ABSTRACT
A dynamic actin cytoskeleton is essential for the remodeling of cell shape during development, but the specific roles of many actin partners remain unclear. Here we characterize a novel actin binding protein, Ciboulot (Cib), which plays a major role in axonal growth during Drosophila brain metamorphosis. Loss of Cib function leads to axonal growth defects in the central brain, while overexpression of the gene during development leads to overgrown projections. The Cib protein displays strong sequence similarity to beta-thymosins but has biochemical properties like profilin: the Cib-actin complex participates in actin filament assembly exclusively at the barbed end, and Cib enhances actin-based motility in vitro. Genetic experiments show that Cib and the Drosophila profilin protein Chickadee (Chic) cooperate in central brain metamorphosis.
Subject(s)
Actins/metabolism , Contractile Proteins , Drosophila/embryology , Gene Expression Regulation, Developmental , Metamorphosis, Biological/physiology , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Animals , Brain/cytology , Brain/embryology , Brain/metabolism , Cell Differentiation/physiology , Cell Size/physiology , Drosophila Proteins , Listeria monocytogenes/metabolism , Molecular Sequence Data , Movement , Mutagenesis/physiology , Nerve Tissue Proteins , Neurons/physiology , Profilins , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Thymosin/geneticsABSTRACT
The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Cytoskeletal Proteins , Listeria monocytogenes/physiology , Microfilament Proteins/antagonists & inhibitors , Actin Cytoskeleton/chemistry , Actin Depolymerizing Factors , Actin-Related Protein 2 , Actin-Related Protein 3 , Actins/antagonists & inhibitors , Actins/ultrastructure , Animals , Biopolymers/chemistry , Biopolymers/metabolism , Destrin , Gelsolin/metabolism , Kinetics , Microfilament Proteins/metabolism , Microscopy, Electron , Models, Biological , Movement , Nerve Tissue Proteins/metabolism , Rabbits , Solutions , Wiskott-Aldrich Syndrome Protein, NeuronalABSTRACT
Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.
Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing , Cytoskeletal Proteins , Proteins/metabolism , Signal Transduction , Actin-Related Protein 2 , Actin-Related Protein 3 , Animals , ErbB Receptors/metabolism , GRB2 Adaptor Protein , Humans , Rabbits , Recombinant Proteins/metabolism , Wiskott-Aldrich Syndrome , Wiskott-Aldrich Syndrome ProteinABSTRACT
Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.
