Ric-8A and Giα 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 ␣ (G␣) 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␣ 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␣ i , thus preventing its GEF activity for G␣ i . Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G␣ 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.

Nucleotide exchange factor RIC‐8 is indispensable in mammalian early development

“…Although, the RIC-8 expression in the embryonic tissues is clearly detectable by immunohistochemistry only from the beginning of the gastrulation, the earlier growth retardation of the Ric-8 À/À mutant embryos, compared to the control littermates, may be due to the interfered mitotic division. This speculation is in line with the experiments in HeLa cells, where the reduced expression of RIC-8A was shown to prolong mitosis, cause occasional mitotic arrest, and decreased movement of the spindle (Woodard et al, 2010).…”

“…This tertiary complex initially localizes at the cell cortex, where the dynamic release of NuMA from LGN may promote aster microtubule pulling during cell division (Du et al, 2002;Du and Macara, 2004). A very recent study demonstrated that RIC-8A is necessary for the assembly of a cortical signaling complex that orients the mitotic spindle during mammalian cell division (Woodard et al, 2010). The expression pattern of RIC-8 characterized in our study lets us assume that it is involved in the cell division in developing ectoderm during mouse gastrulation.…”

“…1 D). Recently, it was shown that, depending on the phase of mitosis, RIC-8A localized at the cell cortex, spindle poles, centromeres, central spindle, or midbody in mammalian cells (Woodard et al, 2010). Biochemical studies suggests that RIC-8A acts on a tertiary complex of GDP-Ga i , LGN (mammalian Pins homolog) and NuMA (nuclear mitotic apparatus protein) causing release of GTP-Ga i and dissociation of NuMA from LGN (Tall and Gilman, 2005).…”

“…Biochemical studies of mammalian RIC-8 function suggest that it regulates the microtubule pulling forces on centrosomes during cell division (Tall and Gilman, 2005). Recent studies demonstrate that mammalian RIC-8 participates in the assembly of a cortical signalling complex that orients the mitotic spindle (Woodard et al, 2010). The ric-8 reduction-offunction mutants of C. elegans exhibited embryonic lethality , whereas ric-8 mutants of Drosophila had various defects during the asymmetric cell division and in gastrulation (Hampoelz et al, 2005;Wang et al, 2005).…”

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

“…In cells with supernumerary centrosomes that exhibit clustering, the net force exerted on each centrosome by motor proteins may point to one or the other of two poles. Nonhomogenous distribution of dynein/dynactin, as has been found in HeLa cells, 116,117 and/or cortical cues 91 could orient the force vectors toward one or the other pole. Clustering would then be accomplished by virtue of the various centrosomes having a common final destination.…”