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Cytoskeletal architecture

Functions of polymeric cytoskeletal assemblies depend not only on their positional, but also directional organization. For example, axonal microtubules are always oriented with their (+)-ends outward toward the axon terminus. To achieve cell division, coordination of the polarity of actin bundles in the contractile ring is required. The structural basis of how these assemblies function are difficult to reveal, because the methods of imaging the directional organization of cytoskeletal filaments in live cells are inadequate. Using fluorescence polarization and computational algorithms, I have studied ordered organization of cytoskeletal assemblies in live cells in collaboration with resident and visiting scientists at Marine Biological Laboratory.

Actin

We analyzed flow and orientation of F-actin at the leading edge of a migrating skin cell using instantaneous fluorescence PolScope. Before our work, the molecular orientation of actin filaments in such complex networks could be visualized only with electron microscopy in fixed cells. We have reported (Reference 1) the first analysis of molecular orientation of F-actin network relative to the local flow.


Septin

Septins are intermediate filaments that play critical role in cell division, cell migration, and cell shape determination. Unlike microtubules and F-actin, they form non-polar assemblies in live cells and exhibit unique modes of polymerization that lead to assemblies of diverse shapes and functions. The rules by which the nanoscale protofilaments form higher-order assemblies is an active area of research. I have addressed the challenge of analyzing polymerization kinetics of septin intermediate filaments at the single molecule scale (References 1 and 2) in collaboration with Molly McQuilken, Andrew Bridges, Rudolf Oldenbourg, Tomomi Tani, and Amy Gladfelter. Tracking the position and orientation of septin subunits in the filamentous fungus model, Ashbya, led to a finding that septin-GFP subunits undergo constrained diffusion - both in position and orientation - in live cells. We have found septin assemblies to be more labile than previously reported. 


Integrin

Integrins are transmembrane receptors that transmit forces produced by actin retrograde flow to more static extracellular matrix, and provide a crucial mechanism for generating traction. They are activated in response to diverse biochemical/mechanical cues and mediate cell migration during immune response, wound healing, and metastasis. The 'molecular clutch' hypothesis posits that integrins are activated by their engagement with actin retrograde flow and extracellular ligands. An important prediction of this hypothesis is that integrin molecules will be aligned relative to the retrograde flow of actin when activated. To test this prediction, we employed fluorescence polarization microscopes (References 3 and 4), including instantaneous fluorescence polscope. We measured the degree of alignment among integrin-GFP chimera using fluorescence polarization as a function of perturbations to the retrograde flow and the extra cellular ligands.  Our data show that ligand-bound integrins are aligned by actin retrograde flow. Analysis of such 'active ordering' of a protein complex by cytoskeletal force is an apt illustration of the ability of fluorescence polarization to reveal molecular order in live cells. This research is a result of multi-institutional collaboration among MBL resident scientists, Clare Waterman's lab at NHLBI, Jity Mayor's  lab at NCBS, and Timothy Springer's lab at Harvard Medical School.



References

*,#,^ Equal contribution
  1. S. B. Mehta, M. McQuilken, P. La Riviere, P. Occhipinti, A. Verma, R. Oldenbourg, A. S. Gladfelter, and T. Tani, "Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells," Proceedings of the National Academy of Sciences PNAS Plus, E6352–E6361 (2016). Preprint: http://biorxiv.org/content/early/2016/08/12/068767.
  2. A. A. Bridges, H. Zhang, S. B. Mehta, P. Occhipinti, T. Tani, and A. S. Gladfelter, "Septin assemblies form by diffusion-driven annealing on membranes," Proceedings of the National Academy of Sciences 111, 2146–2151 (2014).
  3. *V. Swaminathan, *J. K. Mathew, *S. B. Mehta, #P. Nordenfelt, #T. I. Moore, N. Koga, D. Baker, R. Oldenbourg, T. Tani, ^S. Mayor, ^T. A. Springer, and ^C. M. Waterman, "Actin retrograde flow orients and aligns ligand-engaged integrins in focal adhesions.," In review (2016). Preprint: http://biorxiv.org/content/early/2016/08/27/071852.
  4. *P. Nordenfelt, *T. I. Moore, *S. B. Mehta, #J. K. Mathew, #V. Swaminathan, N. Koga, T. J. Lambert, D. Baker, J. C. Waters, R. Oldenbourg, T. Tani, ^S. Mayor, ^C. M. Waterman, and ^T. A. Springer, "Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration," In review (2016). Preprint: http://biorxiv.org/content/early/2016/08/27/071936.




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