Biology subjects

Parkhurst, S. M.

Publications and source records attributed to Parkhurst, S. M..

9 recordsLinked to original sources

bioRxiv2026

Spatiotemporal Dynamics of Protein Recruitment During Cell Wound Repair

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

cell biology↗

bioRxiv2026

Reversible Actin modifications by Mical and SelR regulate dynamic actomyosin ring functions during cell wound repair

Cell wound repair requires rapid and coordinated remodeling of the actin cytoskeleton to restore cortex integrity. Here, we show that a Rab35-Mical-SelR pathway regulates actomyosin ring dynamics through reversible actin redox. We find that Rab35 is recruited to wounds and is essential for proper actin ring assembly and disassembly. Rab35 regulates the recruitment of Mical, an actin-oxidizing enzyme, and SelR, a reductase that reverses oxidation, to the cell wound. Mical and SelR knockdowns disrupt actin ring formation and wound closure, whereas double knockdown partially rescues these defects, indicating a balanced redox cycle is required. Super-resolution microscopy reveals that Mical and SelR differentially regulate F-actin architecture and orientation. Mutation of actin at Methionine 44 does not fully recapitulate Mical knockdown phenotypes, suggesting the presence of additional targets and enzymes. Taken together, our results indicate that reversible actin modifications dynamically regulate F-actin architecture and orientation for actin ring assembly and disassembly.

cell biology↗

bioRxiv2023

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds

To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, we find that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, our results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair. SummaryCells have rapid and robust repair systems to survive daily damage. This study shows that calcium influx regulates the three distinct Drosophila Annexin recruitment patterns to the cell wound in order to organize an actomyosin ring for efficient wound closure.

cell biology↗

bioRxiv2023

Two Septin Complexes Mediate Actin Dynamics During Cell Wound Repair

Cells have robust wound repair systems to prevent further damage or infection and to quickly restore cell cortex integrity when exposed to mechanical and chemical stress. Actomyosin ring formation and contraction at the wound edge are major events during closure of the plasma membrane and underlying cytoskeleton during cell wound repair. Here, we show that all five Drosophila Septins are required for efficient cell wound repair. Based on their different recruitment patterns and knockdown/mutant phenotypes, two distinct Septin complexes, Sep1-Sep2-Pnut and Sep4-Sep5-Pnut, are assembled to regulate actin ring assembly, contraction, and remodeling during the repair process. Intriguingly, we find that these two Septin complexes have different F-actin bending activities. In addition, we find that Anillin regulates the recruitment of only one of two Septin complexes upon wounding. Our results demonstrate that two functionally distinct Septin complexes work side-by-side to discretely regulate actomyosin ring dynamics during cell wound repair.

cell biology↗

bioRxiv2022

The Centralspindlin proteins Pavarotti and Tumbleweed work with WASH to regulate Nuclear Envelope budding

Nuclear envelope (NE) budding is a nuclear pore independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott-Aldrich Syndrome protein Wash, its regulatory complex (SHRC), and Arp2/3, and requires Washs actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin bundling activities of Wash and Pav are required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development.

cell biology↗

bioRxiv2022

Cell wound repair requires the coordinated action of linear and branched actin nucleation factors

Cells are subjected to a barrage of daily insults that often lead to its cortex being ripped open and requiring immediate repair. An important component of the cell s repair response is the formation of an actomyosin ring at the wound periphery to mediate its closure. Inhibition of linear actin nucleation factors and myosin result in a disrupted contractile apparatus and delayed wound closure. Here we show that branched actin nucleators function as a scaffold to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and slow wound closure. Removing linear and branched actin results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear actin filaments that undergo a chiral swirling movement to close the wound. These results provide insight into actin organization in contractile actomyosin rings and uncover a new mechanism of wound closure. SummaryHui et al. find that branched actin is required during cell wound repair to serve as a scaffold to anchor the contractile actomyosin cable at the wound periphery. Inhibition of branched actin and myosin results in parallel linear filaments that swirl to close the wound, uncovering a new mechanism for cell wound repair.

cell biology↗

bioRxiv2020

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find 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. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

developmental biology↗

bioRxiv2019

Wash and the WASH Regulatory Complex function in Nuclear Envelope budding

Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes can be packaged inside the nucleus and be extruded through the nuclear membranes, completely bypassing nuclear pores. While factors have been identified both as cargos or actively involved in this process, much remains unknown about the molecules that generate the forces and membrane deformations which appear inherent. Using fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model, we identify Wash, its regulatory complex, and Arp2/3 as novel players in NE-budding. Surprisingly, Washs role in this process is bipotent and, independent of SHRC/Arp2/3, its perturbation disrupts the normal homotypic Lamin A/B meshworks that are necessary for NE-budding to occur. In addition to NE-budding emerging as important in additional cellular processes and organisms, its incredible similarity to herpesvirus egress suggests new avenues for exploration in both normal and disease biology.

cell biology↗

bioRxiv2019

The kinesin-like protein Pavarotti functions non-canonically to regulate actin dynamics

Pavarotti, the Drosophila MKLP1 ortholog, is a kinesin-like protein that with Tumbleweed (MgcRacGAP) works together as the centralspindlin complex. This complex is essential for cytokinesis where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. We previously showed that Tumbleweed and Pebble are required for the cell wound repair process. Here, we show that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are utilized during this cellular repair, it is not as the conserved centralspindlin complex. Surprisingly, in vitro and in vivo work show that the classically microtubule-associated Pavarotti binds directly to actin and has a non-canonical role directly regulating actin dynamics. We show that Pavarotti also works independently from Tumbleweed in several actin-related processes during the normal developmental process of oogenesis.

cell biology↗