EPI64 regulates microvillar subdomains and structure.
EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.
Hanono
Abraham
A
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Garbett
Damien
D
Reczek
David
D
Chambers
David N
DN
Bretscher
Anthony
A
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism* / Q000502:physiology; D000199:Actins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism; D000099149:Ezrin
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C425805:TBC1D10A protein, human; C093376:sodium-hydrogen exchanger regulatory factor; C000715348:ARF6 protein, human