Tuba stimulates intracellular N-WASP-dependent actin assembly.
Tuba is a multidomain scaffolding protein that links cytoskeletal dynamics and membrane trafficking pathways. The N-terminus of Tuba binds dynamin1, and the C-terminus contains domains that can interact with signaling pathways and cytoskeletal regulatory elements. We investigated Tuba localization, distribution and function in B16 melanoma cells. Tuba overexpression stimulated dorsal ruffles that occurred independently of dynamin function. Tuba expression induced actin-driven motility of small puncta that required the C-terminal SH3, GEF and BAR domains. Additionally, Tuba was recruited to lipid vesicles generated by overexpression of phosphatidylinositol-4-phosphate 5-kinase type Ialpha (PIP5Kalpha), localizing prominently to the head of the comets and at lower levels along the actin tail. We propose that Tuba facilitates dorsal ruffling of melanoma cells through direct interaction with actin-regulatory proteins and the recruitment of signaling molecules to lipid microdomains for the coordinated assembly of a cytoskeletal network. Knockdown of Tuba by RNA interference (RNAi) attenuated PIP5Kalpha-generated comet formation and the invasive behavior of B16 cells, implying that Tuba function is required for certain aspects of these processes. These results suggest first that Tuba-stimulated dorsal ruffling might represent a novel mechanism for the coordination of N-WASP-dependent cytoskeletal rearrangements and second that Tuba function is implicated in motility processes.
Kovacs
Eva M
EM
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Makar
Robert S
RS
Gertler
Frank B
FB
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D021961:Cell Membrane Structures; D002465:Cell Movement; D002478:Cells, Cultured; D051356:Cortactin / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005353:Fibronectins / Q000378:metabolism; D020868:Gene Silencing; D006133:Growth Substances / Q000494:pharmacology; D008546:Melanoma, Experimental; D051379:Mice; D015778:Minor Histocompatibility Antigens; D009361:Neoplasm Invasiveness; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D051356:Cortactin; C495869:Cttn protein, mouse; D003598:Cytoskeletal Proteins; C102177:Evl protein, mouse; C482527:FNBP1L protein, human; D005353:Fibronectins; D006133:Growth Substances; D015778:Minor Histocompatibility Antigens; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D014404:Tubulin; C495772:Wasl protein, mouse; C491671:Waspip protein, mouse; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D020764:cdc42 GTP-Binding Protein