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Tension on actin filaments alters the affinity of calponin homology domains
Andrew R. Harris, Postdoctoral Researcher
Fletcher Lab for Visualization & Control of Biological Assembly,
Department of Bioengineering, University of California Berkeley
The actin cytoskeleton is organized into distinct structures that transmit forces during cell movements, shape change, and internal rearrangements.
Differences in filament nucleotide state, oxidative state, and nucleator have been shown to influence the binding of accessory proteins in a shared cytoplasm, but a general role for force in localizing actin-binding proteins to specific actin structures is unclear.
I have shown that mechanical loading of individual actin filaments can change the affinity of proteins containing tandem calponin-homology domains (CH1-CH2), a family of actin binding proteins that include the crosslinkers filamin B, β-spectrin, and BPAG1, providing a direct mechanism for altering network composition in response to tension.
Despite high sequence homology, we find that CH1-CH2 domains from multiple actin-binding proteins show different patterns of binding in live cells compared to utrophin CH1-CH2, a commonly-used label of filamentous actin in live cells.
By making point mutations in and around the actin binding regions, I have shown that mutants of the utrophin CH1-CH2 can be made to localize preferentially to either contractile networks or to lamellipodial networks.
In vitro measurements of the mutant CH1-CH2 domain binding reveal that tension on single filaments and filament networks is sufficient to alter affinity, with both negative and positive stress dependence in different mutants.
Using ratiometric imaging of actin-binding domains with different force-dependent affinities, one can conceive a framework to screen for force-dependent binding proteins and map relative stress distributions in live cells.
This work provides insight into how mechanical loads can directly organize actin cytoskeletal structures by altering binding protein affinity and offers a tool for visualization of stresses within the actin cytoskeleton.
Andrew Harris’ research interests lie at the interface between the physical and life sciences, using tools from engineering, biology and biochemistry to identify fundamental determinants of cytoskeletal organization (as an EMBO/HFSP Postdoctoral Fellow with Prof. Daniel Fletcher at UC Berkeley) and to characterize cell and tissue mechanical properties (as an EPSRC Ph.D. student with Prof. Guillaume Charras at UCL).
His future research interests focus on understanding the molecular basis by which cells generate and respond to mechanical forces, and on using reverse-engineered molecular tools to design tissues with custom morphologies. Together, this ‘bottom-up’ approach to tissue engineering – and the basic science on which it is based – has the potential to enable fundamentally new regenerative and therapeutic strategies.