Scalable Imaging of Molecular Order

Seminar | February 26 | 12-1 p.m. | 489 Minor Hall

 Shalin Mehta, PhD, Platform Leader, Advanced Optical Microscopy, Chan Zuckerberg Biohub

 Neuroscience Institute, Helen Wills

Abstract: Nanoscale alignment of molecules, or molecular order, underpins the directed functions of cells. Cells have the fascinating capacity of creating and sustaining molecular order at the expense of chemical energy, as illustrated by the planar organization of the lipid membrane and the three-dimensional organization of chromatin, cytoskeleton, and extracellular matrix. The molecular order within cells is too fine to visualize even with super-resolution fluorescence microscopy. Although electron microscopy directly visualizes molecular order, it is limited to fixed cells. Many outstanding structure-function questions in biology require precise, high-resolution, and scalable technologies of imaging molecular order in live cells.

In this talk, I will discuss utility of fluorescence polarization imaging, combined with robust analysis algorithms, for measurement of molecular order across biological scales. When a fluorescent probe is attached to a biomolecule at a specific site, the intrinsic anisotropy of fluorophores reports rotational diffusion, alignment, and orientation of the biomolecules. We designed instantaneous fluorescence PolScope to measure concentration, alignment, and orientation of biomolecules simultaneously. The instantaneous fluorescence PolScope revealed the dynamic orientation of nanoscale actin filaments within microscale actomyosin network in migrating skin cells1, previously accessible only with electron microscopy in fixed cells. Instantaneous fluorescence PolScope enabled analysis of active alignment of integrin transmembrane receptors at the surface of live cells. Integrins in migrating white blood cells2 and fibroblasts3 align and orient when engaged with actin flow inside the cells and a rigid ligand outside the cell. These two integrins are structurally very different, yet both types are oriented in a direction dictated by the intracellular actin flow when activated.

At CZ Biohub, my group pursues measurement of molecular order in dynamic three-dimensional specimens to analyze mechanisms of virus biogenesis and life cycle. These approaches promise insights in the mechanism of infection and discovery of new therapeutic opportunities.

References:

* Equal contribution

1. Mehta, S. B. et al. Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells. Proc. Natl. Acad. Sci. 113, E6352–E6361 (2016).

2. *Nordenfelt, P. et al. Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration. Nat. Commun. 8, 2047 (2017).

3. *Swaminathan, V. et al. Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc. Natl. Acad. Sci. 114, 10648–10653 (2017).

 nrterranova@berkeley.edu