Membrane Budding Research Articles

Sodium Proton Exchanger NHE9 pHine-Tunes Exosome Production by Impairing Rab7 Activity.

Cell-to-cell communication is mediated by vesicles ranging from 30 to 150 nanometers, known as exosomes. These exosomes shuttle bioactive molecules such as proteins, lipids, and nucleic acids, thus playing crucial roles in both health and disease mechanisms. Exosomes form within the endocytic pathway through the process of inward budding of the endosomal membrane, facilitated by the progressive acidification of the endosomal lumen. Although endosomal pH is known to be critical for exosome production, the precise molecular mechanisms involved remain poorly defined. Maintaining optimal endosomal pH involves meticulous coordination between proton pumping and leakage mechanisms. The sodium-proton exchanger NHE9, located on the endosomal membrane, modulates endosomal pH by transporting protons out of the endosomes in exchange for sodium or potassium ions. Here, we use genetic engineering, biochemistry, and advanced microscopy to demonstrate that the sodium-proton exchanger NHE9 significantly affects exosome production by regulating endosomal pH. NHE9-mediated endosomal alkalization impairs Rab7 activation, thereby disrupting the delivery of multivesicular endosomes (MVEs) to lysosomes. Moreover, luminal alkalization promotes the recruitment of Rab27b. This enhances the targeting of MVEs to the cell periphery, their fusion with the plasma membrane, and subsequent exosome secretion. Our findings reveal the detailed molecular mechanisms through which endosomal pH regulates exosome production. Additionally, we identify NHE9 as a potential target for therapeutic strategies aimed at controlling exosome dynamics.

Exosomes in Dermatological Research: Unveiling Their Multifaceted Role in Cellular Communication, Healing, and Disease Modulation

Exosomes, a subtype of extracellular vehicles (EVs), play a pivotal role in cellular communication and have gained considerable attention in dermatological research. Formed through the inward budding of the endosomal membrane, exosomes facilitate the transfer of proteins, lipids, and nucleic acids, including microRNAs (miRNAs), thereby influencing the behavior and function of recipient cells. These vesicles are secreted by various cell types, including keratinocytes, and are crucial for maintaining skin homeostasis, regulating immune responses, and promoting wound healing. Exosomes have demonstrated therapeutic potential in addressing dermatological conditions such as hair disorders, skin cancers and photoaging through enhanced regeneration and reduced oxidative stress. However, they are also implicated in disease progression, with pathogens utilizing exosome release to evade host immune responses. Recent studies highlight the diverse origins and functions of exosomes, suggesting their promise as innovative therapeutic agents in dermatology. As research continues to elucidate their multifaceted roles, exosomes represent a frontier in understanding intercellular communication and developing novel treatments for skin-related diseases, underscoring their potential impact on both health and clinical applications. This review synthesizes the existing literature on exosome biology and isolation with a focus on their implications in dermatological contexts.

Recent Advances in Microfluidics for Nucleic Acid Analysis of Small Extracellular Vesicles in Cancer.

Small extracellular vesicles (sEVs) are membranous vesicles released from cellular structures through plasma membrane budding. These vesicles contain cellular components such as proteins, lipids, mRNAs, microRNAs, long-noncoding RNA, circular RNA, and double-stranded DNA, originating from the cells they are shed from. Ranging in size from ≈25 to 300 nm and play critical roles in facilitating cell-to-cell communication by transporting signaling molecules. The discovery of sEVs in bodily fluids and their involvement in intercellular communication has revolutionized the fields of diagnosis, prognosis, and treatment, particularly in diseases like cancer. Conventional methods for isolating and analyzing sEVs, particularly their nucleic acid content face challenges including high costs, low purity, time-consuming processes, limited standardization, and inconsistent yield. The development of microfluidic devices, enables improved precision in sorting, isolating, and molecular-level separation using small sample volumes, and offers significant potential for the enhanced detection and monitoring of sEVs associated with cancer. These advanced techniques hold great promise for creating next-generation diagnostic and prognostic tools given their possibility of being cost-effective, simple to operate, etc. This comprehensive review explores the current state of research on microfluidic devices for the detection of sEV-derived nucleic acids as biomarkers and their translation into practical point-of-care and clinical applications.

Role of ubiquitin-proteasome pathway in budded virus egress and GP64 surface distribution in Bombyx mori nucleopolyhedrovirus.

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that affects the silkworm, B. mori, causing substantial economic losses in sericulture. This study investigates the mechanisms underlying budded virus egress, focusing on the roles of the ubiquitin-proteasome pathway (UPP) machinery. BmNPV produces two virion types: budded virions (BVs) and occlusion-derived virions (ODVs), which differ in their envelope origins and functions. Recent findings suggest similarities in the budding pathways of BmNPV and Autographa californica multiple nucleopolyhedrovirus (AcMNPV), involving plasma membrane budding and multivesicular body (MVB) pathways. The study reveals that specific UPP-related proteins, including 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), polyubiquitin, proteasome alpha subunit 6 (PSMA6) and proteasome zeta subunit (PSMZ), are involved in BV egress. Using recombinant viruses and UPP inhibitors, we demonstrate the necessity of these proteins for GP64 secretion and effective BV release. RNA interference and cell surface display of GP64 analyses further validate the critical role of UPP in BmNPV BV egress and protein secretion. This research enhances our understanding of the mechanisms behind BmNPV MVB budding and GP64 secretion while also identifying potential targets for controlling the virus in sericulture.

Budding of Asymmetric Lipid Bilayers: Effects of Cholesterol, Anionic Lipid, and Electric Field.

Membrane budding is vital for various cellular processes such as synaptic activity regulation, vesicle transport and release, and endocytosis/exocytosis. Although protein-mediated membrane budding has been extensively investigated, the effects of the lipid asymmetry of the two leaflets and the asymmetrically electrical environments of the cellular membrane on membrane budding remain elusive. In this work, using coarse-grained molecular dynamics simulations, we systematically investigate the impacts of lipid bilayer asymmetry and external electric fields mimicking the asymmetric membrane potential on the membrane budding. The results show that the differential stress induced by the asymmetric distribution of lipids in the two leaflets is a crucial factor for the membrane budding. The unidirectional flip of cholesterol induced by the membrane curvature and the asymmetric ion adsorption induced by the anionic lipids promote the budding process. Furthermore, the external electric field applied perpendicularly to the bilayer plane increases the transmembrane potential and produces an additional differential stress across the leaflets by imposing an asymmetric torque on the lipid headgroups in the two leaflets, facilitating the membrane budding. These findings offer insights into how the structural and the environmental asymmetry in natural cellular membranes influence membrane budding in cellular processes.

Chemically Driven Division of Protocells by Membrane Budding.

Division is crucial for replicating biological compartments and, by extension, a fundamental aspect of life. Current studies highlight the importance of simple vesicular structures in prebiotic conditions, yet the mechanisms behind their self-division remain poorly understood. Recent research suggests that environmental factors can induce phase transitions in fatty acid-based protocells, leading to vesicle fission. However, using chemical energy to induce vesicle division, similar to the extant of life, has been less explored. This study investigates a mechanism of vesicle division by membrane budding driven by chemical energy without complex molecular machinery. We demonstrate that, in response to chemical fuel, simple fatty acid-based vesicles can bud off smaller daughter vesicles. The division mechanism is finely controlled by adjusting fuel concentration, offering valuable insights into primitive cellular dynamics. We showcase the robustness of self-division across different fatty acids, retaining encapsulated materials during division and suggesting protocell-like behavior. These results underscore the potential for chemical energy to drive autonomous replication in protocell models, highlighting a plausible pathway for the emergence of life. Furthermore, this study contributes to the development of synthetic cells, enhancing our understanding of the minimal requirements for cellular life and providing a foundation for future research in synthetic biology and the origins of life.

Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair.

The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist-a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems.

Endochondral ossification: Insights into the cartilage mineralization processes achieved by an anhydrous freeze substitution protocol

Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5 % uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500–1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100–200 nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined. Statement of SignificanceWe do not fully understand the process by which epiphyseal cartilage mineralizes - a vital step in endochondral bone formation. Previous work has proposed that mitochondria and intracellular vesicles are storage sites for the delivery of mineral to collagen fibrils. However, these concepts are founded on results from in vitro models of mineralization; no prior work has observed mineral-containing intracellular vesicles or mitochondria in developing epiphyseal cartilage. Here we developed a new cryofixation preparation route for transmission electron microscopy (TEM) imaging that has disclosed a cell-regulated process of mineralization in epiphyseal cartilage. High resolution TEM images revealed an involvement of mitochondria and intracellular and extracellular vesicles in delivering transient mineral phases to the collagen fibrils to promote cartilage mineralization.

Endosomal membrane budding patterns in plants

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis, endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets.

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation.

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group Im3m incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes.

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically-active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group incorporate into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time-resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life-like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Static magnetic field-modulated mesenchymal stem cell-derived mitochondria-containing microvesicles for enhanced intervertebral disc degeneration therapy

Intervertebral disc degeneration (IVDD) is characterized by the senescence and declining vitality of nucleus pulposus cells (NPCs), often driven by mitochondrial dysfunction. This study elucidates that mesenchymal stem cells (MSCs) play a crucial role in attenuating NPC senescence by secreting mitochondria-containing microvesicles (mitoMVs). Moreover, it demonstrates that static magnetic fields (SMF) enhance the secretion of mitoMVs by MSCs. By distinguishing mitoMV generation from exosomes, this study shifts focus to understanding the molecular mechanisms of SMF intervention, emphasizing cargo transport and plasma membrane budding processes, with RNA sequencing indicating the potential involvement of the microtubule-based transport protein Kif5b. The study further confirms the interaction between Rab22a and Kif5b, revealing Rab22a’s role in sorting mitoMVs into microvesicles (MVs) and potentially mediating subsequent plasma membrane budding. Subsequent construction of a gelatin methacrylate (GelMA) hydrogel delivery system further addresses the challenges of in vivo application and verifies the substantial potential of mitoMVs in delaying IVDD. This research not only sheds light on the molecular intricacies of SMF-enhanced mitoMV secretion but also provides innovative perspectives for future IVDD therapeutic strategies.

Delineating the shape of COat Protein complex-II coated membrane bud.

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Virus-Mimetic Extracellular-Vesicle Vaccine Boosts Systemic and Mucosal Immunity via Immune Recruitment.

Mucosal vaccines can prevent viruses from infecting the respiratory mucosa, rather than only curtailing infection and protecting against the development of disease symptoms. The SARS-CoV-2 spike receptor-binding domain (RBD) is a compelling vaccine target but is undermined by suboptimal mucosal immunogenicity. Here, we report a SARS-CoV-2-mimetic extracellular-vesicle vaccine developed using genetic engineering and dendritic cell membrane budding. After mucosal immunization, the vaccine recruits antigen-presenting cells rapidly initiating a strong innate immune response. Notably, it obviates the need for adjuvants and can induce germinal center formation through both intramuscular and intratracheal vaccination. It not only elicits high levels of RBD-specific antibodies but also stimulates extensive cellular immunity in the respiratory mucosa. A sequential immunization strategy, starting with an intramuscular injection followed by an intratracheal booster, significantly bolsters mucosal immunity with high levels of IgA and tissue-resident memory T cell responses, thereby establishing a formidable defense against pseudovirus infection.

Clathrin mediates membrane fission and budding by constricting membrane pores

Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles’ pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles’ pore closure and that clathrin around Ω-profiles’ base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles’ base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin’s well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents’ function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin’s pivotal role in pore constriction/closure.

Comparing Multifunctional Viral and Eukaryotic Proteins for Generating Scission Necks in Membranes.

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is M2 viroporin, a proton pump from the influenza A virus that is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging small-angle X-ray scattering structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemifission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

Vesicle protrusion induced by antimicrobial peptides suggests common carpet mechanism for short antimicrobial peptides

Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs—BP100, Decoralin, Neurokinin-1, and Temporin L—take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.

Current Insights into the Maturation of Epstein-Barr Virus Particles.

The three subfamilies of herpesviruses (alphaherpesviruses, betaherpesviruses, and gammaherpesviruses) appear to share a unique mechanism for the maturation and egress of virions, mediated by several budding and fusion processes of various organelle membranes during replication, which prevents cellular membrane disruption. Newly synthesized viral DNA is packaged into capsids within the nucleus, which are subsequently released into the cytoplasm via sequential fusion (primary envelopment) and budding through the inner and outer nuclear membranes. Maturation concludes with tegumentation and the secondary envelopment of nucleocapsids, which are mediated by budding into various cell organelles. Intracellular compartments containing mature virions are transported to the plasma membrane via host vesicular trafficking machinery, where they fuse with the plasma membrane to extracellularly release mature virions. The entire process of viral maturation is orchestrated by sequential interactions between viral proteins and intracellular membranes. Compared with other herpesvirus subfamilies, the mechanisms of gammaherpesvirus maturation and egress remain poorly understood. This review summarizes the major findings, including recently updated information of the molecular mechanism underlying the maturation and egress process of the Epstein-Barr virus, a ubiquitous human gammaherpesvirus subfamily member that infects most of the population worldwide and is associated with a number of human malignancies.

Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.