Curated Optogenetic Publication Database

Abstract: The myocardin‐related transcription factor A (MRTF‐A) controls the transcriptional activity of the serum response factor (SRF) in a tightly controlled actin‐dependent manner. In turn, MRTF‐A is crucial for many actin‐dependent processes including adhesion, migration, and contractility and has emerged as novel targets for anti‐tumor strategies. MRTF‐A rapidly shuttles between cytoplasmic and nuclear compartment via dynamic actin interactions within its N‐terminal RPEL domain. Here, optogenetics is used to spatiotemporally control MRTF‐A nuclear localization by blue light using the light‐oxygen‐voltage‐sensing domain 2‐domain based system LEXY (light‐inducible nuclear export system). It is found that light‐regulated nuclear export of MRTF‐A occurs within 10–20 min. Importantly, MRTF‐A‐LEXY shuttling is independent of perturbations of actin dynamics. Furthermore, light‐regulation of MRTF‐A‐LEXY is reversible and repeatable for several cycles of illumination and its subcellular localization correlates with SRF transcriptional activity. As a consequence, optogenetic control of MRTF‐A subcellular localization determines subsequent cytoskeletal dynamics such as non‐apoptotic plasma membrane blebbing as well as invasive tumor‐cell migration through 3D collagen matrix. This data demonstrate robust optogenetic regulation of MRTF as a powerful tool to control SRF‐dependent transcription as well as cell motile behavior.

Abstract: De novo formation of epithelial cell-cell contacts relies on actin-based protrusions as well as tightly controlled turnover of junctional actin once cells encounter each other and adhesion complexes assemble. The specific contributions of individual actin regulators on either protrusion formation or junctional actin turnover remain largely unexplored. Based on our previous findings of Formin-like 2 (FMNL2)-mediated control of junctional actin dynamics, we investigated its potential role in membrane protrusions and impact on newly forming epithelial contacts. CRISPR/Cas9-mediated loss of FMNL2 in human MCF10A cells combined with optogenetic control of Rac1 activity confirmed its critical function in the establishment of intercellular contacts. While lamellipodial protrusion rates remained unaffected, FMNL2 knockout cells were characterized by impaired filopodia formation similar to depletion of the Rho GTPase Cdc42. Silencing of Cdc42, however, failed to affect FMNL2-mediated contact formation. Hence, we propose a cell-cell contact-specific and Rac1-mediated function of FMNL2 entirely independent of Cdc42. Consistent with this, direct visualizations of native epithelial junction formation revealed a striking and specifically Rac1- and not Cdc42-dependent recruitment of FMNL2 to newly forming junctions as well as established cell-cell contacts within epithelial sheets.

Abstract: Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.

Abstract: Formins are potent activators of actin filament assembly in the cytoplasm. In turn, cytoplasmic actin polymerization can promote release of actin from megakaryocytic acute leukemia (MAL) protein for serum response factor (SRF) transcriptional activity. We found that formins polymerized actin inside the mammalian nucleus to drive serum-dependent MAL-SRF activity. Serum stimulated rapid assembly of actin filaments within the nucleus in a formin-dependent manner. The endogenous formin mDia was regulated with an optogenetic tool, which allowed for photoreactive release of nuclear formin autoinhibition. Activated mDia promoted rapid and reversible nuclear actin network assembly, subsequent MAL nuclear accumulation, and SRF activity. Thus, a dynamic polymeric actin structure within the nucleus is part of the serum response.