The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Gαi; in addition, Gβ13F is also required. However, the role of Gαi and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Gαi mutants and show that Gαi and Gβ13F play distinct roles. Gαi is required for Pins to localize to the cortex, and the effects of loss of Gαi or pins are highly similar, supporting the idea that Pins/Gαi act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gβ13F appears to regulate the asymmetric localization/stability of all apical components, and Gβ13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.
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 G13F 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...
Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cryb. The period mutant perL manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of perL flies, because a perL; cryb strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.
Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.
Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila,asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6,DaPKC and Gαi. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. We have isolated a mouse homologue of pins (Pins) and characterized its expression profile. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc,and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. We further show that Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. Our results suggest a conservation of function between the fly and mammalian Pins homologues.
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