Polarity mediates asymmetric trafficking of the G beta heterotrimeric G-protein subunit GPB-1 in C. elegans embryos

Asymmetric 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 alpha 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 G beta 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 G beta trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.

Asymmetric Spindle Positioning and Intracellular Trafficking in C. Elegans Embryos

Kalyani Thyagarajan

Asymmetric cell division is critical for generating daughter cells that possess distinct fates during development and in stem cell lineages. Correct orientation of the mitotic spindle with respect to the polarity axis is crucial to this process. In the one-cell stage embryo of the nematode C. elegans, polarity cues define an anterior-posterior axis, and the mitotic spindle is positioned asymmetrically towards the posterior of the embryo in response to these cues, resulting in daughter cells that differ not only in fates, but also in sizes. Although the way by which force is generated has becoming increasingly well understood, the mechanisms by which polarity cues regulate the asymmetric distribution of active cortical force generators that pull on astral microtubules to position the spindle remained unclear at the onset of my thesis. During the course of our work, we found that intracellular trafficking plays a crucial role in the distribution and levels of proteins involved in asymmetric spindle positioning. Firstly, we found that the distribution of the Gβ protein GPB-1, a negative regulator of pulling forces, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. GPB-1 transits through the endosomal network in a dynamin-, clathrin- and RAB-5-dependent manner. We also found 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, and thus symmetric pulling forces during mitosis. Secondly, we found that the clathrin heavy chain modulates spindle-positioning forces as well as the cortical distribution of positive (GPR-1/2 and DHC-1) and negative (GPB-1 and LET-99) regulators of pulling forces. Although the mechanisms by which it does so remain unclear, our data suggests that clathrin plays multiple roles in the one-cell stage C. elegans embryos, including in recycling GPB-1 to the plasma membrane. Overall, our results lead us to propose that the clathrin heavy chain and the modulation of Gβγ trafficking play a critical role during asymmetric division of one-cell stage C. elegans embryos.

EPFL2011

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Pierre Gönczy

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.

2005

Probing the distribution of cortical components, including PtdIns(4,5)P2, during asymmetric division of C. elegans embryos

Melina Jasmin Scholze

Asymmetric cell division is crucial for embryonic development and stem cell lineages. In the one-cell C. elegans embryo, a contractile cortical actomyosin network contributes to asymmetric division by segregating PAR proteins to discrete cortical domains. The anterior-posterior (AP) polarity established in this manner is maintained thereafter and translated into mechanisms that lead to asymmetric spindle positioning during mitosis, thus ensuring proper segregation of cell fate determinants and fate specification of the daughter cells. Here, performing cortical live imaging using confocal spinning disk microscopy, we discovered that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; PIP2) forms polarized dynamic structures at the cell membrane of C. elegans zygotes, which distribute in a PAR-dependent manner along the AP embryonic axis. PIP2 cortical structures overlap with filamentous actin (F-actin), and coincide with the actin regulators RHO-1, CDC-42 as well as ECT-2. Particle image velocimetry analysis revealed that PIP2 and F-actin cortical movements are coupled, with PIP2 structures moving slightly ahead of polymerizing F-actin bundles. Importantly, we established that the formation of PIP2 cortical structure depends on F-actin and their movement on actin polymerization. Conversely, we found that decreasing or increasing the level of PIP2 results in severe F-actin disorganization, revealing interdependence between these components. Moreover, increasing or decreasing the levels of PIP2 revealed that the formation of CDC-42 and RHO-1 cortical structures depends on PIP2, while PIP2 structures formation is mostly independent of RHO-1 and CDC-42. Further-more, we uncovered that PIP2 and F-actin regulate the sizing of PAR cortical domains during the establishment and maintenance phases of polarization. Overall, our work establishes that a lipid membrane component, PIP2, modulates actin organization and cell polarity in C. elegans embryos. Furthermore, we found that the force generation mediators GPR-1 and LIN-5, as well as the negative force regulators CSNK-1 and GPB-1, distribute in cortical structures. While CSNK-1 and GPB-1 form elongated cortical structures during pseudo-cleavage overlapping with PIP2 cortical structures, GPR-1 and LIN-5 form mostly cortical foci not overlapping with GPB-1 and PIP2 cortical structures. The formation of GPB-1 cortical structures is independent of GPR-1 and vice versa. However, the formation and localization of GPB-1 cortical structures depends on PIP2. Overexpressing GPR-1 increases the formation of GPR-1 cortical structures and PIP2/GPB-1 cortical structures, which overlap in large part. CSNK-1 regulates PPK-1 localization to the embryo posterior and is required for the formation of PIP2 cortical structures on the anterior side. Overall, this work reveals that many important players of asymmetric cell division in the one-cell stage C. elegans embryo distribute unevenly on the cortex in dynamic and polarized elongated structures and foci. Moreover, for the first time, the contribution of a plasma membrane lipid, PIP2, to proper AP polarity establishment and maintenance is demonstrated.