@article{appalabhotla_butler_bear_haugh_2023, title={G-actin diffusion is insufficient to achieve F-actin assembly in fast-treadmilling protrusions}, volume={122}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2023.08.022}, abstractNote={<h2>Abstract</h2> To generate forces that drive migration of a eukaryotic cell, arrays of actin filaments (F-actin) are assembled at the cell's leading membrane edge. To maintain cell propulsion and respond to dynamic external cues, actin filaments must be disassembled to regenerate the actin monomers (G-actin), and transport of G-actin from sites of disassembly back to the leading edge completes the treadmilling cycle and limits the flux of F-actin assembly. Whether or not molecular diffusion is sufficient for G-actin transport has been a long-standing topic of debate, in part because the dynamic nature of cell motility and migration hinders the estimation of transport parameters. In this work, we applied an experimental system in which cells adopt an approximately constant and symmetrical shape; they cannot migrate but exhibit fast, steady treadmilling in the thin region protruding from the cell. Using fluorescence recovery after photobleaching, we quantified the relative concentrations and corresponding fluxes of F- and G-actin in this system. In conjunction with mathematical modeling, constrained by measured features of each region of interest, this approach revealed that diffusion alone cannot account for the transport of G-actin to the leading edge. Although G-actin diffusion and vectorial transport might vary with position in the protruding region, good agreement with the fluorescence recovery after photobleaching measurements was achieved by a model with constant G-actin diffusivity ∼2 <i>μ</i>m<sup>2</sup>/s and anterograde G-actin velocity less than 1 <i>μ</i>m/s.}, number={18}, journal={BIOPHYSICAL JOURNAL}, author={Appalabhotla, Ravikanth and Butler, Mitchell T. and Bear, James E. and Haugh, Jason M.}, year={2023}, month={Sep}, pages={3816–3829} }
 @article{baldwin_van bruggen_koelbl_appalabhotla_bear_haugh_2021, title={Microfluidic devices fitted with "flowver" paper pumps generate steady, tunable gradients for extended observation of chemotactic cell migration}, volume={15}, ISSN={["1932-1058"]}, DOI={10.1063/5.0054764}, abstractNote={Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.}, number={4}, journal={BIOMICROFLUIDICS}, author={Baldwin, Scott A. and Van Bruggen, Shawn M. and Koelbl, Joseph M. and Appalabhotla, Ravikanth and Bear, James E. and Haugh, Jason M.}, year={2021}, month={Jul} }