Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.actin-thin filaments | cardiomyopathy | cytoskeletal dynamics S triated muscle cells contain arrays of protein filaments assembled into contractile units that are nearly crystalline in structure. Efficient contraction at the molecular level is predicated upon accurate overlap of actin-containing thin and myosin-containing thick filaments. Therefore, proper control of filament assembly is absolutely critical.In striated muscle it is currently thought that the thin-filament pointed end capping protein tropomodulin (Tmod) is the predominant regulator of thin filament length, with Tmod1 being the sole isoform expressed in cardiomyocytes (1). Extensive in vitro work has revealed that Tmod1 uses two actin-and two tropomyosin-binding sites to associate with the end of the thin filament and to prevent addition or loss of actin monomers, thereby controlling length of the thin filament (2-7). Tmod1 is essential for life; Tmod1-KO mice are embryonic lethal because of cardiac defects (8-11).Identification of additional but structurally different members of the Tmod family of proteins, the leiomodins (Lmods), raises the possibility that thin filament lengths are not regulated solely by Tmod at thin filament pointed ends (12). Although there are three Lmod genes (Lmod1-3), Lmod2 and 3 are expressed in striated muscle with Lmod2 being the predominant isoform in cardiac muscle and Lmod3 the predominant isoform in skeletal muscle (12-16). The Lmods share ∼40% sequence identity at the protein level with the Tmods but do not contain a recognizable second tropomyosin-binding domain and have an additional C-terminal extension that includes a proline-rich region and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain (12, 17). Lmod2 has been proposed to be the long-sought muscle actin filament nucleator because it robustly nucleates actin filament formation in ...

Highlights Probabilistic filter with active contours to automate cerebrovascular segmentation. Geometric features of vessel network to study cerebrovascular diseases. Quantitative comparison of stroke and healthy cerebral vasculature. Vascular changes with aging and cerebrovascular disease. Comparison of CTA and MRA imaging modalities.

Capillary morphogenesis is a multistage, multicellular activity that plays a pivotal role in various developmental and pathological situations. In-depth understanding of the regulatory mechanism along with the capability of controlling the morphogenic process will have direct implications on tissue engineering and therapeutic angiogenesis. Extensive research has been devoted to elucidate the biochemical factors that regulate capillary morphogenesis. The roles of geometric confinement and cell-matrix mechanical interactions on the capillary architecture, nevertheless, remain largely unknown. Here, we show geometric control of endothelial network topology by creating physical confinements with microfabricated fences and wells. Decreasing the thickness of the matrix also results in comparable modulation of the network architecture, supporting the boundary effect is mediated mechanically. The regulatory role of cell-matrix mechanical interaction on the network topology is further supported by alternating the matrix stiffness by a cell-inert PEG-dextran hydrogel. Furthermore, reducing the cell traction force with a Rho-associated protein kinase inhibitor diminishes the boundary effect. Computational biomechanical analysis delineates the relationship between geometric confinement and cell-matrix mechanical interaction. Collectively, these results reveal a mechanoregulation scheme of endothelial cells to regulate the capillary network architecture via cell-matrix mechanical interactions.

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.