Research

Research

Overview

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Cells are organized on length scales of angstroms to microns, and on timescales of picoseconds to hours. Organization at the shortest and longest ends of these scales is increasingly well understood through work in structural biology/biophysics and cell biology, respectively.

Actin Phallodin
Droplets with actin phalloidin

However, the mechanisms by which the properties of individual molecules give rise to cellular architecture and function remain poorly understood. Research in the Rosen lab is focused on understanding the physical mechanisms of cell organization across scales, primarily through studies of the signaling pathways that control the actin cytoskeleton. Our work is directed toward both understanding the structure and dynamics of individual proteins and their signaling complexes, as well as discovering how and why these discrete entities produce cellular organization and activities at longer length and time scales.

Dynamics of actin play a central role in numerous physiologic and pathologic processes, including cell motility and adhesion, vesicle trafficking, tumor metastasis and bacterial/viral pathogenesis. During these processes, cytoskeletal dynamics are regulated on length scales ranging from angstroms to microns, and we seek to understand them in this context. On the molecular scale, we study how individual actin filaments are formed and destroyed in response to upstream signaling networks, and how molecules in these pathways fluctuate on picosecond to millisecond timescales in order to receive and transmit information, often through allosteric changes. On the cellular scale, we work to understand how these filaments and their upstream regulators are organized into higher-order assemblies to produce the micron-size structures observed in vivo such as lamellipodia, focal adhesions and budding vesicles, which dynamically rearrange on much longer timescales relevant to cell physiology. Critically, we also strive to bridge between these realms through discovering how structure and dynamics at the molecular scale produce those at the cellular scale. We use a wide array of conceptual approaches and technologies to achieve these goals.

WAVE Regulatory Complex (WRC)
WAVE Regulatory Complex (WRC)

One component of our work is directed toward understanding how actin nucleation factors, including the Arp2/3 complex, formin proteins and so-called WH2 based nucleators, generate new actin filaments de novo from actin monomers. We also study how upstream regulators of these factors, particularly proteins in the WASP family, receive and integrate diverse signals to control actin nucleation. In each case we are interested in the structure and dynamics of the relevant molecules, and how these physical properties control biochemical and cellular activities. For example, we study how nucleation factors bind and organize actin monomers, how these complexes dynamically rearrange as a nascent filament grows, and how such rearrangements affect filament elongation rates and nucleation efficiency. We also study how WASP proteins bind multiple upstream ligands, and how thermodynamic cooperativity between these ligands is achieved and yields high specificity of WASP activation in vitro and in cells. This research utilizes biochemical and biophysical tools ranging from NMR spectroscopy and x-ray crystallography to fluorescence techniques and ultracentrifugation.

A second component is directed toward understanding how signaling molecules, including actin regulatory factors, are organized into higher-order, micron sized assemblies in vitro and in cells. This work is based on our recent discovery that interactions between multivalent proteins and their multivalent ligands produce switch like-formation of macroscopic polymers. These polymers often undergo macroscopic phase separation, resulting in liquid droplets suspended in aqueous solution or high density puncta on membranes. We hypothesize that this behavior may contribute to the formation, regulation and function of signaling clusters that control actin, as well as a variety of additional so-called cellular bodies that function in diverse processes. This work is directed by concepts in polymer chemistry and soft matter physics, and utilizes tools derived from those areas, including light scattering, electron microscopy and various in vitro and in vivo imaging modalities.

Research in the lab is designed to deeply understand structure, energetics and function on the molecular scale, and, through applying concepts from chemistry and physics, discover how molecular properties generate organization and activity on the cellular scale.

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