ABSTRACT
Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.
Subject(s)
Actins/metabolism , Adaptor Proteins, Vesicular Transport , Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , cdc42 GTP-Binding Protein/metabolism , Animals , Cell Line , Genes, Reporter/genetics , Humans , Microscopy, Confocal , Models, Biological , Neutrophils/metabolism , Phalloidine/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Wiskott-Aldrich Syndrome Protein, NeuronalABSTRACT
Mammalian Son-of-sevenless (mSos) functions as a guanine nucleotide exchange factor for Ras and Rac, thus regulating signaling to mitogen-activated protein kinases and actin dynamics. In the current study, we have identified a new mSos-binding protein of 50 kDa (p50) that interacts with the mSos1 proline-rich domain. Mass spectrometry analysis and immunodepletion studies reveal p50 as PACSIN 1/syndapin I, a Src homology 3 domain-containing protein functioning in endocytosis and regulation of actin dynamics. In addition to PACSIN 1, which is neuron-specific, mSos also interacts with PACSIN 2, which is expressed in neuronal and nonneuronal tissues. PACSIN 2 shows enhanced binding to the mSos proline-rich domain in pull-down assays from brain extracts as compared with lung extracts, suggesting a tissue-specific regulation of the interaction. Proline to leucine mutations within the Src homology 3 domains of PACSIN 1 and 2 abolish their binding to mSos, demonstrating the specificity of the interactions. In situ, PACSIN 1 and mSos1 are co-expressed in growth cones and actin-rich filopodia in hippocampal and dorsal root ganglion neurons, and the two proteins co-immunoprecipitate from brain extracts. Moreover, epidermal growth factor treatment of COS-7 cells causes co-localization of PACSIN 1 and mSos1 in actin-rich membrane ruffles, and their interaction is regulated through epidermal growth factor-stimulated mSos1 phosphorylation. These data suggest that PACSINs may function with mSos1 in regulation of actin dynamics.
Subject(s)
Carrier Proteins/metabolism , Cytoskeleton , Endocytosis , Son of Sevenless Proteins/metabolism , rac GTP-Binding Proteins/metabolism , ras Proteins/metabolism , Actins/metabolism , Animals , Cells, Cultured , Cytoskeletal Proteins , Cytoskeleton/metabolism , Rats , Signal TransductionABSTRACT
Our understanding of the means by which the left-right axis is patterned is not fully understood, although a number of key intermediaries have been recently described. We report here that retinoic acid (RA) excess affects heart situs concomitant with alterations in the expression of genes implicated in the establishment of the left-right axis. Specifically, RA exposure during a specific developmental window evoked bilateral expression of lefty-1, lefty-2, nodal, and pitx-2 in the lateral plate mesoderm. Time course experiments, together with analysis of midline markers, suggest that nascent mesoderm constitutes a predominant RA target involved in this process. These events are likely to underlie the perturbations of heart looping provoked by excess RA and suggest a means by which retinoids influence the early steps in establishment of the left-right embryonic axis.
Subject(s)
Heart/embryology , Nuclear Proteins , Tretinoin/pharmacology , Animals , Female , Gene Expression Regulation/drug effects , Homeodomain Proteins/genetics , Left-Right Determination Factors , Male , Mice , Nodal Protein , Organ Culture Techniques , Paired Box Transcription Factors , Pregnancy , Transcription Factors/genetics , Transforming Growth Factor beta/genetics , Homeobox Protein PITX2ABSTRACT
Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.
