Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Ectosomal PKM2 Promotes HCC by Inducing Macrophage Differentiation and Remodeling the Tumor Microenvironment.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

The expanding family of neutrophil-derived extracellular vesicles.

Neutrophils are immune cells involved in several inflammatory and homeostatic processes. Their capacity to release cargo can be classified based on whether the cargo is released on its own, or in conjunction with plasma membrane structures. Examples of plasma membrane-free secretion modes are degranulation, neutrophil extracellular trap (NET) release, and cytokine release through inflammasome formation. The most studied membrane-covered neutrophil-derived structures are exosomes and ectosomes that are collectively called extracellular vesicles (EV). Apoptotic vesicles are another recognized EV subtype. Over the last decade, additional membrane-covered neutrophil-derived structures were characterized: migratory cytoplasts, migrasomes, and elongated neutrophil-derived structures (ENDS). All these structures are smaller than the neutrophils, cannot reproduce themselves, and thus meet the latest consensus definition of EVs. In this review, we focus on the less well-studied neutrophil EVs: apoptotic vesicles, cytoplasts, migrasomes, and ENDS.

Cilia-derived vesicles: An ancient route for intercellular communication.

Extracellular vesicles (EVs) provide a mechanism for intercellular communication that transports complex signals in membrane delimited structures between cells, tissues and organisms. Cells secrete EVs of various subtypes defined by the pathway leading to release and by the pathological condition of the cell. Cilia are evolutionarily conserved organelles that can act as sensory structures surveilling the extracellular environment. Here we discuss the secretory functions of cilia and their biological implications. Studies in multiple species - from the nematode Caenorhabditis elegans and the chlorophyte alga Chlamydomonas reinhardtii to mammals - have revealed that cilia shed bioactive EVs (ciliary EVs or ectosomes) by outward budding of the ciliary membrane. The content of ciliary EVs is distinct from that of other vesicles released by cells. Peptides regulate numerous aspects of metazoan physiology and development through evolutionarily conserved mechanisms. Intriguingly, cilia-derived vesicles have recently been found to mediate peptidergic signaling. C. reinhardtii releases the peptide α-amidating enzyme (PAM), bioactive amidated products and components of the peptidergic signaling machinery in ciliary EVs in a developmentally regulated manner. Considering the origin of cilia in early eukaryotes, it is likely that release of peptidergic signals in ciliary EVs represents an alternative and ancient mode of regulated secretion that cells can utilize in the absence of dedicated secretory granules.

The Characterization and Cytotoxic Evaluation of Chondrosia reniformis Collagen Isolated from Different Body Parts (Ectosome and Choanosome) Envisaging the Development of Biomaterials.

Chondrosia reniformis is a collagen-rich marine sponge that is considered a sustainable and viable option for producing an alternative to mammalian-origin collagens. However, there is a lack of knowledge regarding the properties of collagen isolated from different sponge parts, namely the outer region, or cortex, (ectosome) and the inner region (choanosome), and how it affects the development of biomaterials. In this study, a brief histological analysis focusing on C. reniformis collagen spatial distribution and a comprehensive comparative analysis between collagen isolated from ectosome and choanosome are presented. The isolated collagen characterization was based on isolation yield, Fourier-transformed infrared spectroscopy (FTIR), circular dichroism (CD), SDS-PAGE, dot blot, and amino acid composition, as well as their cytocompatibility envisaging the development of future biomedical applications. An isolation yield of approximately 20% was similar for both sponge parts, as well as the FTIR, CD, and SDS-PAGE profiles, which demonstrated that both isolated collagens presented a high purity degree and preserved their triple helix and fibrillar conformation. Ectosome collagen had a higher OHpro content and possessed collagen type I and IV, while the choanosome was predominately constituted by collagen type IV. In vitro cytotoxicity assays using the L929 fibroblast cell line displayed a significant cytotoxic effect of choanosome collagen at 2 mg/mL, while ectosome collagen enhanced cell metabolism and proliferation, thus indicating the latter as being more suitable for the development of biomaterials. This research represents a unique comparative study of C. reniformis body parts, serving as a support for further establishing this marine sponge as a promising alternative collagen source for the future development of biomedical applications.

Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism.

Extracellular vesicles in the treatment of neurological disorders.

Extracellular vesicles (EVs) are small, cell-derived membranous particles containing various nucleic acids, proteins, and lipids that play essential roles in intercellular communication. Evidence indicating that part of the regenerative benefit from stem cell therapy arises through EVs released from transplanted cells created interest in using EVs for clinical applications. EVs from various cellular sources, including mesenchymal stem cells, neural stem cells, and glia, are efficacious in models of neurological disease. In these models, EVs attenuate reactive gliosis, neuronal death, pro-inflammatory signaling, as well as reduce cognitive, behavioral, and motor deficits. EVs are naturally permeable to the blood-brain barrier and can be modified to contain molecules of interest, thereby also serving as a vehicle to transport therapeutics into the brain. This review summarizes the current state of research using EVs as a treatment in models of neurological disorders and highlights considerations for future research.

Extracellular vesicle interplay in cardiovascular pathophysiology.

Extracellular vesicles (EVs) are nanosized lipid bilayer-delimited particles released from cells that mediate intercellular communications and play a pivotal role in various physiological and pathological processes. Subtypes of EVs may include plasma membrane ectosomes or microvesicles and endosomal origin exosomes, although functional distinctions remain unclear. EVs carry cargo proteins, nucleic acids (RNA and DNA), lipids, and metabolites. By presenting or transferring this cargo to recipient cells, EVs can trigger cellular responses. We summarize contemporary understanding of EV biogenesis, composition, and function, with an emphasis on the role of EVs in the cardiovascular system. In addition, we outline the functional relevance of EVs in cardiovascular pathophysiology, further highlighting their potential for diagnostic and therapeutic applications.

Cross-Talk among Polymorphonuclear Neutrophils, Immune, and Non-Immune Cells via Released Cytokines, Granule Proteins, Microvesicles, and Neutrophil Extracellular Trap Formation: A Novel Concept of Biology and Pathobiology for Neutrophils.

Polymorphonuclear neutrophils (PMNs) are traditionally regarded as professional phagocytic and acute inflammatory cells that engulf the microbial pathogens. However, accumulating data have suggested that PMNs are multi-potential cells exhibiting many important biological functions in addition to phagocytosis. These newly found novel activities of PMN include production of different kinds of cytokines/chemokines/growth factors, release of neutrophil extracellular traps (NET)/ectosomes/exosomes and trogocytosis (membrane exchange) with neighboring cells for modulating innate, and adaptive immune responses. Besides, PMNs exhibit potential heterogeneity and plasticity in involving antibody-dependent cellular cytotoxicity (ADCC), cancer immunity, autoimmunity, inflammatory rheumatic diseases, and cardiovascular diseases. Interestingly, PMNs may also play a role in ameliorating inflammatory reaction and wound healing by a subset of PMN myeloid-derived suppressor cells (PMN-MDSC). Furthermore, PMNs can interact with other non-immune cells including platelets, epithelial and endothelial cells to link hemostasis, mucosal inflammation, and atherogenesis. The release of low-density granulocytes (LDG) from bone marrow initiates systemic autoimmune reaction in systemic lupus erythematosus (SLE). In clinical application, identification of certain PMN phenotypes may become prognostic factors for severe traumatic patients. In the present review, we will discuss these newly discovered biological and pathobiological functions of the PMNs.

Biogenesis and function of ESCRT-dependent extracellular vesicles.

From bacteria to humans, cells secrete a large variety of membrane-bound extracellular vesicles. Only relatively recently has it however started to become clear that the exovesicular transport of proteins and RNAs is important for normal physiology and numerous pathological conditions. Extracellular vesicles can be formed through the release of the intralumenal vesicles of multivesicular endosomes as so-called exosomes, or through direct, ectosomal, budding from the cell surface. Through their ability to promote the bending of membranes away from the cytoplasm, the components of the Endosomal Sorting Complex Required for Transport (ESCRT) have been implicated in both exo- and ectosomal biogenesis. Studies of the ESCRT machinery may therefore provide important insights into the formation and function of extracellular vesicles. In the present review, we first describe the cell biological mechanisms through which ESCRT components contribute to the biogenesis of different types of extracellular vesicles. We then discuss how recent functional studies have started to uncover important roles of ESCRT-dependent extracellular vesicles in a wide variety of processes, including the transport of developmental signaling molecules and embryonic morphogenesis, the regulation of social behavior and host-pathogen interactions, as well as the etiology and progression of neurodegenerative pathologies and cancer.

Therapeutic Potential of Cathelicidin Peptide LL-37, an Antimicrobial Agent, in a Murine Sepsis Model.

Among the mechanisms put-up by the host to defend against invading microorganisms, antimicrobial peptides represent the first line. In different species of mammals, the cathelicidin family of antimicrobial peptides AMPs has been identified, and in humans, LL-37 is the only type of cathelicidin identified. LL-37 has many different biological activities, such as regulation of responses to inflammation, besides its lipopolysaccharide (LPS)-neutralizing and antimicrobial and activities. Recently, employing a murine septic model that involves cecal ligation and puncture (CLP), we examined the effect of LL-37. The results indicated that LL-37 exhibits multiple protective actions on septic mice; firstly, the survival of CLP mice was found to be improved by LL-37 by the suppression of the macrophage pyroptosis that induces the release of pro-inflammatory cytokines (such as IL-1ß) and augments inflammatory reactions in sepsis; secondly, the release of neutrophil extracellular traps (NETs), which have potent bactericidal activity, is enhanced by LL-37, and protects mice from CLP-induced sepsis; thirdly, LL-37 stimulates neutrophils to release antimicrobial microvesicles (ectosomes), which improve the pathological condition of sepsis. These findings indicate that LL-37 protects CLP septic mice through at least three mechanisms, i.e., the suppression of pro-inflammatory macrophage pyroptosis and the release of antimicrobial NETs (induction of NETosis) and ectosomes from neutrophils. Thus, LL-37 can be a potential therapeutic candidate for sepsis due to its multiple properties, including the modulation of cell death (pyroptosis and NETosis) and the release of antimicrobial NETs and ectosomes as well as its own bactericidal and LPS-neutralizing activities.

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

Regulation of apical blebbing in the porcine epididymis.

Apical blebbing, a non-classical secretion mechanism, occurs in the mature porcine epididymis as part of its normal function. Proteins secreted by this mechanism contribute to the modification of the sperm plasma membrane during epididymal transit and are thought to contribute to acquisition of fertilizing ability. However, little is known about the regulation of this secretion mechanism in an in vivo model. Previous work demonstrated apical blebbing in the epididymis developed pubertally, suggesting androgens, sperm or other luminal factors regulated this process. Hence, the objective was to evaluate the hypothesized regulation of apical blebbing in the epididymis of pubertal boars by androgens and luminal factors. Androgen receptor blockade (flutamide) and surgical interventions (efferent duct ligation, orchidectomy or transection of the caput epididymis) were used to alter signaling, and the subsequent effects on apical blebbing were evaluated histologically. Apical blebbing was not altered by androgen receptor blockade with flutamide, but was significantly reduced 24 h after efferent duct ligation and after orchidectomy, treatments that eliminated luminal flow from the testis (P < 0.05). Like efferent duct ligation, epididymal transection altered luminal flow without removing the androgen source and significantly reduced the appearance of apical blebbing (P < 0.05). In conclusion, apical blebbing in the porcine epididymis appears to be regulated by luminal factors.

Ciliary Extracellular Vesicles: Txt Msg Organelles.

Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.

Focus on Extracellular Vesicles: Introducing the Next Small Big Thing.

Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These "extracellular vesicles" (EV) were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The "Focus on extracellular vesicles" series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition.

Cilia Provide a Platform for the Generation, Regulated Secretion, and Reception of Peptidergic Signals.

Cilia are microtubule-based cellular projections that act as motile, sensory, and secretory organelles. These structures receive information from the environment and transmit downstream signals to the cell body. Cilia also release vesicular ectosomes that bud from the ciliary membrane and carry an array of bioactive enzymes and peptide products. Peptidergic signals represent an ancient mode of intercellular communication, and in metazoans are involved in the maintenance of cellular homeostasis and various other physiological processes and responses. Numerous peptide receptors, subtilisin-like proteases, the peptide-amidating enzyme, and bioactive amidated peptide products have been localized to these organelles. In this review, we detail how cilia serve as specialized signaling organelles and act as a platform for the regulated processing and secretion of peptidergic signals. We especially focus on the processing and trafficking pathways by which a peptide precursor from the green alga Chlamydomonas reinhardtii is converted into an amidated bioactive product-a chemotactic modulator-and released from cilia in ectosomes. Biochemical dissection of this complex ciliary secretory pathway provides a paradigm for understanding cilia-based peptidergic signaling in mammals and other eukaryotes.

Unraveling multilayered extracellular vesicles: Speculation on cause.

Extracellular vesicles (EVs) are cell-released, heterogenous nanoparticles that play important roles in (patho)physiological processes through intercellular communication. EVs are often depicted as having a single lipid bilayer, but many studies have demonstrated the existence of multilayered EVs. There has been minimal inquiry into differences between unilamellar and multilamellar EVs in terms of biogenesis mechanisms and functional effects. This commentary speculates on potential causes and roles of multilamellar EVs and serves as a call to action for the research community to unravel the complex layers of EVs.

Food-Derived Extracellular Vesicles as Multi-Bioactive Complex and Their Versatile Health Effects.

Extracellular vesicles (EVs) are membrane-bound organelles that are generally released by eukaryotic cells and enclose various cellular metabolic information, such as RNA, meta-proteins, and versatile metabolites. The physiological properties and diverse functions of food-derived EVs have been extensively elucidated, along with a recent explosive upsurge in EV research. Therefore, a concise review of the health effects of food-derived EVs is necessary. This review summarizes the structural stability and uptake pathways of food-derived EVs to target cells and their health benefits, including antioxidant, anti-inflammatory, and anticarcinogenic effects, gut microbiome modulation, and intestinal barrier enhancement.

Extracellular vesicles highlight many cases of photoreceptor degeneration.

The release of extracellular vesicles is observed across numerous cell types and serves a range of biological functions including intercellular communication and waste disposal. One cell type which stands out for its robust capacity to release extracellular vesicles is the vertebrate photoreceptor cell. For decades, the release of extracellular vesicles by photoreceptors has been documented in many different animal models of photoreceptor degeneration and, more recently, in wild type photoreceptors. Here, I review all studies describing extracellular vesicle release by photoreceptors and discuss the most unifying theme among them-a photoreceptor cell fully, or partially, diverts its light sensitive membrane material to extracellular vesicles when it has defects in the delivery or morphing of this material into the photoreceptor's highly organized light sensing organelle. Because photoreceptors generate an enormous amount of light sensitive membrane every day, the diversion of this material to extracellular vesicles can cause a massive accumulation of these membranes within the retina. Little is known about the uptake of photoreceptor derived extracellular vesicles, although in some cases the retinal pigment epithelial cells, microglia, Müller glia, and/or photoreceptor cells themselves have been shown to phagocytize them.