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Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-[beta]1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy., Introduction Proper wound healing depends on effective hemostasis, which allows regeneration and reconstitution of defective tissue function (1). Coronary thrombosis causing myocardial infarction (MI) (2) leads to irreversible tissue injury, [...]

In pemphigus vulgaris, a life-threatening autoimmune skin disease, epidermal blisters are caused by autoantibodies primarily targeting desmosomal cadherins desmoglein 3 (DSG3) and DSG1, leading to loss of keratinocyte cohesion. Due to limited insights into disease pathogenesis, current therapy relies primarily on nonspecific long-term immunosuppression. Both direct inhibition of DSG transinteraction and altered intracellular signaling by p38 MAPK likely contribute to the loss of cell adhesion. Here, we applied a tandem peptide (TP) consisting of 2 connected peptide sequences targeting the DSG adhesive interface that was capable of blocking autoantibody-mediated direct interference of DSG3 transinteraction, as revealed by atomic force microscopy and optical trapping. Importantly, TP abrogated autoantibody-mediated skin blistering in mice and was effective when applied topically. Mechanistically, TP inhibited both autoantibody-induced p38 MAPK activation and its association with DSG3, abrogated p38 MAPK-induced keratin filament retraction, and promoted desmosomal DSG3 oligomerization. These data indicate that p38 MAPK links autoantibody-mediated inhibition of DSG3 binding to skin blistering. By limiting loss of DSG3 transinteraction, p38 MAPK activation, and keratin filament retraction, which are hallmarks of pemphigus pathogenesis, TP may serve as a promising treatment option., Introduction Pemphigus vulgaris (PV) is a life-threatening blistering disease histopathologically characterized by acantholysis, i.e., cleft formation within the epidermis or mucous membranes (1). The disease is caused by autoantibodies predominantly [...]

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in bio-medical research., Introduction A variety of different imaging strategies have been utilized to study immune cell activity and migration in animal models of human disease. Imaging techniques such as MRI, PET, and [...]

In the human autoimmune blistering skin disease pemphigus vulgaris autoantibodies (PV-IgG), which are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and Dsg1, cause keratinocyte cell dissociation (acantholysis). Recent studies reported that loss of keratinocyte cell adhesion was accompanied by profound alterations of the actin cytoskeleton. Nevertheless, the relevance of actin reorganization in this process is unclear at present. In this study, we provide evidence for an important role of actin reorganization in pemphigus pathogenesis. In parallel to loss of cell adhesion and fragmentation of Dsg3 staining along cell borders, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization. Moreover, in experiments using fluorescence recovery after photobleaching (FRAP), PV-IgG were detected to interfere with actin dynamics. Therefore, we investigated whether pharmacological manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments via jasplakinolide significantly blocked cell dissociation and Dsg3 fragmentation, whereas cytochalasin D-induced actin depolymerization strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Cytotoxic necrotizing factor-1 (CNF-1)-mediated activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions via addition of latrunculin B, the protective effects of CNF-1 were abrogated. Taken together, these experiments indicate that reorganization of cortical actin filaments is a critical step in PV-IgG-induced keratinocyte dissociation. cytoskeleton; Rho-GTPase; cytotoxic necrotizing factor-1; desmoglein doi: 10.1152/ajpcell.00075.2010.

Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 μm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular “fruiting bodies” of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium, we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21. [ABSTRACT FROM AUTHOR]