent être ajoutés au milieu à des concentrations bien spécifiques. J’ai donc optimisé les conditions de croissance du pollen d’Arabidopsis thaliana et de Camellia japonica qui ont été utilisés avec le pollen de Lilium longiflorum comme modèles pour mes expériences. J’ai développé une méthode rapide et efficace de fixation et de marquage du tube pollinique basée sur la technologie des microondes. J’ai aussi utilisé des outils pharmacologiques, mécaniques et moléculaires couplés à différentes techniques de microscopie pour comprendre le rôle du cytosquelette d’actine lors de la croissance et le tropisme du tube pollinique. J’ai trouvé que le cytosquelette d’actine et plus précisément l’anneau d’actine localisé dans la partie sub-apicale du tube est fortement impliqué dans la croissance et le maintien de l’architecture du tube à travers le contrôle de la livraison des vésicules de sécrétion. J’ai construit une chambre galvanotropique qui peut être montée sur un microscope inversé et qui sert à envoyer des signaux tropistiques bien précis à des tubes polliniques en croissance. J’ai trouvé que les filaments d’actine sont impliqués dans la capacité du tube pollinique à changer de direction. Ce comportement tropistique dépend de la concentration du calcium dans le milieu de croissance et du flux de calcium à travers des canaux calciques. Le gradient de calcium établi dans le tube pollinique affecte l’activité de certaines protéines qui se lient à l’actine et dont le rôle est la réorganisation des filaments d’actine. Parmi ces protéines, il y a celles de dépolymérisation de l’actine (ADF) dont deux spécifiquement exprimées dans le gamétophyte mâle d’Arabidopsis (ADF7 et ADF10). Par marquage avec des proteins fluorescents, j’ai trouvé que l’ADF7 et l’ADF10 ont des expressions différentielles pendant la microsporogenèse et la germination et croissance du tube pollinique et qu’elles partagent entre elles des rôles importants durant ces différents stades.

Theileria annulata promotes Src kinase-dependent host cell polarization by manipulating actin dynamics in podosomes and lamellipodia

Theileria annulata is an intracellular protozoan parasite that infects B cells and macrophages of ruminants. Macrophages infected with T. annulata are de-differentiated and display tumour cell properties and a metastatic behaviour. How parasitized cells adapt their morphology, motility and invasive behaviour has not yet been addressed in detail. In this study, I investigated the regulation of host cell actin dynamics in T. annulata-transformed macrophages and how this affects host cell morphology and motility. T. annulata was found to promote the formation of filamentous-actin-rich podosome-type adhesions (PTAs) and lamellipodia, and to establish a polarized morphology of the infected cell. Characteristic for parasite-dependent host cell polarization is that infected cells display a single, persistent lamellipodium. Src kinases--in particular Hck--are required for the polar extension of this lamellipodium. Hck does so by promoting the clustered assembly of PTAs and accumulation of proteins of the Ezrin, Radixin, Moesin (ERM) family in lamellipodia. Polar accumulation of PTAs and ERM proteins correlates with focal matrix degradation underneath lamellipodia. These findings suggest that T. annulata equips its host cell with properties to adhere and invade. These properties are likely to promote the motile behaviour required for dissemination of infected cells in vivo.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho

Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5′-[γ-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.

An autonomous folding unit mediates the assembly of two-stranded coiled coils

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil “trigger” sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 μM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.