Mario Gimona

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Assembly and biological role of podosomes and invadopodia

Regulated tissue invasion via motile and lytic events is critical for physiological processes such as immune system function and inflammatory responses, wound healing, and organ development, but pathological subversion of this process drives tumour cell invasion and metastasis. Cell migration and invasion require the integration of several processes that include: first, the local modulation of cytoskeleton structure and contractile forces; second, the turnover of substrate adhesions and their associated microfilaments; and third, the generation of specialised, transient domains that mediate the protease-dependent focal degradation of the extracellular matrix. Recent work has re-discovered prominent actin-based cellular structures, termed invadopodia and podosomes, as unique structural and functional modules through which major invasive mechanisms are regulated. The stage is now set to unravel their roles in the physiology and pathology of tissue plasticity and repair.

Functional plasticity of CH domains

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross‐linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Smooth muscle specific expression of calponin

Calponin is an actin‐, calmodulin‐. and tropomyosin‐binding protein that has been isolated from smooth muscle tissue. Using a monoclonal antibody specific for avian calponin, we demonstrate a differentiation‐linked increase in calponin expression in embryonic chick gizzard. Cultivation of gizzard smooth muscle cells in vitro resulted in a down‐regulation of calponin expression after the first 48 h that was paralleled by a loss of synthesis of metavinculin and the high molecular weight isoform of caldesmon. In early cultures of smooth muscle cells calponin was localised in the actin‐containing stress fibres but labelling was restricted to the central parts of the actin cytoskeleton. Calponin expression is suggested as a potentially useful index of smooth muscle differentiation.

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.

Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains.

Phorbol ester triggers the conversion of focal adhesions into podosomes in A7r5 smooth muscle cells. Here we followed the dynamics of podosome formation using dual fluorescence live video and confocal microscopy, as well as interference reflection and evanescent wave microscopy. We show that podosomes form at the outer region of stress fiber bundles, at specialized sites where they are embedded in adhesion plaques at the basal surface of the plasma membrane, and that cortactin resides constitutively at these microdomains. We further demonstrate that the formation of podosomes requires Arp2/3-dependent actin polymerization at the stress fiber-focal adhesion interface. Concentration of Arp2/3 coincides with podosome formation and precedes the engagement of SM22 and alpha-actinin, while the focal adhesion components zyxin and vinculin redistribute only at later stages of podosome development. We thus suggest that the genesis of podosomes includes two steps, one requiring the early de novo polymerization of actin filaments, and a second, late phase characterized by the recruitment of focal adhesion components. Moreover, we provide evidence for the existence of an as yet unidentified region in close proximity to the focal adhesion-stress fiber interface, which marks the site of actin cytoskeleton remodeling and is a novel site of Arp2/3-dependent F-actin polymerization.

Mammalian calponin. Identification and expression of genetic variants.

Calponin is a smooth muscle specific, actin‐, tropomyosin‐ and calmodulin‐binding protein thought to be involved in some way in the regulation or modulation of contraction. Here we describe the cloning and bacterial expression of two calponin species from murine and porcine smooth muscle tissues. Primary and secondary structural analyses of the deduced amino acid sequences revealed a high degree of homology to avian calponin with the exception of a short and variable C‐terminal segment. The sequence data demonstrate that the two mammalian calponin variants do not arise via alternative splicing but are encoded by different genes.

Normalization of nomenclature for peptide motifs as ligands of modular protein domains

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer‐readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains

Activation of conventional protein kinase C by phorbol ester triggers the Src-dependent remodelling of the actin cytoskeleton and the formation of podosomes in vascular smooth muscle cells. Rearrangement of actin cytoskeleton in response to phorbol-12,13-dibutyrate is characterised by the simultaneous disassembly of peripheral actin stress fibres and focal adhesions, focal de novo actin polymerisation and actomyosin contraction in the cell center, indicating a spatially and temporally segregated, differential modulation of actin-cytoskeleton stability and turnover. Taking advantage of the prominent actin cytoskeleton in A7r5 cells we show here, that the molecular basis for the local inhibition of contractility is the specific recruitment of p190RhoGAP to specialised microdomains at the focal adhesion/stress fibre interface, which are constitutively enriched in cortactin. The microdomains contain structurally altered actin filaments inaccessible to phalloidin. However, the filaments remain decorated with high molecular weight tropomyosins. Clustering of cortactin during podosome formation causes the rapid, local dispersion of myosin and tropomyosin, and interferes with the F-actin binding of h1calponin, consistent with a RhoGAP-mediated reduction of contractility. Phorbol ester-induced podosome formation is efficiently blocked by expression of constitutively active Dia1, which leads to the dispersion of cortactin. The results provide direct evidence for the spatially restricted inhibition of contractility via the recruitment and accumulation of cortactin and p190RhoGAP.