Biology subjects

Raposo, G.

Publications and source records attributed to Raposo, G..

16 recordsLinked to original sources

bioRxiv2025

A novel mechanism for centrosome expulsion ensures metabolic activity in polyploid cells

Programmed polyploidy is often linked to increase cell size to support enhanced metabolism, barrier function or regeneration. In certain polyploid cells, the cytoskeleton is drastically remodeled-most notably by eliminating centrosomes. However, the purpose and mechanisms underlying centrosome elimination have remained unclear. We investigated this question in Drosophila acentrosomal salivary glands (SGs), a physiological polyploid model where cells reach high chromosome content through endoreplication. Using genetic tools, live imaging approaches, super-resolution microscopy combined with tissue clearing and electron microscopy, our study uncovers a novel centrosome elimination pathway in vivo. This process requires non-muscle myosin II (MyoII) activity and the macroautophagic machinery to drive centrosome release into the lumen of the salivary glands via autolysosomal exocytosis. Failure to eliminate centrosomes disrupts the mitochondria network, impairing respiration and ATP production. Our findings reveal a previously unknown mechanism that removes centrosomes through the secretory autophagy pathway to protect mitochondrial function and support the high metabolic demands of polyploid cells.

cell biology↗

bioRxiv2025

Keratin intermediate filaments mechanically position melanin pigments for genome photoprotection

Melanin pigments block genotoxic agents by positioning on the sun-exposed side of human skin keratinocytes nucleus. How this position is regulated and its role in genome photoprotection remains unknown. By developing a model of human keratinocytes internalizing extracellular melanin into pigment organelles, we show that keratin 5/14 intermediate filaments mechanically control the 3D perinuclear position of pigments, shielding DNA from photodamage. Imaging and microrheology in human disease-related model identify structural keratin cages surrounding pigment organelles to stiffen their microenvironment and maintain their 3D position. Optimum pigment spatialization is required for DNA photoprotection and rely on the interplay between intermediate filaments and microtubules bridged by plectin cytolinkers. Thus, the mechanically-driven proximity of pigment organelles to the nucleus is a key photoprotective parameter. Uncovering how human skin counteracts solar radiation by positioning the melanin microparasol next to the genome anticipates that dynamic spatialization of organelles is a physiological UV stress response. Short summaryMelanin pigments shield DNA from photodamage by positioning atop nuclei in skin keratinocytes. We show keratin 5/14 intermediate filaments control this 3D spatialization, forming protective cages around pigments. This positioning, together with microtubule function, optimizes genome protection, revealing cytoskeletons and organelle dynamics as a UV stress response.

cell biology↗

bioRxiv2024

Caveolin-1 and mechanical stress control the release of a pro-metastatic subpopulation of small extracellular vesicles

Mechanical forces within the tumor microenvironment critically influence cancer progression, in part by modulating extracellular vesicle (EV) dynamics. However, the molecular mechanisms by which EVs respond to mechanical cues remain poorly understood. Here, we identify caveolin-1 (Cav1), a core structural component of caveolae, specialized membrane invaginations that act as mechanosensors, as a key regulator of EV release and composition under mechanical stress. We demonstrate that both 2D osmotic shock and 3D mechanical compression significantly enhance EV secretion from cancer cells in a Cav1-dependent manner. These EVs exhibit a distinct lipid profile and are selectively enriched in proteins involved in cell adhesion, motility, wound healing and extracellular matrix remodeling, cargo that is lost upon Cav1 deletion. Functionally, EVs secreted from mechanically stressed cells preferentially accumulate in the liver in vivo and promote breast cancer cell migration and invasion in vitro, effects abolished in the absence of Cav1. Together, these findings reveal a previously unrecognized role for Cav1 in coupling mechanical stress to EV biogenesis and cargo loading, thereby promoting metastatic traits. This study underscores a novel mechanism by which mechanical cues within tumor environment regulate EV-mediated communication and suggests that Cav1-enriched EVs may contribute to pre-metastatic niche formation.

cancer biology↗

bioRxiv2023

Extracellular vesicles released by keratinocytes regulate melanosome maturation, melanocyte dendricity and pigment transfer

Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids and genetic material molecules between cells, and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthetize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes (Lo Cicero et al., Nat. Comm. 2015). However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites, mature melanosomes and their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulate pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors of cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication. SIGNIFICANCE STATEMENTOur work uncovers how keratinocyte-derived EVs control melanocyte physiology and functions. By promoting the growth of melanocyte dendrites, maturation, accumulation and peripheral positioning of pigmented melanosomes within the dendrites, and transfer of melanin to keratinocytes, EVs released by keratinocytes control crucial processes in skin photo protection. Importantly, given that dysregulation of these pathways could underlie pigment disorders, melanoma or skin carcinoma, our results open avenues to exploit keratinocyte EVs as tools for the design of new therapies to enhance the ability of melanocytes to provide skin photoprotection, and thus decrease the incidence pigmentary disorders and skin cancers.

cell biology↗

bioRxiv2022

Golgi apposition promotes the generation of specialized lysosomes in differentiated keratinocytes

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling in lysosomes and the Golgi apparatus. However, the functional significance and the molecular link between lysosome and Golgi remodeling were unknown. Here, we show that in differentiated keratinocytes, the Golgi apparatus redistributes as ministacks leading to a significant increase in total protein secretion. The Golgi ministacks establish contact with lysosomes facilitated by Golgi tethering protein GRASP65, the depletion of which is associated with the loss of Golgi-lysosome contact and malformation of lysosomes defined by their aberrant morphology, size, and function. Strikingly, these lysosomes receive secretory Golgi cargoes, contribute to the protein secretion from the Golgi, and are critically maintained by the secretory function of the Golgi apparatus. We uncovered a novel mechanism of lysosome specialization through unique Golgi-lysosome contact that likely supports high secretion from differentiated keratinocytes. Key pointsO_LICalcium induced differentiation of human keratinocytes accompanies the dispersal of functional Golgi stacks and lysosomes. C_LIO_LIDispersed Golgi stacks establish contact/physical apposition with the lysosomes. C_LIO_LIGolgi tether GRASP65 surrounds keratinocyte lysosomes and facilitates Golgi-lysosome apposition. C_LIO_LIGRASP65 depletion abolishes Golgi-lysosome apposition and accumulates morphologically altered non-degradative lysosomes. C_LIO_LILysosomes of differentiated keratinocytes receive secretory Golgi cargo and contribute to the protein secretion from the Golgi. C_LIO_LISpecialized lysosomes are maintained by the secretory function of the Golgi apparatus. C_LI

cell biology↗

bioRxiv2021

Human Cytomegalovirus modifies placental small extracellular vesicle secretion and composition towards a proviral phenotype to enhance infection of fetal recipient cells

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during human cytomegalovirus (hCMV) congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of hCMV infection on sEVs production, composition and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from either an ex vivo model of infected histocultures from early placenta or from the amniotic fluid of patients naturally infected by hCMV at the beginning of pregnancy. Based on these findings, we propose that placental sEVs could be key actors favoring viral dissemination to the fetal brain during hCMV congenital infection. Significance StatementHuman cytomegalovirus (hCMV) infection is a major issue during pregnancy, affecting 1% of births in western countries. Despite extensive research, the pathophysiology of this congenital infection remains unclear. Recently, increasing evidence point to the key role of placental small extracellular vesicles (sEVs) in materno-fetal communication during pregnancy. Here, we examined the impact of hCMV infection on the protein composition and function of placental sEVs. We observe that hCMV infection leads to major changes in placental sEV protein content. Functional studies show the ability of sEVs produced by placental infected cells to facilitate further infection of naive recipient fetal cells, notably human neural stem cells. Our study demonstrates that placental sEVs are key players of hCMV pathophysiology during congenital infection.

cell biology↗

bioRxiv2021

PI4P and BLOC-1 remodel endosomal membranes into tubules

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.

cell biology↗

bioRxiv2021

Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes

Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KII and PI4KII{beta}) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KII or PI4KII{beta} with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KII and PI4KII{beta} function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation towards melanosomes by generating local pools of PtdIns4P. The data show that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are downstream of likely redundant functions in BLOC-1-dependent tubule initiation. SUMMARYContents are delivered to maturing melanosomes from early endosomal intermediates through tubular transport carriers. Zhu et al show that two type II phosphatidylinositol kinases, PI4KII and PI4KII{beta}, sequentially generate phosphatidylinositol-4-phosphate during tubule initiation and elongation for ultimate melanosome content delivery.

cell biology↗

bioRxiv2021

Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments

Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and, second, how their catabolism is intracellularly processed. Using scanning-transmission electron microscopy, electron tomography and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.

cell biology↗

bioRxiv2021

Dynlt3, a novel fundamental regulator of melanosome movement, distribution, maturation and transfer

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, maturation and transfer. In melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional (convective) motion leading to their peripheral location in the cell, and are not fully matured. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on {beta}-catenin activity, revealing a novel function of the Wnt/{beta}-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.

cell biology↗

bioRxiv2020

Consequences of human cytomegalovirus infection on small extracellular vesicles isolated from first trimester placental histocultures

Currently, research on the use of non-invasive biomarkers as diagnosis and prognosis tools during pathological pregnancies is in full development. Among these, placenta-derived small extracellular vesicles (sEVs) are considered as serious candidates, since their composition is modified during many pregnancy pathologies. Moreover, sEVs are found in maternal serum and can thus be easily purified from a simple blood sample. In this study, we describe the isolation of sEVs from a histoculture model of first trimester placental explants. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these sEVs and defined their associated markers and ultrastructure. We next examined the consequences of infection by human cytomegalovirus on sEVs secretion and characteristics. We observed that infection led to increased levels of expression of several surface markers, without any impact on the secretion and integrity of sEVs. Our findings open the prospect for the identification of new predictive biomarkers for the severity and outcome of this congenital infection early during pregnancy, which are still sorely lacking.

cell biology↗

bioRxiv2020

Microvilli-derived Extracellular Vesicles Govern Morphogenesis in Drosophila wing epithelium

The regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo.

cell biology↗

bioRxiv2020

HPM Live μ for full CLEM workflow

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

biophysics↗

bioRxiv2020

Centrosome amplification mediates small extracellular vesicles secretion via lysosome disruption

Bidirectional communication between cells and their surrounding environment is critical in both normal and pathological settings. Extracellular vesicles (EVs), which facilitate the horizontal transfer of molecules between cells, are recognized as an important constituent of cell-cell communication. In cancer, alterations in EV secretion contribute to the growth and metastasis of tumor cells. However, the mechanisms underlying these changes remain largely unknown. Here, we show that centrosome amplification is associated with and sufficient to promote small extracellular vesicle (SEV) secretion in pancreatic cancer cells. This is a direct result due of lysosomal dysfunction, caused by increased reactive oxygen species (ROS) downstream of extra centrosomes. Defects in lysosome function promotes multivesicular body fusion with the plasma membrane, thereby enhancing SEV secretion. Furthermore, we find that SEVs secreted in response to amplified centrosomes are functionally distinct and activate pancreatic stellate cells (PSCs). These activated PSCs promote the invasion of pancreatic cancer cells in heterotypic 3-D cultures. We propose that SEVs secreted by cancer cells with amplified centrosomes influence the bidirectional communication between the tumor cells and the surrounding stroma to promote malignancy.

cell biology↗

bioRxiv2019

Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced by melanocytes into signaling cues to promote their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we found that caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released by keratinocytes. Preventing caveolae formation in melanocytes results in increased production of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, our data establish that, in melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and intercellular communication, skin pigmentation and tissue homeostasis.

cell biology↗

bioRxiv2018

Live tracking of inter-organ communication by endogenous exosomes in vivo

Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.\n\nHighlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin\n- In the YSL, syntenin regulates exosome release into the blood for their propagation\n- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells\n- Uptake is scavenger receptor and dynamin-dependent and provides trophic support\n\n\nBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

cell biology↗