Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active beta 1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells. Single-molecule localisation microscopy is limited by low labeling and detection efficiencies of the molecular probes. Here the authors report a framework to obtain absolute molecular quantities on a true molecular scale; the data reveal a ternary adhesion complex underlying cell-matrix adhesion

Ena/VASP is required for endothelial barrier function in vivo

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia

Probabilistic movement modeling for intention inference in human-robot interaction.

Intention inference can be an essential step toward efficient humanrobot interaction. For this purpose, we propose the Intention-Driven Dynamics Model (IDDM) to probabilistically model the generative process of movements that are directed by the intention. The IDDM allows to infer the intention from observed movements using Bayes ’ theorem. The IDDM simultaneously finds a latent state representation of noisy and highdimensional observations, and models the intention-driven dynamics in the latent states. As most robotics applications are subject to real-time constraints, we develop an efficient online algorithm that allows for real-time intention inference. Two human-robot interaction scenarios, i.e., target prediction for robot table tennis and action recognition for interactive humanoid robots, are used to evaluate the performance of our inference algorithm. In both intention inference tasks, the proposed algorithm achieves substantial improvements over support vector machines and Gaussian processes.

Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure.

Epithelial fusion is a key process of morphogenesis by which tissue connectivity is established between adjacent epithelial sheets. A striking and poorly understood feature of this process is "zippering," whereby a fusion point moves directionally along an organ rudiment. Here, we uncover the molecular mechanism underlying zippering during mouse spinal neural tube closure. Fusion is initiated via local activation of integrin β1 and focal anchorage of surface ectoderm cells to a shared point of fibronectin-rich basement membrane, where the neural folds first contact each other. Surface ectoderm cells undergo proximal junction shortening, establishing a transitory semi-rosette-like structure at the zippering point that promotes juxtaposition of cells across the midline enabling fusion propagation. Tissue-specific ablation of integrin β1 abolishes the semi-rosette formation, preventing zippering and causing spina bifida. We propose integrin-mediated anchorage as an evolutionarily conserved mechanism of general relevance for zippering closure of epithelial gaps whose disturbance can produce clinically important birth defects

Fast Ionic Conductivity in the Most Lithium-Rich Phosphidosilicate Li14SiP6.

Solid electrolytes with superionic conductivity are required as a main component for all-solid-state batteries. Here we present a novel solid electrolyte with three-dimensional conducting pathways based on "lithium-rich" phosphidosilicates with ionic conductivity of σ > 10-3 S cm-1 at room temperature and activation energy of 30-32 kJ mol-1 expanding the recently introduced family of lithium phosphidotetrelates. Aiming toward higher lithium ion conductivities, systematic investigations of lithium phosphidosilicates gave access to the so far lithium-richest compound within this class of materials. The crystalline material (space group Fm3m), which shows reversible thermal phase transitions, can be readily obtained by ball mill synthesis from the elements followed by moderate thermal treatment of the mixture. Lithium diffusion pathways via both tetrahedral and octahedral voids are analyzed by temperature-dependent powder neutron diffraction measurements in combination with maximum entropy method and DFT calculations. Moreover, the lithium ion mobility structurally indicated by a disordered Li/Si occupancy in the tetrahedral voids plus partially filled octahedral voids is studied by temperature-dependent impedance and 7Li NMR spectroscopy

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates

Analysis of symmetries in models of multi-strain infections

In mathematical studies of the dynamics of multi-strain diseases caused by antigenically diverse pathogens, there is a substantial interest in analytical insights. Using the example of a generic model of multi-strain diseases with cross-immunity between strains, we show that a significant understanding of the stability of steady states and possible dynamical behaviours can be achieved when the symmetry of interactions between strains is taken into account. Techniques of equivariant bifurcation theory allow one to identify the type of possible symmetry-breaking Hopf bifurcation, as well as to classify different periodic solutions in terms of their spatial and temporal symmetries. The approach is also illustrated on other models of multi-strain diseases, where the same methodology provides a systematic understanding of bifurcation scenarios and periodic behaviours. The results of the analysis are quite generic, and have wider implications for understanding the dynamics of a large class of models of multi-strain diseases

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia