title: Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle.
abstract: In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.
authors.affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Disease, 10 Center Drive, MSC 1888, Bethesda, MD 20892-1752, USA.
authors.forename: Geoffrey E
authors.initials: GE
authors.lastname: Woodard
authors.forename: Ning-Na
authors.initials: NN
authors.lastname: Huang
authors.forename: Hyeseon
authors.initials: H
authors.lastname: Cho
authors.forename: Toru
authors.initials: T
authors.lastname: Miki
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Tall
authors.forename: John H
authors.initials: JH
authors.lastname: Kehrl
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism*; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004285:Dogs; D004398:Dyneins / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008677:Metaphase / Q000502:physiology; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C102754:GPSM2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D034741:RNA, Small Interfering; D037342:Pertussis Toxin; D004398:Dyneins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go