Resistance to inhibitors of cholinesterase (Ric)-8A and Gαi contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity.
Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.
B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Cedric
C
Boularan
B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Olena
O
Kamenyeva
B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Hyeseon
H
Cho
B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
John H
JH
Kehrl
D045744:Cell Line, Tumor; D058546:Class III Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D048749:Cytokinesis / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology
D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C561938:Ric8A protein, human; D058546:Class III Phosphatidylinositol 3-Kinases