Drosophila immune cells undergo four stereotypical developmental migrations to populate the embryo. Wash is a downstream effector of Rho1 and establishes Rho1>Wash>Arp2/3 as the regulatory pathway controlling the cytoskeleton during one of these developmental hemocyte migrations in a WASH regulatory complex–independent manner.
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.
Although high strain and strain-rate impacts to the human body have been the subject of substantial research at both the systemic and tissue levels, little is known about the celllevel ramifications of such assaults. This is largely due to the lack of high throughput, dynamic compression devices capable of simulating such traumatic loading conditions on individual cells. To fill this gap, we developed and characterized a high speed, high actuation force, magnetically driven MEMS chip to apply stress to biological cells at unprecedented strain (10% to 90%), strain rate (30,000 to 200,000 s −1), and throughput (12,000 cells/min). To demonstrate the capabilities of the µHammer, we applied biologically relevant strains and strain rates to human leukemic K562 cells and then monitored their viability for up to 8 days. We observed significantly repressed proliferation of the hit cells compared to both unperturbed and sham-hit control cells, accompanied by minimal cell death. This indicates success in applying cellular damage without compromising the overall viability of the population, allowing us to conclude that this device is well suited to study the subtle effects of impact on large populations of inherently heterogeneous cells.
We report the application of stress to biological cells at unprecedented strain (50%), strain rate (180,000 s -1 ), and throughput (1,800 cells/min) using a high-speed, high actuation force magnetically-driven MEMS chip. This device is uniquely suited to study the effects of impact on large populations of inherently heterogeneous cells, enabling statistical analysis that can elucidate the cell-level ramifications of Traumatic Brain Injury (TBI). To demonstrate the capabilities of the µHammer, we applied TBIrelevant strains and strain rates to human leukemic K562 cells then monitored their proliferation for 9 days. We observed significantly repressed proliferation of the hit cells compared to both the negative and sham controls, indicating success in applying sublethal cellular damage.
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