Cytoskeleton最新文献

The platelet integrin α_IIbβ₃ undergoes long-range conformational transitions between bent and extended conformations to regulate platelet aggregation during hemostasis and thrombosis. However, how exactly α_IIbβ₃ transitions between conformations remains largely elusive. Here, we studied how transitions across bent and extended-closed conformations of α_IIbβ₃ integrin are regulated by effective interactions between its functional domains. We first carried out μs-long equilibrium molecular dynamics (MD) simulations of full-length α_IIbβ₃ integrins in bent and intermediate conformations, the latter characterized by an extended headpiece and closed legs. Then, we built heterogeneous elastic network models, perturbed inter-domain interactions, and evaluated their relative contributions to the energy barriers between conformations. Results showed that integrin extension emerges from: (i) changes in interfaces between functional domains; (ii) allosteric coupling of the head and upper leg domains with flexible lower leg domains. Collectively, these results provide new insights into integrin conformational activation based on short- and long-range interactions between its functional domains and highlight the importance of the lower legs in the regulation of integrin allostery.

A primary cilium, made of nine microtubule doublets enclosed in a cilium membrane, is a mechanosensing organelle that bends under an external mechanical load and sends an intracellular signal through transmembrane proteins activated by cilium bending. The nine microtubule doublets are the main load-bearing structural component, while the transmembrane proteins on the cilium membrane are the main sensing component. No distinction was made between these two components in all existing models, where the stress calculated from the structural component (nine microtubule doublets) was used to explain the sensing location, which may be totally misleading. For the first time, we developed a microstructure-based primary cilium model by considering these two components separately. First, we refined the analytical solution of bending an orthotropic cylindrical shell for individual microtubule, and obtained excellent agreement between finite element simulations and the theoretical predictions of a microtubule bending as a validation of the structural component in the model. Second, by integrating the cilium membrane with nine microtubule doublets and simulating the tip-anchored optical tweezer experiment on our computational model, we found that the microtubule doublets may twist significantly as the whole cilium bends. Third, besides being cilium-length-dependent, we found the mechanical properties of the cilium are also highly deformation-dependent. More important, we found that the cilium membrane near the base is not under pure in-plane tension or compression as previously thought, but has significant local bending stress. This challenges the traditional model of cilium mechanosensing, indicating that transmembrane proteins may be activated more by membrane curvature than membrane stretching. Finally, we incorporated imaging data of primary cilia into our microstructure-based cilium model, and found that comparing to the ideal model with uniform microtubule length, the imaging-informed model shows the nine microtubule doublets interact more evenly with the cilium membrane, and their contact locations can cause even higher bending curvature in the cilium membrane than near the base.

ON THE FRONT COVER: Tissue level cytoskeletal architecture of the inner medullary region of a mouse kidney. Green: Phalloidin fluorescence of F-actin filaments along cell membranes of the renal thin loops and collecting ducts. Red: CD34 immunofluorescence of capillaries. Blue: DAPI fluorescence of the cell nuclei.

Credit: Girishkumar K. Kumaran (Ariel University, Israel; University of Oxford, UK) & Israel Hanukoglu (Ariel University, Israel)

ON THE INNER FRONT COVER: Hs27 fibroblasts stained for Vinculin (green) and F-actin/phalloidin (red) on etched grooved quartz, illustrating the concept of a focal adhesion confinement mechanism in contact guidance at shallow groove depths.

Credit: Jinny L. Liu & Michael C. Robitaille (U.S. Naval Research Laboratory, USA)

ON THE INNER BACK COVER: Hs27 fibroblasts stained for Vinculin (green) and F-actin/phalloidin (red) on etched grooved quartz, illustrating the concept of a focal adhesion confinement mechanism in contact guidance at shallow groove depths.

Credit: Jinny L. Liu & Michael C. Robitaille (U.S. Naval Research Laboratory, USA)

ON THE BACK COVER: Control MDA-MB-231 cells seen in confocal (60X objective) max intensity projections with alpha-tubulin (red), DNA (blue), and Golgi marker GM130 (green) labeled.

Credit: Laura A. Zahn (Vanderbilt University Medical Center, Nashville, TN, USA)

Vasodilator-stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly-Pro region, a G-actin-binding domain (GAB), and an F-actin-binding domain (FAB). Together, the poly-Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin–G-actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly-Pro region and GAB domain of VASP with profilin–actin, has been solved over a decade ago; however, a detailed structure of the FAB-F-actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F-actin. Here, in order to develop our understanding of the VASP–actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain-actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain-level binding–unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side-binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP–actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k _cat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.