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Characterizing the functions of WHIMP as a new WASP‐family actin nucleation factor
Author(s) -
Kabrawala Shail,
Zimmer Margaret,
Campellone Kenneth
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.860.1
Subject(s) - microbiology and biotechnology , cytoplasm , biology , actin , actin binding protein , green fluorescent protein , pleckstrin homology domain , cytoskeleton , actin cytoskeleton , cell , biochemistry , gene , signal transduction
Actin nucleation factors from the Wiskott‐Aldrich Syndrome Protein (WASP) family cooperate with the Arp2/3 complex to assemble branched filament networks during cell migration, vesicle trafficking, and many other vital cellular functions. To initiate actin assembly, conserved WH2‐Connector‐Acidic (WCA) domains within the eight known mammalian WASP‐family proteins bind both to actin monomers and the Arp2/3 complex. In the current study, based on sequence similarity to WCA domains, we have identified a new protein in the family, WHIMP (WAVE Homology In Membrane Protrusions). Immunoblotting of mouse tissue extracts indicated that WHIMP was expressed in multiple organs, including heart and kidney. Additionally, RT‐PCR and immunoblotting demonstrated that WHIMP was expressed in several different mouse cell lines. Separation of NIH3T3 cells into their nuclear, membrane, and cytosolic fractions revealed that endogenous WHIMP was found predominantly in the cytosol. A further examination of WHIMP localization by fluorescence microscopy confirmed that GFP‐tagged WHIMP was dispersed throughout the cytoplasm. However, after treatment of cells with epidermal growth factor, WHIMP became enriched at membrane ruffles. Similar recruitment of WHIMP near the plasma membrane was observed when cells adhered to a substrate. Overexpression of WHIMP also caused a small, yet statistically significant increase in cytoplasmic actin filaments. Additional biochemical analyses using purified proteins in pyrene‐actin assembly assays suggested that recombinant WHIMP was a weaker nucleation‐promoting factor than other WASP‐family proteins. Taken together, these results provide insight into the expression, localization, and actin assembly properties of WHIMP as a new member of the WASP family.