Biology subjects

Galbraith, J. A.

Publications and source records attributed to Galbraith, J. A..

3 recordsLinked to original sources

bioRxiv2024

Compartmentalized Cytoplasmic Flows Direct Protein Transport to the Cell's Leading Edge

Inside the cell, proteins essential for signaling, morphogenesis, and migration navigate complex pathways, typically via vesicular trafficking or microtubule-driven mechanisms 1-3. However, the process by which soluble cytoskeletal monomers maneuver through the cytoplasms ever-changing environment to reach their destinations without using these pathways remains unknown. 4-6 Here, we show that actin cytoskeletal treadmilling leads to the formation of a semi-permeable actin-myosin barrier, creating a specialized compartment separated from the rest of the cell body that directs proteins toward the cell edge by advection, diffusion facilitated by fluid flow. Contraction at this barrier generates a molecularly non-specific fluid flow that transports actin, actin-binding proteins, adhesion proteins, and even inert proteins forward. The local curvature of the barrier specifically targets these proteins toward protruding edges of the leading edge, sites of new filament growth, effectively coordinating protein distribution with cellular dynamics. Outside this compartment, diffusion remains the primary mode of protein transport, contrasting sharply with the directed advection within. This discovery reveals a novel protein transport mechanism that redefines the front of the cell as a pseudo-organelle, actively orchestrating protein mobilization for cellular front activities such as protrusion and adhesion. By elucidating a new model of protein dynamics at the cellular front, this work contributes a critical piece to the puzzle of how cells adapt their internal structures for targeted and rapid response to extracellular cues. The findings challenge the current understanding of intracellular transport, suggesting that cells possess highly specialized and previously unrecognized organizational strategies for managing protein distribution efficiently, providing a new framework for understanding the cellular architectures role in rapid response and adaptation to environmental changes.

cell biology↗

bioRxiv2024

Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of "glitter bomb" labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

cell biology↗

bioRxiv2017

A Cautionary Tail: Changes in Integrin Behavior with Labeling

Genetic expression of fluorescently labeled proteins is essential to visualizing dynamic behavior within live cells. Recent advances in microscopy have increased resolution to the level where it is now possible to capture individual molecules interacting. However, the criteria for determining whether a fluorescent label perturbs protein function have not undergone a corresponding increase in resolution. The effects of protein labeling on cell function are still judged by whether populations of protein localize and interact with known binding partners. Here we use integrins, bidirectional signal adhesion molecules that regulate interactions between the extracellular matrix and the cytoskeleton through a well-defined series of conformational changes to show that not all labeling strategies are the same. We found that labeling the beta subunit decreased the mobility of individual integrin molecules and the protrusive activity of the entire cell. While integrins with labeled alpha subunits behaved similarly to unlabeled integrins, labeling the beta subunit increased the size of adhesions by elevating integrin affinity and exposing the ligand induced binding domain to change the molecule conformation. Thus, our single molecule and cellular data indicate that the ability of labeled proteins to localize and interact with known binding partners does not guarantee it does not alter protein function. We propose that the behaviors of individual molecules rather than the ensemble behavior of populations need to be considered as criteria to determine if a probe is non-perturbative.

cell biology↗