We use optogenetic methods to spatially confine critical molecular to selected regions of a single cell. This approach allows processes like cell migration and cytokinesis to be optically directed. The spectral selectivity of optogenetic tools allows intracellular molecules to be quantitatively imaged simultaneously using fluorescent proteins or fluorophores while orchestrating cell behavior. Using this approach we deconstruct complex cell behaviors to understand the mechanisms at their basis.
Below are our most recent results:
O’Neill PR, Castillo-Badillo JA, Meshik X, Kalyanaraman V, Melgarejo K, Gautam N. (2018) Membrane Flow Drives an Adhesion-Independent Amoeboid Cell Migration Mode. Dev Cell. 46(1):9-22.
Cells migrate by applying rearward forces against extracellular media. It is unclear how this is achieved in amoeboid migration, which lacks adhesions typical of lamellipodia-driven mesenchymal migration. To address this question, we developed optogenetically controlled models of lamellipodia-driven and amoeboid migration. On a two-dimensional surface, migration speeds in both modes were similar. However, when suspended in liquid, only amoeboid cells exhibited rapid migration accompanied by rearward membrane flow. These cells exhibited increased endocytosis at the back and membrane trafficking from back to front. Genetic or pharmacological perturbation of this polarized trafficking inhibited migration. The ratio of cell migration and membrane flow speeds matched the predicted value from a model where viscous forces tangential to the cell-liquid interface propel the cell forward. Since this mechanism does not require specific molecular interactions with the surrounding medium, it can facilitate amoeboid migration observed in diverse microenvironments during immune function and cancer metastasis.
RAW cell transfected with optogenetic RhoA GEF (red) and RhoA activity sensor (green) undergoing RhoA-driven migration. White rectangle represents the area of photoactivation.Movie from O’Neill et al, Dev Cell (2018).
Clathrin (red) and trans-Golgi (green) dynamics during RhoA-driven migration suggest polarized endocytosis and trafficking. Images are maximum intensity projections from a z stack. Movie from O’Neill et al, Dev Cell (2018).
Optical RhoA vs. GPCR activation in cells suspended in Ficoll solution.Movie from O’Neill et al, Dev Cell (2018).
Meshik X, O’Neill PR, Gautam N. (2019) Physical Plasma Membrane Perturbation Using Subcellular Optogenetics Drives Integrin-Activated Cell Migration. ACS Synth Biol. 8(3):498-510.
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.
RAW cell migration driven by polarized accumulation of mCherry (red) and resulting decrease in membrane tension. Low tension sensor Venus-FBP17 is in green.
Movie from Meshik et al, ACS Synth Biol (2019).
Distribution of activated integrin β1 during protein accumulation-driven migration. HUTS4- AlexaFluor488 (green), an antibody for activated integrin β1, was added to the dish at t=7:20.
Movie from Meshik et al, ACS Synth Biol (2019).
Castillo-Badillo JA, Bandi AC, Harlalka S, Gautam N. (2020) SRRF-Stream Imaging of Optogenetically Controlled Furrow Formation Shows Localized and Coordinated Endocytosis and Exocytosis Mediating Membrane Remodeling. ACS Synth Biol. 9(4):902-919.
Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension, and endocytosis. FRAP, 4-D imaging, and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.
RAW cell undergoing furrow formation through RhoA-driven actomyosin contractility.
Movie from Castillo-Badillo et al, ACS Synth Biol (2020).
Visualization of directional vesicle trafficking to the bridge-like structure through FRAP. Membrane marker mCh-GL-GPI (red) was photobleached (red rectangle) at t=10:45.
Movie from Castillo-Badillo et al, ACS Synth Biol (2020).
SRRF-stream TIRF imaging shows membrane incorporation at the front edge of the daughter cells (red arrows). Cells were incubated with membrane dye FM4-64 (left) for 30 min and washed.
Movie from Castillo-Badillo et al, ACS Synth Biol (2020).