RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery.
Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.
Engqvist-Goldstein
Asa E Y
AE
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
Zhang
Claire X
CX
Carreno
Sebastien
S
Barroso
Consuelo
C
Heuser
John E
JE
Drubin
David G
DG
D008841:Actin Cytoskeleton / Q000737:chemistry; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D051356:Cortactin; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D034281:Dynamins / Q000737:chemistry; D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011419:Propidium / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014168:Transferrin / Q000737:chemistry / Q000493:pharmacokinetics; D033921:Vesicular Transport Proteins
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C495868:CTTN protein, human; D051356:Cortactin; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C073368:HIP1 protein, human; C489537:HIP1R protein, human; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D014168:Transferrin; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D011419:Propidium; D012313:RNA; D004247:DNA; D034281:Dynamins