Membrane curvature as a signal to ensure robustness of diverse cellular processes.

An increasing corpus of research has demonstrated that membrane shape, generated either by the external environment of the cell or by intrinsic mechanisms such as cytokinesis and vesicle or organelle formation, is an important parameter in the control of diverse cellular processes. In this review we discuss recent findings that demonstrate how membrane curvature (from nanometer to micron length-scales) alters protein function. We describe an expanding toolkit for experimentally modulating membrane curvature to reveal effects on protein function, and discuss how membrane curvature - far from being a passive consequence of the physical environment and the internal protein activity of a cell - is an important signal that controls protein affinity and enzymatic activity to ensure robust forward progression of key processes within the cell.

Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin.

During clathrin-mediated endocytosis (CME), flat plasma membrane is remodeled to produce nanometer-scale vesicles. The mechanisms underlying this remodeling are not completely understood. The ability of clathrin to bind membranes of distinct geometries casts uncertainty on its specific role in curvature generation/stabilization. Here, we used nanopatterning to produce substrates for live-cell imaging, with U-shaped features that bend the ventral plasma membrane of a cell into shapes resembling energetically unfavorable CME intermediates. This induced membrane curvature recruits CME proteins, promoting endocytosis. Upon AP2, FCHo1/2, or clathrin knockdown, CME on flat substrates is severely diminished. However, induced membrane curvature recruits CME proteins in the absence of FCHo1/2 or clathrin and rescues CME dynamics/cargo uptake after clathrin (but not AP2 or FCHo1/2) knockdown. Induced membrane curvature enhances CME protein recruitment upon branched actin assembly inhibition under elevated membrane tension. These data establish that membrane curvature assists in CME nucleation and that the essential function of clathrin during CME is to facilitate curvature evolution, rather than scaffold protein recruitment.

Mechanosensing through Direct Binding of Tensed F-Actin by LIM Domains.

Mechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators. Here, we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding as a mechanosensing mechanism by which cytoskeletal tension can govern nuclear localization.

Spatio-temporal regulation of RAG2 following genotoxic stress.

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr(490). In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.

Cytoskeletal modulation of lipid interactions regulates Lck kinase activity.

The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C(16))-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C(16)-anchored DiO with didodecyl (C(12))-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation.