Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the “zippering” of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability

A Role for Rebinding in Rapid and Reliable T Cell Responses to Antigen

Experimental work has shown that T cells of the immune system rapidly and specifically respond to antigenic molecules presented on the surface of antigen-presenting-cells and are able to discriminate between potential stimuli based on the kinetic parameters of the T cell receptor-antigen bond. These antigenic molecules are presented among thousands of chemically similar endogenous peptides, raising the question of how T cells can reliably make a decision to respond to certain antigens but not others within minutes of encountering an antigen presenting cell. In this theoretical study, we investigate the role of localized rebinding between a T cell receptor and an antigen. We show that by allowing the signaling state of individual receptors to persist during brief unbinding events, T cells are able to discriminate antigens based on both their unbinding and rebinding rates. We demonstrate that T cell receptor coreceptors, but not receptor clustering, are important in promoting localized rebinding, and show that requiring rebinding for productive signaling reduces signals from a high concentration of endogenous pMHC. In developing our main results, we use a relatively simple model based on kinetic proofreading. However, we additionally show that all our results are recapitulated when we use a detailed T cell receptor signaling model. We discuss our results in the context of existing models and recent experimental work and propose new experiments to test our findings

A hidden Markov model for single particle tracks quantifies dynamic interactions between LFA-1 and the actin cytoskeleton.

The extraction of hidden information from complex trajectories is a continuing problem in single-particle and single-molecule experiments. Particle trajectories are the result of multiple phenomena, and new methods for revealing changes in molecular processes are needed. We have developed a practical technique that is capable of identifying multiple states of diffusion within experimental trajectories. We model single particle tracks for a membrane-associated protein interacting with a homogeneously distributed binding partner and show that, with certain simplifying assumptions, particle trajectories can be regarded as the outcome of a two-state hidden Markov model. Using simulated trajectories, we demonstrate that this model can be used to identify the key biophysical parameters for such a system, namely the diffusion coefficients of the underlying states, and the rates of transition between them. We use a stochastic optimization scheme to compute maximum likelihood estimates of these parameters. We have applied this analysis to single-particle trajectories of the integrin receptor lymphocyte function-associated antigen-1 (LFA-1) on live T cells. Our analysis reveals that the diffusion of LFA-1 is indeed approximately two-state, and is characterized by large changes in cytoskeletal interactions upon cellular activation

Actin Cytoskeleton Reorganization by Syk Regulates Fcγ Receptor Responsiveness by Increasing Its Lateral Mobility and Clustering

Clustering of immunoreceptors upon association with multivalent ligands triggers important responses including phagocytosis, secretion of cytokines, and production of immunoglobulins. We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR). While the receptors exist as monomers in resting macrophages, two distinct populations were discernible based on their mobility: some diffuse by apparent free motion, while others are confined within submicron boundaries that reduce the frequency of spontaneous collisions. Src-family and Syk kinases determine the structure of the actin cytoskeleton, which is fenestrated, accounting for the heterogeneous diffusion of the FcγR. Stimulation of these kinases during phagocytosis induces reorganization of the cytoskeleton both locally and distally in a manner that alters receptor mobility and clustering, generating a feedback loop that facilitates engagement of FcγR at the tip of pseudopods, directing the progression of phagocytosis

Actin Cytoskeleton Reorganization by Syk Regulates Fcγ Receptor Responsiveness by Increasing Its Lateral Mobility and Clustering

Clustering of immunoreceptors upon association with multivalent ligands triggers important responses including phagocytosis, secretion of cytokines, and production of immunoglobulins. We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR). While the receptors exist as monomers in resting macrophages, two distinct populations were discernible based on their mobility: some diffuse by apparent free motion, while others are confined within submicron boundaries that reduce the frequency of spontaneous collisions. Src-family and Syk kinases determine the structure of the actin cytoskeleton, which is fenestrated, accounting for the heterogeneous diffusion of the FcγR. Stimulation of these kinases during phagocytosis induces reorganization of the cytoskeleton both locally and distally in a manner that alters receptor mobility and clustering, generating a feedback loop that facilitates engagement of FcγR at the tip of pseudopods, directing the progression of phagocytosis

Sequence Note: nef sequences of primary HIV type 1 isolates from Northern India

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Systematic in silico analysis of clinically tested drugs for reducing amyloid‐beta plaque accumulation in Alzheimer's disease

INTRODUCTION: Despite strong evidence linking amyloid beta (Aβ) to Alzheimer's disease, most clinical trials have shown no clinical efficacy for reasons that remain unclear. To understand why, we developed a quantitative systems pharmacology (QSP) model for seven therapeutics: aducanumab, crenezumab, solanezumab, bapineuzumab, elenbecestat, verubecestat, and semagacestat. METHODS: Ordinary differential equations were used to model the production, transport, and aggregation of Aβ; pharmacology of the drugs; and their impact on plaque. RESULTS: The calibrated model predicts that endogenous plaque turnover is slow, with an estimated half-life of 2.75 years. This is likely why beta-secretase inhibitors have a smaller effect on plaque reduction. Of the mechanisms tested, the model predicts binding to plaque and inducing antibody-dependent cellular phagocytosis is the best approach for plaque reduction. DISCUSSION: A QSP model can provide novel insights to clinical results. Our model explains the results of clinical trials and provides guidance for future therapeutic development