TheDrosophila melanogasterRab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function

Abstract

To identify novel regulators of non-muscle myosin II (NMII) we performed an image-based RNAi screen using stable Drosophila melanogaster S2 cells expressing EGFP-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1, and a decrease in phosphomyosin-positive cells by immunostaining while expression of constitutively active Rho or Rho-kinase (Rok) rescues the puncate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as over-expression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility. [Media: see text] [Media: see text]

From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway

“…To this end, cells were treated with either control, Oya, or Sqh RNAi for seven days and then challenged to contract by perfusion of Fog-enriched media. We then immunostained the cells with an antibody raised against a synthetic phosphopeptide mimicking phosphorylated NMII [ 39 ] as well as fluorescently-labeled phalloidin to visualize actin filaments (Fig. 3 A-C).…”

“…Following Fog treatment in S2R + cells the NMII network reorganizes assembling into ordered peri-nuclear structures such as rings which are highly reminiscent of medioapical polarization observed in epithelial cells in vivo [ 40 ]. In order to quantify NMII distribution we used a previously established coalescence index [ 39 , 41 ]. The higher coalescence index value the more organized (or less diffuse) the actomyosin network.…”

“…The observed hypocontractility, when viewed in conjunction with the increased abundance of phosphorylated myosin, suggests that Flw depleted cells might display defects in spatial or temporal regulation NMII. In order to explore this latter possibility we decided to quantify the spatial distribution of the phosphorylated regulatory light chain using our coalescence index [ 39 , 41 ]. Again, the greater the coalescence index the more punctate the distribution which in turn indicates a more organized phosphomyosin network.…”

From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway

“…The cell contractility assay was performed as described in (Peters et al, 2018;and Platenkamp et al, 2020). The number of constricted and relaxed cells were counted per image frame, excluding ambiguous cell states.…”

“…The coalescence index was used to measure the distribution of phosphorylated NMII Sqh at Ser21 in each treatment (Platenkamp et al, 2020). The coalescence index was adapted from a punctate/diffuse index (Bouchier-Hayes et al, 2008) used to compare the progression of cytochrome c dispersal across a cell during apoptosis.…”