Polarity mediates asymmetric trafficking of the Gβ heterotrimeric G-protein subunit GPB-1 inC. elegansembryos

Abstract

SUMMARYAsymmetric cell division is an evolutionarily conserved process that gives rise to daughter cells with different fates. In one-cell stage C. elegans embryos, this process is accompanied by asymmetric spindle positioning, which is regulated by anterior-posterior (A-P) polarity cues and driven by force generators located at the cell membrane. These force generators comprise two G proteins, the coiled-coil protein LIN-5 and the GoLoco protein GPR-1/2. The distribution of GPR-1/2 at the cell membrane is asymmetric during mitosis, with more protein present on the posterior side, an asymmetry that is thought to be crucial for asymmetric spindle positioning. The mechanisms by which the distribution of components such as GPR-1/2 is regulated in time and space are incompletely understood. Here, we report that the distribution of the Gb subunit GPB-1, a negative regulator of force generators, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. Furthermore, we uncover that GPB-1 trafficks through the endosomal network in a dynamin-and RAB-5-dependent manner, which is most apparent during mitosis. We find that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis. Overall, our results lead us to propose that modulation of Gb trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.

Mechanisms of spindle positioning: cortical force generators in the limelight

“…In C. elegans, GaÁGDP levels are also modulated by Gbg, since depletion of Gbg results in excess pulling forces, presumably because excess GaÁGDP is available for interaction with GPR-1/2 [44,46]. Intriguingly, intracellular trafficking of the Gb protein GPB-1 is modulated in time and space in C. elegans embryos in a manner consistent with it dictating cortical GPR-1/2 localization [47]. This raises the possibility that intracellular trafficking is crucial for proper spindle positioning, as is the case for other aspects of asymmetric cell division, for instance in Drosophila sensory organ precursor cells [48,49].…”

Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos

“…First, endocytosis in general affects the actin cytoskeleton and reciprocally, making it challenging to analyze the two processes separately (reviewed by Galletta and Cooper, 2009). Second, inhibiting endocytosis in one-cell C. elegans embryos using a dynamin mutant results in lower levels of cortical GPB-1, thus probably influencing force generation via increased cortical dynein (Thyagarajan et al, 2011). This could explain the clathrin-like centration/rotation phenotype observed upon depletion of the early endosomal protein RAB-5 (Hyenne et al, 2012).…”

“…Primary mouse antibodies against α-tubulin (1/300; DM1A, Sigma), GFP (1/100; MAB3580, Millipore) were used together with rabbit antibodies against GPB-1 (1/200; Thyagarajan et al, 2011), LIN-5 (1/300;Nguyen-Ngoc et al, 2007), DHC-1 (1/100; Kotak et al, 2012), PGL-1 (1/300; Kawasaki et al, 1998) or TAC-1 (1/500; Bellanger and Gönczy, 2003). Secondary antibodies were Alexa488-conjugated goat anti-mouse (1:500; Life Technologies, A11001) and Cy3-conjugated goat anti-rabbit (1/1000; Jackson ImmunoResearch, 711-165-152).…”

“…More general cellular processes are also harnessed to modulate forces pulling on centrosomes and spindle poles. Thus, endosomal trafficking of GPB-1 occurs in a cell-cycle-regulated manner, and it has been proposed that trafficking-dependent downregulation of cortical GPB-1 during mitosis is important to allow association of GPR-1/2 with the Gα proteins (Thyagarajan et al, 2011). Moreover, interfering with the actin cytoskeleton during mitosis results in increased pulling forces on the anterior side (Afshar et al, 2010;Berends et al, 2013), although whether this occurs via an impact on ternary complex components, and whether a similar relationship pertains to centration/rotation are unanswered questions.…”

Polarity Control of Spindle Positioning in the C. elegans Embryo

“…Thus, the asymmetry in Gβγ could contribute to the asymmetry in pulling forces and posterior displacement of the spindle in mitosis. A recent study showed dynamic regulation of cortical GPB-1 levels and trafficking through both early endosomes and recycling endosomes (Thyagarajan et al 2011). In metaphase of the zygote, trafficking rates are higher in the anterior than the posterior, and more GPB-1 remains present in endosomal vesicles in the posterior.…”