RESUMEN
The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.
Asunto(s)
Actinas/fisiología , Axones/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Proteínas de Microfilamentos/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Actinas/genética , Secuencias de Aminoácidos/genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Inactivación de Genes/métodos , Conos de Crecimiento/fisiología , Masculino , Ratones , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/deficiencia , Mutación , Células 3T3 NIH , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/fisiología , Cultivo Primario de CélulasRESUMEN
By using a microarray screen to compare gene responses after sterile laser wounding of wild-type and 'macrophageless' serpent mutant Drosophila embryos, we show the wound-induced programmes that are independent of a pathogenic response and distinguish which of the genes are macrophage dependent. The evolutionarily conserved nature of this response is highlighted by our finding that one such new inflammation-associated gene, growth arrest and DNA damage-inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild-type and serpent mutant embryos also shows a portfolio of 'macrophage-specific' genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound- and macrophage-related genes, our data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a 'pathogen response', which might prime the organism for what is likely to be an increased risk of infection.
Asunto(s)
Drosophila/embriología , Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/fisiología , Animales , Drosophila/genética , Proteínas de Drosophila/fisiología , Embrión no Mamífero , Factores de Transcripción GATA/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Hemocitos/fisiología , Homocigoto , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Arriba , Heridas y Lesiones/etiología , Heridas y Lesiones/genética , Proteinas GADD45RESUMEN
Dorsal closure is a paradigm epithelial fusion episode that occurs late in Drosophila embryogenesis and leads to sealing of a midline hole by bonding of two opposing epithelial sheets. The leading edge epithelial cells express filopodia and fusion is dependent on interdigitation of these filopodia to prime formation of adhesions. Since the opposing epithelia are molecularly patterned there must exist some mechanism for accurately aligning the two sheets across this fusion seam. To address this, we generated a fly in which RFP-Moesin and GFP-Moesin are expressed in mutually exclusive stripes within each segment using the engrailed and patched promoters. We observe mutually exclusive interactions between the filopodia of engrailed and patched cells. Interactions between filopodia from matching cells leads to formation of tethers between them, and these tethers can pull misaligned epithelial sheets into alignment. Filopodial matching also occurs during repair of laser wounds in the ventral epithelium, and so this behaviour is not restricted to leading edge cells during dorsal closure. Finally, we characterise the behaviour of a patched-expressing cell that we observe within the engrailed region of segments A1-A5, and provide evidence that this cell contributes to cell matching.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Desarrollo Embrionario , Seudópodos/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Tipificación del Cuerpo , Proteínas de Drosophila , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Epitelio/embriología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores Patched , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Cicatrización de HeridasRESUMEN
IRSp53 is a scaffold protein that contains an IRSp53/MIM homology domain (IMD) that bundles actin filaments and interacts with the small GTPase Rac. IRSp53 also binds to the small GTPase Cdc42 and to Scar/WAVE and Mena/VASP proteins to regulate the actin cytoskeleton. We have characterised a novel IMD-containing protein, insulin receptor tyrosine kinase substrate (IRTKS), which has widespread tissue distribution, is a substrate for the insulin receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters of short actin bundles rather than filopodia-like protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which resembles a WASP-homology 2 (WH2) motif. Addition of the Ct extension to IRSp53 causes an apparent shortening of bundles induced by the IMD in vitro, and in cultured cells, suggesting that the Ct extension of IRTKS modulates the organising activity of the IMD. Lastly, we could not detect actin monomer sequestration by the Ct extension of IRTKS as would be expected with a conventional WH2 motif, but it did interact with actin filaments.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Chlorocebus aethiops , Cortactina/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
BACKGROUND: The Scar/WAVE family of proteins mediates signals to actin assembly by direct activation of the Arp2/3 complex. These proteins have been characterised as major regulators of lamellipodia formation downstream of Rac activation and as members of large protein complexes. RESULTS: We have investigated the interactions of the three human Scar/WAVE isoforms with several previously described binding partners for Scar/WAVE 1 or 2. We find that all three Scar/WAVE isoforms behave similarly and are likely to participate in the same kinds of protein complexes that regulate actin assembly. CONCLUSION: Differences between Scar/WAVE proteins are therefore likely to be at the level of tissue distribution or subtle differences in the affinity for specific binding partners.
Asunto(s)
Complejos Multiproteicos/química , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Animales , Encéfalo , Ratones , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Sitios de Unión , Neoplasias de la Mama/química , Carcinoma/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Endosomas/química , Endosomas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/análisis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Proteínas del Tejido Nervioso/análisis , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Factores de Intercambio de Guanina Nucleótido Rho , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/análisis , Quinasas p21 ActivadasRESUMEN
The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.
Asunto(s)
Proteínas del Tejido Nervioso/química , Seudópodos/fisiología , Secuencia de Aminoácidos , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Seudópodos/química , Homología de Secuencia de AminoácidoRESUMEN
The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas/metabolismo , Citoesqueleto de Actina/ultraestructura , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Adenosina Trifosfato/metabolismo , Animales , Proteínas del Citoesqueleto/ultraestructura , Humanos , Sustancias Macromoleculares , Ratones , Ratones Noqueados , Microscopía Electrónica , Modelos Moleculares , Morfogénesis , Proteínas de Plantas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína del Síndrome de Wiskott-AldrichRESUMEN
Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain.
Asunto(s)
Calpaína/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Dominio Catalítico/genética , Membrana Celular/metabolismo , Citosol/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Aparato de Golgi/metabolismo , Proteínas de Microfilamentos/fisiología , Cicatrización de Heridas , Células 3T3 , Proteína 2 Relacionada con la Actina , Animales , Astrocitos/metabolismo , Western Blotting , Movimiento Celular , ADN/metabolismo , Eliminación de Gen , Ligandos , Ratones , Proteínas de Microfilamentos/química , Microscopía Fluorescente , Unión Proteica , Estructura Terciaria de Proteína , Factores de Tiempo , Familia de Proteínas del Síndrome de Wiskott-AldrichRESUMEN
Scratch-wound assays are commonly used to study the ability of cells to polarize and migrate. In a previous study we showed that Golgi reorientation in response to a scratch wound is actin-dependent in NIH 3T3 cells but not in astrocytes. In this investigation, to study cell polarity and motility further, we used the polarization of the Golgi and microtubule organizing center (MTOC), as well as the ability of NIH 3T3 cells to migrate, in a scratch-wound assay. Unlike Golgi polarization, MTOC polarization was not dependent on actin, the Arp2/3 complex or Wiskott-Aldrich syndrome protein (WASP)-family proteins. By contrast, disruption of microtubules inhibited MTOC polarity, but not Golgi polarity. Migration was found to be dependent both on actin and microtubules. Expression of the formin-homology 2 (FH2) region of mDia1 inhibited Golgi polarization and migration but not MTOC polarization. Similarly, ST638, a Src inhibitor, inhibited Golgi polarization and migration but not MTOC polarization, whereas expression of the actin regulator IRSp53 only inhibited cell migration. Interestingly, the inhibition of cell migration by the mDia1 FH2 domain could be overcome by addition of Y27632, an inhibitor of ROCK (Rho-associated kinase). In fact, in the presence of ROCK inhibitor, cell migration was accelerated but polarization of both the Golgi and MTOC were inhibited. These data show that, in NIH 3T3 cells, different aspects of cell polarization and migration occur by different mechanisms, and both actin and microtubule networks are required. In addition, this study indicates that MTOC and Golgi polarization events are separately controlled.
Asunto(s)
Movimiento Celular/fisiología , Polaridad Celular/fisiología , Células Eucariotas/metabolismo , Aparato de Golgi/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células 3T3 , Actinas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Inhibidores Enzimáticos/farmacología , Células Eucariotas/ultraestructura , Forminas , Aparato de Golgi/ultraestructura , Péptidos y Proteínas de Señalización Intracelular , Ratones , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína/fisiología , Cicatrización de Heridas/fisiología , Quinasas Asociadas a rhoRESUMEN
The Arp2/3 complex is an actin filament nucleator that activates regulated actin assembly in response to extracellular signals. The mammalian complex is composed of seven subunits, the smallest of which is known as ARPC5 or p16-Arc. We have identified a human cDNA sequence with homology to ARPC5 and here provide evidence that this encodes a novel ARPC5 isoform. Specific antibodies were generated against the novel protein, which we have termed ARPC5B, as well as the previously characterised ARPC5 isoform, henceforth ARPC5A. The presence of both ARPC5 isoforms was detected in Arp2/3 complex affinity purified from human neutrophil extract. The tissue distribution of ARPC5A and B was analysed using the isoform-specific antibodies and it was found that the two isoforms exhibited significant differences; ARPC5A was found to be highly enriched in spleen and thymus, while ARPC5B exhibits a more regular expression, with levels in the brain being highest. Myc-tagged ARPC5A and B co-localised with the Arp2/3 complex when expressed in C2C12 cells and the cellular distribution of the two isoforms could not be distinguished. Our data show for the first time that mammalian cells contain multiple forms of the Arp2/3 complex.