Human cytomegalovirus (HCMV) is a widespread beta-herpesvirus that establishes lifelong infection and can cause severe disease in immunocompromised individuals as well as congenital abnormalities. While HCMV entry into fibroblasts is classically described as plasma membrane fusion, accumulating evidence indicates that a fraction of virions undergo endocytic uptake, notably via macropinocytosis, and traffic through the endosomal system. However, the mechanisms by which internalized viral components reach the nucleus are still being elucidated. Here, we investigated whether HCMV exploits type II nuclear envelope invaginations (NEIs), rare and discrete folds of the nuclear membrane that extend into the nucleoplasm, and the associated VAP-A-ORP3-Rab7 (VOR) complex to mediate nuclear delivery of viral components, a mechanism previously described for HIV-1. Using primary human foreskin fibroblasts (HFFs), we tracked the tegument protein pp65 and immediate-early proteins IE1/2 during early infection. We show that HCMV infection induces a rapid increase in NEI formation within the first hour of infection, accompanied by the accumulation of pp65 within Rab7 ⁺ endosomal structures that localize to NEIs. Pharmacological inhibition of the VOR complex with an ORP3-targeting drug significantly reduced NEI formation, decreased the association of pp65 with NEIs, and impaired its nuclear accumulation by approximately 2.5-fold. In contrast, inhibition of this pathway did not affect immediate-early gene expression at 24 hours post-infection. Functionally, disruption of the VOR complex resulted in a 3-fold reduction in viral replication, highlighting the contribution of this pathway to efficient infection. Together, these findings support a model in which HCMV tegument proteins, but not the viral genome, access the nucleus via a NEI/VOR-dependent trafficking route. This work identifies a previously unrecognized nuclear delivery pathway exploited by HCMV and suggests that targeting nuclear-endosomal communication may represent a novel antiviral strategy.

Extracellular membrane vesicles (EVs) offer promising values in various medical fields, e.g., as biomarkers in liquid biopsies or as native (or bioengineered) biological nanocarriers in tissue engineering, regenerative medicine and cancer therapy. Based on their cellular origin EVs can vary considerably in composition and diameter. Cell biological studies on mammalian prominin-1, a cholesterol-binding membrane glycoprotein, have helped to reveal new donor membranes as sources of EVs. For instance, small EVs can originate from microvilli and primary cilia, while large EVs might be produced by transient structures such as retracting cellular extremities of cancer cells during the mitotic rounding process, and the midbody at the end of cytokinesis. Here, we will highlight the various subcellular origins of prominin-1+ EVs, also called prominosomes, and the potential mechanism(s) regulating their formation. We will further discuss the molecular and cellular characteristics of prominin-1, notably those that have a direct effect on the release of prominin-1+ EVs, a process that might be directly implicated in donor cell reprogramming of stem and cancer stem cells. Prominin-1+ EVs also mediate intercellular communication during embryonic development and adult homeostasis in healthy individuals, while disseminating biological information during diseases.

The primary cilium, a solitary membrane-bound, microtubule-based cellular organelle, has been considered an evolutionary relict for almost a century. Over the past three decades, interest in this protruding, non-motile structure of the plasma membrane has been boosted by the identification of ciliary dysfunctions as the underlying cause of developmental abnormalities and inherited disorders, commonly called ciliopathies. The primary cilium responds to environmental stimuli, such as mechanical, chemical, or light (in the case of the modified cilium of photoreceptors) signals. The membrane of primary cilia host specific sensory complexes and/or receptors associated with various pathways, predisposing them to transmit (or convert) spatiotemporal environmental information into cellular response. These dual mechanochemical aspects led to the recognition that primary cilia are multifunctional sensory organelles that act as "cellular antennae". Beyond their established role in signal transduction, primary cilia are newly recognized as important hubs for short- and long-distance intercellular communication due to their ability to release and, perhaps, selectively take up extracellular vesicles, which are biological carriers exchanged between cells. In addition, the physical contact of the primary cilium with other cilia, cytonemes or with nerve cell axons adds another layer of complexity to the mechanisms of sensory and/or intercellular communication between neighboring cells that needs to be further explored. In this review, we focus on these new and less-explored ciliary properties and processes, which can affect cell communication and signaling and thus have a direct impact on development, tissue homeostasis, and pathological conditions.

Physical exercise affects multiple physiological processes, including those regulated by hormones, such as energy balance, stress responses, and pain perception. The prohormone precursor proopiomelanocortin (POMC) gives rise to β-endorphin, adrenocorticotropic hormone, β-lipotropin, and the melanocyte-stimulating hormones (MSH), which act through melanocortin (MC1-5) and opioid receptors to influence these key physiological responses. Here, we identified a mechanism by which full-length POMC interacts with small extracellular vesicles (sEVs) in the circulation. In healthy adults, the fraction of plasma POMC associated with sEVs increased fourfold following intense exercise, despite unchanged POMC and sEV concentrations. POMC-positive sEVs contained leukocyte or megakaryocyte/platelet markers, suggesting hematopoietic origin. A substantial subset of circulating sEVs expressed MC1–MC5 and μ-opioid receptors, and coimmunoisolation assays demonstrated binding of unprocessed POMC to MC1 and MC3 receptors. Molecular modeling indicated that POMC undergoes pH-dependent conformational changes that favor MC binding, a finding corroborated by in vitro binding assays under acidic conditions. Functionally, addition of POMC-positive sEVs to B16-F10 melanoma cells induced melanogenesis, consistent with POMC-derived MSH activity. The sEV-associated POMC had higher rates of transport than unbound POMC in assays mimicking the blood–brain barrier. These findings establish that exercise-induced, pH-dependent binding of POMC to circulating sEVs may facilitate hormone precursor transport and potentially influence transfer across the blood–brain barrier. This mechanism represents a separate paradigm for endocrine regulation, providing insight into how exercise modulates sEV interactions with systemic hormone distribution and signaling.

Intercellular communication is an essential hallmark of multicellular organisms for their development and adult tissue homeostasis. Over the past two decades, attention has been focused on communication mechanisms based on various membrane structures, as illustrated by the burst of scientific literature in the field of extracellular vesicles (EVs). These lipid bilayer-bound nano- or microparticles, as vehicle-like devices, act as regulators in various biological and physiological processes. When EVs are internalized by recipient cells, their membrane and cytoplasmic cargoes can interfere with cellular activities, affecting pathways that regulate cell proliferation, differentiation, and migration. In cancer, EVs can transfer oncogenic factors, stimulate neo-angiogenesis and immunosuppression, reprogram stromal cells, and confer drug resistance traits, thereby remodeling the surrounding microenvironment. Although the mechanisms underlying EV biogenesis and uptake are now better understood, little is known about the spatiotemporal mechanism(s) of their actions after internalization. In this respect, we have shown that a fraction of endocytosed EVs reaches the nuclear compartment via the VOR (VAP-A-ORP3-Rab7) complex-mediated docking of late endosomes to the outer nuclear membrane in the nucleoplasmic reticulum, positioning and facilitating the transfer of EV cargoes into the nucleoplasm via nuclear pores. Here, we highlight the EV heterogeneity, the cellular pathways governing EV release and uptake by donor and recipient cells, respectively, and focus on a novel intracellular pathway leading to the nuclear transfer of EV cargoes. We will discuss how to intercept it, which could open up new avenues for clinical applications in which EVs and other small extracellular particles (e.g., retroviruses) are implicated.

Background The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues. Results We report here a new type of large CD133 + EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised. Conclusion Given the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133 + EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.

Prominin-1 (CD133) is a cholesterol-binding membrane glycoprotein selectively associated with highly curved and prominent membrane structures. It is widely recognized as an antigenic marker of stem cells and cancer stem cells and is frequently used to isolate them from biological and clinical samples. Recent progress in understanding various aspects of CD133 biology in different cell types has revealed the involvement of CD133 in the architecture and dynamics of plasma membrane protrusions, such as microvilli and cilia, including the release of extracellular vesicles, as well as in various signaling pathways, which may be regulated in part by posttranslational modifications of CD133 and its interactions with a variety of proteins and lipids. Hence, CD133 appears to be a master regulator of cell signaling as its engagement in PI3K/Akt, Src-FAK, Wnt/β-catenin, TGF-β/Smad and MAPK/ERK pathways may explain its broad action in many cellular processes, including cell proliferation, differentiation, and migration or intercellular communication. Here, we summarize early studies on CD133, as they are essential to grasp its novel features, and describe recent evidence demonstrating that this unique molecule is involved in membrane dynamics and molecular signaling that affects various facets of tissue homeostasis and cancer development. We hope this review will provide an informative resource for future efforts to elucidate the details of CD133’s molecular function in health and disease.

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure-activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-Gpseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70’s key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell–cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication. Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes. Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment.

Metastases are responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing the metastatic process. Traditionally, cancer metastasis is attributed to the dissemination of neoplastic cells from the primary tumor to distant organs through blood and lymphatic circulation. A thorough understanding of the metastatic process is essential to develop new therapeutic strategies that improve cancer survival. Since Paget’s original description of the “Seed and Soil” hypothesis over a hundred years ago, alternative theories and new players have been proposed. In particular, the role of extracellular vesicles (EVs) released by cancer cells and their uptake by neighboring cells or at distinct anatomical sites has been explored. Here, we will outline and discuss these alternative theories and emphasize the horizontal transfer of EV-associated biomolecules as a possibly major event leading to cell transformation and the induction of metastases. We will also highlight the recently discovered intracellular pathway used by EVs to deliver their cargoes into the nucleus of recipient cells, which is a potential target for novel anti-metastatic strategies.

Abstract The contribution of Prominin‐1 (aka CD133) to male fertility has recently been (re)investigated, with contradictory results. Early findings, essential for deciphering its role, have unfortunately been neglected. Here, the authors present what is currently known about its expression in the male reproductive system of rodents and men so that its involvement in male fertility can be re‐examined and discussed in the light of these elements.

The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4+ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7+ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4+ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle.

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand–receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.

Protein tyrosine O-sulfation is a posttranslational modification that takes place in the trans-Golgi network and occurs for a wide spectrum of transmembrane and secretory proteins found in all animals and in the plant kingdom. The sulfate transfer reaction to tyrosine residues is catalyzed by one of two tyrosylprotein sulfotransferases (EC 2.8.2.20) which use the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate. Protein tyrosine O-sulfation promotes extracellular protein–protein interactions occurring in various biological processes, ranging from receptor binding of regulated peptides to the interaction of viral envelope proteins with cell surface proteins. An overview of our knowledge of protein tyrosine sulfation is presented.

Fragestellung Das Mammakarzinom ist die zweithäufigste Todesursache für Frauen weltweit. Im Jahr 2020 starben ca. 685.000 Patienten. Anders als andere Krebsarten bilden bestimmte Brustkrebssubtypen, wie luminal A und B, in bis zu ca. 60% der Fällen Knochenmetastasen aus. Dabei stellt sich die Frage, warum bestimmte Subtypen eine höhere Affinität zum Knochenmark aufweisen. In vorangegangenen Untersuchungen hat sich gezeigt, dass Zellmembranausläufer (z.B. sog. Lamellipodium, Magnupodium, Filopodium etc.) eine erhebliche Rolle in der Interaktion mit den Komponenten des Knochenmarks spielen.

In the present minireview, we intend to provide a brief history of the field of CD9 involvement in oncogenesis and in the metastatic process of cancer, considering its potential value as a tumor-associated antigenic target. Over the years, CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type. To understand its implications in cancer beside its use as an antigenic biomarker, it is essential to know its physiological functions, including its molecular partners in a given cell system. Moreover, the discovery that CD9 is one of the most specific and broadly expressed markers of extracellular membrane vesicles, nanometer-sized entities that are released into extracellular space and various physiological body fluids and play a role in intercellular communication under physiological and pathological conditions, notably the establishment of cancer metastases, has added a new dimension to our knowledge of CD9 function in cancer. Here, we will discuss these issues as well as the possible cancer therapeutic implications of CD9, their limitations, and pitfalls.

ABSTRACT Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro‐metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP‐A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy‐ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP‐A complexes, leading to inhibition of EV‐mediated pro‐metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre‐metastatic niche, small‐sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.