Swedha Sidharthan


Regulation of Rho1 by RhoGAPs during Drosophila Germ Band Extension

Morphogenesis is an intricate process where epithelial cells reorganize themselves to form differentiated, three-dimensional tissues and organs. These cell shape changes are driven by the contractile actomyosin network. One such morphogenetic process is the Drosophila ectodermal Germ Band Extension (GBE), where cell intercalation results in cell-neighbour exchange and subsequent extension of the dorsal-lateral ectoderm in the anterior-posterior axis. This intercalation is brought about by the Myosin-II (MyoII) molecular motors within the ectoderm. Drosophila ectoderm displays two distinct pools of MyoII, a pulsatile medio-apical pool and a planar polarized junctional pool. The MyoII is activated by the conserved Rho1-Rok pathway. Rho1 is a GTPase and functions like a molecular switch that shifts between its active GTP-bound form to its inactive GDP bound form and this transition is regulated by the GEFs (Guanine nucleotide Exchange Factors) and GAPs (GTPase Activating Proteins) respectively. Two distinct modules of Rho1 regulators activate the two pools of MyoII in the ectoderm. A junction specific RhoGEF- Dp114RhoGEF activates the junctional Rho1 and a medial specific RhoGEF- RhoGEF2 activates the medial Rho1. Surprisingly, the medial specific RhoGEF2 is present medially and planar polarized at the junctions while the junction specific Dp114RhoGEF is present uniformly at all junctions lacking any planar polarity. Strikingly, the distribution of these GEFs do not correspond to their role in regulating MyoII activity. Therefore, RhoGEFs did not fully explain the observed planar polarized Rho1 signaling. In this context, we wanted to study the RhoGAPs that negatively regulate Rho1 in the ectoderm during GBE. We hypothesized that a differential distribution of RhoGAPs could contribute to the planar polarity of Rho1 observed.
Through this project, I performed a functional screen of 6 RhoGAPs expressed in the early embryo. I identified two RhoGAPs that regulate Rho1 and hence MyoII activity during GBE. I found GRAF regulates only the junctional pool of MyoII and is localized at the junctions. I identified that GRAF regulates MyoII in the ectoderm in a dose-dependent manner. Like several other RhoGAPs, GRAF is a multidomain protein with BAR, PH, RhoGAP and SH3 domains. The BAR and PH domains are known to play roles in membrane localization. Additionally, the BAR domains are also known to play autoregulatory roles where they bind to and inhibit the catalytic GAP domain.
I identified that the membrane localization of GRAF is independent of the BAR and PH domains and that the BAR domain does not play any essential role in regulating the activity of the GAP domain. I also identified that GRAF is localized in the ectoderm and mesoderm but is not functional in the mesoderm. Thus uncovering a tissue specific role for GRAF exclusively in the ectoderm.
I further discovered CdGAPr that specifically regulates Rho1 medially. I also identified that CdGAPr is localized medially and planar polarized at the junctions.  Through a yeast two-hybrid screen, we discovered CdGAPr binds to MyoII, specifically the heavy chain of MyoII called Zipper. Thus CdGAPr colocalizes with MyoII in the ectoderm both at the junctions in a planar polarized manner and medially as pulses.
Collectively, I have identified that there exist 2 distinct pools of Rho1 signaling, a junctional and medial and both these modules of Rho1 are separately regulated by their own pair of GAPs. Another interesting finding in this study is the similarity between the localization of the junction specific regulators and the medial specific regulators. This proposes a mechanism where Rho1 is present in a dynamic range of activity rather than the conventional ON/OFF.