Most cells of the body are subjected to physiological events during normal functions that can lead to disruption of the cell’s plasma membrane. The capacity of single cells to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Upon disruption of the plasma membrane, an influx of calcium signals the deployment of vesicles that fuse with each other and with the plasma membrane to plug the hole. After the membrane has been sealed, the wound closes followed by remodeling of the cell’s cortex (plasma membrane and cortical cytoskeleton) to reestablish normal cyto-architecture and function.

As the first thirteen nuclear divisions (NC1-NC13) in the Drosophila embryo are not accompanied by cytokinesis, the early fly embryo can be considered as a giant single cell. The early embryo’s multinucleate nature is not unlike that of muscle cells; one of the major mammalian cell types undergoing continuous membrane tearing and employing single cell repair mechanisms. We employ 4D in vivo microscopy and fluorescently-tagged reporters, along with pharmacological and genetic manipulations, to define the series of changes that occur in response to wounding using this early stage (NC4-6) Drosophila embryo as a model.

Cell Wound Repair. Analysis of single cell wound repair in NC4 staged embryos allowed us to divide single cell wound repair into four major steps: i) Expansion, ii) Contraction, iii) Closure, and iv) Remodeling. During the first 30-60s post-wounding, the plasma membrane and cortical actin disappears and the initial wound area rapidly expands. Actin then accumulates in a dense ring at the wound periphery surrounded by a less dense region (actin halo). The wound diameter starts to reduce progressively with the initiation of the Contraction phase, followed by a slower Closure phase that closes the breach. Following wound closure, cells must remodel their cortex to restore its pre-wounded state in terms of composition, structural organization, attachments, and functions.

We are taking both global (genetic screens) and specific approaches to identify the molecules, machineries, and pathways involved.

Global approaches: We have performed a visual screen to identify genes that are recruited to or depleted from wounds, and a microarray screen to examine the role of transcription in cell wound repair and to potentially identify start signals for the process. The visual screen has identified genes with specific spatial and temporal recruitment patterns upon wounding that are allowing us to define the earliest acting genes, as well as those required at all other steps in the repair process. From our microarray screen, we have found that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. Interestingly, also we found that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair.

Specific molecules or steps: To investigate specific steps in the repair process or to look at the roles of specific molecules, we are analyzing the effects of perturbations on and the dynamic expression of fluorescent reporters for specific molecules – including actin, myosin, Rho family GTPases and their regulators, microtubules, E-cadherin, and plasma membrane. For example, we find that E-cadherin accumulates at the wound edge during single cell repair and wound expansion is excessive in E-cadherin mutants, indicating a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. These studies are allowing us to define specific molecular mechanisms required for different steps of the cell wound repair process, as well as to establish the molecular pathway underpinning the cell wound repair process, which is currently not available for any cell wound repair model.

Nakamura M, Verboon JM and Parkhurst SM (2017). Pre-patterning by RhoGEFs governs Rho GTPase spatiotemporal dynamics during wound repair.  J. Cell Biol. 216: 3959-3969.

Nakamura M, Dominguez AN, Decker JR, Hull AJ, Verboon JM and Parkhurst SM (2018). Into the breach: How cells cope with wounds. (invited review) Open Biology 8: 180135.                  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223217/

Nakamura M, Verboon JM, Allen, TE, Abreu-Blanco MT, Liu R, Dominguez AN, Delrow JJ and Parkhurst SM (2020). Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair. PLoS Genetics 16(12): e1009186. https://doi.org/10.1371/journal.pgen.1009186 [preprint posted on BioRxiv]

Stjepić V*, Nakamura M*, Hui J* and Parkhurst SM. (2024). Two Septin Complexes Mediate Actin Dynamics during Cell Wound Repair. Cell Reports 43: 114215.  https://doi.org/10.1016/j.celrep.2024.114215. [preprint posted on BioRxiv]