Single-cell wound repair in Drosophila involves mechanistically distinct expansion, contraction, and closure phases.

Summary Background Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. Here we describe how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. Results We find that Rho, Rac and Cdc42 rapidly accumulate around the wound and segregate into dynamic, partially overlapping, zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation leading to its association with actin. Rho1, along with Cdc42, are necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization towards the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. We find Rho1 GTPase uses several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. Conclusions Our results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single cell wound repair in the Drosophila embryo. Wounding triggers the formation of Rho GTPases arrays that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, are required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell’s needs.

SummaryThe repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular, which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process, expansion, coalescence, contraction and closure, and describe the molecular dynamics of each phase. Specifically, we find that myosin, E-cadherin, Echinoid, the plasma membrane, microtubules and the Cdc42 small GTPase respond dynamically during wound repair. We demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular protrusions and actomyosin ring contraction along the leading edge of the wound.

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE,the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. We recently identified a third WAS family member, called Wash, with Arp2/3-mediated actin nucleation activity. We show that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures.

Wound repair on the cellular and multicellular levels is essential to the survival of complex organisms. In order to avoid further damage, prevent infection, and restore normal function, cells and tissues must rapidly seal and remodel the wounded area. The cytoskeleton is an important component of wound repair, needed for actomyosin contraction, recruitment of repair machineries, and cell migration. Recent use of model systems and high-resolution microscopy has provided new insight into molecular aspects of the cytoskeletal response during wound repair. Here we discuss the role of the cytoskeleton in single cell, embryonic, and adult repair, as well as the striking resemblance of these processes to normal developmental events and many diseases.

Summary Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as Rigosertib and Paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.

Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

The Ras-ERK (extracellular signal–regulated kinase) pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors for treating cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends upon multifunctional-autoprocessing repeats-in-toxin (MARTX), a toxin that releases bacterial effector domains into host cells. One of the effector domains delivered by this toxin is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance (NMR) analysis and nucleotide exchange assays, we determined that RRSP processing did not release any fragments of KRAS or cause KRAS to dissociate from its bound nucleotide, but instead only locally impacted the structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor (GEF)-mediated nucleotide exchange and RAF binding. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance to its target.