We are studying the principles by which the actin cytoskeleton self-assembles and adapts to load at sites of mammalian endocytosis.
We think that by studying force-producing, membrane-reshaping processes, we will gain insights into the general logic of self-assembly and force responsiveness for living cellular processes in the stochastic environment of cells. Our approach is strongly influenced by theories of collective and emergent subcellular behavior.
We focus on a couple of core questions:
- What are the mechanisms by which the endocytic actin cytoskeleton adapts to load?
Endocytic actin cytoskeletal protein often exhibit force-dependent behavior at the molecular scale. How does this molecular-scale behavior influence collective assembly of cytoskeletal components? In previous work, we found that endocytic actin networks self-organize, bend to store elastic energy, and actively respond to loads by growing and producing more force. Based on these findings, we now wonder: how does that work? Given that so many individual cytoskeletal proteins have load-dependent binding or activity, how do these molecular-scale force-dependent properties influence and give rise to collective assembly and adaptive mechanical responsiveness?
- What are the conserved and divergent features of endocytic actin mechanical function between different membrane trafficking processes?
The actin cytoskeleton participates in several membrane trafficking processes including clathrin-dependent and clathrin-independent endocytosis. What are the universal features of endocytic actin assembly and operation in cellular membrane bending, and which features are specialized to each cellular process?
To address these questions, we use an integrated approach that blends biophysical modeling with live-cell fluorescence microscopy of gene-edited human stem cells.
The models represent our hypotheses of the minimal actin machinery necessary to participate in endocytosis. Simulations allow us to propose physically plausible and experimentally constrained explanations of cytoskeletal self-assembly and load adaptation.
We constrain and test the models using CRISPR gene editing of human induced pluripotent stem cells and quantitative fluorescence microscopy.
Iterating back and forth between the models and experiments allows us to propose and test physically plausible and experimentally grounded hypotheses of cytoskeletal self-assembly, mechanical function, and load responsiveness in endocytosis. Ultimately, this “bottom-up” approach to studying cytoskeletal self-organization and mechanical function will scale up to include reciprocal membrane trafficking processes, whole-cell membrane trafficking, cell polarization, differentiation, and assembly into higher-order tissues.