Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions

Abstract

Heterotrimeric G proteins mediate asymmetric division of Drosophila neuroblasts. Free G␤␥ appears to be crucial for the generation of an asymmetric mitotic spindle and consequently daughter cells of distinct size. However, how G␤␥ is released from the inactive heterotrimer remains unclear. Here we show that Locomotion defects (Loco) interacts and colocalizes with G␣i and, through its GoLoco motif, acts as a guanine nucleotide dissociation inhibitor (GDI) for G␣i. Simultaneous removal of the two GoLoco motif proteins, Loco and Pins, results in defects that are essentially indistinguishable from those observed in G␤13F or G␥1 mutants, suggesting that Loco and Pins act synergistically to release free G␤␥ in neuroblasts. Furthermore, the RGS domain of Loco can also accelerate the GTPase activity of G␣i to regulate the equilibrium between the GDP-and the GTP-bound forms of G␣i. Thus, Loco can potentially regulate heterotrimeric G-protein signaling via two distinct modes of action during Drosophila neuroblast asymmetric divisions. Asymmetric cell division is a universal mechanism used to generate cellular diversity during development. The Drosophila embryonic central nervous system (CNS) derives largely from neural progenitors called neuroblasts (NBs). NBs delaminate from the neuroectoderm and undergo asymmetric cell division along the apical/basal axis to give rise to two daughters of distinct fate and size. The larger apical daughter cell retains a NB identity and undergoes repeated asymmetric divisions, whereas the smaller basal daughter differentiates into a ganglion mother cell (GMC) that divides only once to generate two neurons/glia (Campos-Ortega 1997). Three wellcharacterized features of the NB asymmetric divisions (Jan and Jan 2001; Knoblich 2001; Wodarz and Huttner 2003) are (1) asymmetric localization and segregation of cell fate determinants and their adaptor proteins Numb/ Partner of Numb (Pon), Prospero (Pros)/Miranda (Mira) into the basal GMC; (2) reorientation of the mitotic spindle along the apical/basal axis at metaphase; (3) generation of an apically biased asymmetric mitotic spindle (Kaltschmidt et al. 2000;Kaltschmidt and Brand 2002) and the displacement of the spindle toward the basal cortex during ana/telophase as well as asymmetric formation of astral microtubules (MTs) (Giansanti et al. 2001), which lead to the generation of two unequal-sized daughter cells.These features of the NB asymmetric division are controlled by an apically localized complex of proteins that include the Drosophila homologs (Doe and Bowerman 2001) of the conserved Par3 (Bazooka, Baz)/Par6 (DmPar6)/aPKC (DaPKC) protein cassette first identified in Caenorhabditis elegans (Kemphues 2000), the novel protein Inscuteable (Insc), G␣i, a subunit of heterotrimeric G proteins (Schaefer et al. 2001;Yu et al. 2003), and an evolutionarily conserved molecule, Partner of Insc (Pins) (Parmentier et al. 2000;Schaefer et al. 2000;Yu et al. 2000) that acts as a guanine nucleotide dissociation inhibitor (GDI) for G␣i. Loss of single member...

Insights into neural stem cell biology from flies

“…The apical complex (figure 3b) consists of the evolutionarily conserved Par proteins, Bazooka (Baz, Par3) and DmPar6, and the Drosophila homologue of the atypical protein kinase C, DaPKC (Kuchinke et al 1998;Schober et al 1999;Wodarz et al 1999;. In neuroblasts, the Baz/DmPar6/DaPKC complex binds through Baz to Inscuteable (Insc), and Insc, in turn, recruits Partner of Inscuteable (Pins) and Locomotion defective (Loco), which are two GDPdissociation inhibitors (GDIs) that interact with the heterotrimeric G protein subunit, Gai (Kraut & Campos-Ortega 1996;Parmentier et al 2000;Yu et al 2000Yu et al , 2003Yu et al , 2005Schaefer et al 2001).…”

“…Loco function is required for the apical localization of Gai and acts in parallel to the Par pathway to mediate unequal daughter cell size ( Yu et al 2005). Yu et al combine these new data with previous knowledge to propose an interesting model whereby activated Gbg, maintained through the GDI activity of Pins and Loco, is required for the generation of unequal daughter cells; the equilibrium between GDP-Gai and GTP-Gai, at least partially regulated by the GAP activity of Loco, is required for the localization of Insc/Pins/Loco at the apical cortex in neuroblasts ( Yu et al 2005).…”

Regulation of Glia Number in Drosophila by Rap/Fzr, an Activator of the Anaphase-Promoting Complex, and Loco, an RGS Protein

“…In Drosophila, however, Yu et al (2005) have shown that, through its interaction with Gai, Loco may function as a guanine nucleotide inhibitor and as a GAP. Disruption of loco gene function leads to multiple defects during development in Drosophila (Granderath et al 1999;Pathirana et al 2001;Yu et al 2005). In loco loss-of-function mutant embryos, failure of glial differentiation leads to improper ensheathing of neuronal axons and, consequently, the blood-brain barrier is disrupted (Granderath et al 1999;Schwabe et al 2005).…”

“…In mammalian systems, RGS domain proteins function as GTPaseactivating proteins (GAPs) and modulate G-protein signaling by promoting GTP hydrolysis (Han et al 2006). In Drosophila, however, Yu et al (2005) have shown that, through its interaction with Gai, Loco may function as a guanine nucleotide inhibitor and as a GAP. Disruption of loco gene function leads to multiple defects during development in Drosophila (Granderath et al 1999;Pathirana et al 2001;Yu et al 2005).…”

Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gβγ

“…The latter group of proteins includes activator of G protein signaling (AGS) 1-4, which were identified in a yeast-based functional screen for receptor-independent activators of G protein signaling as an extension of earlier biochemical characterization of such bioactivity in cell and tissue extracts (1-3, 11-13). The interaction of AGS1, AGS3, and AGS3-related proteins with heterotrimeric G proteins has subsequently been shown to regulate a diverse set of functions including asymmetric cell division, addictive behavior, and the circadian rhythm (1,(3)(4)(5)(6)(7)(14)(15)(16)(17)(18).…”