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Proteolytic degradation of the extracellular matrix by metastatic tumor cells is initiated by the formation of invadopodia, i.e., actin-driven filopodia-like membrane protrusions endowed with matrix-degradative activity. A signaling cascade involving neural Wiskott-Aldrich syndrome protein and the Arp2/3 actin nucleating complex is involved in actin assembly at invadopodia. Yet, the mechanism of invadopodia formation is poorly understood. Based on their role as actin nucleators in cytoskeletal rearrangements, including filopodia formation, we examined the function of Diaphanous-related formins (DRF) in invadopodia formation and invasion by breast tumor cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that three members of the DRF family (DRF1–DRF3) are required for invadopodia formation and two-dimensional matrix proteolysis. We also report that invasion of a three-dimensional Matrigel matrix involves filopodia-like protrusions enriched for invadopodial proteins, including membrane type 1 matrix metalloproteinase, which depend on DRFs for their formation. These data identify DRFs as critical components of the invasive apparatus of tumor cells in two-dimensional and three-dimensional matrices and suggest that different types of actin nucleators cooperate during the formation of invadopodia. [Cancer Res 2009;69(7):2792–800]

Supplementary Figures 1-5 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Video 6 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Video 7 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Video 4 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Video 1 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Tables 1-6 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Video 3 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Supplementary Methods, Figure Legends 1-5, Video Legends 1-7 from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.

Extracellular vesicles are now considered as active contributors to melanoma progression through their capacity to modify the tumor microenvironment and to favor the formation of a pre-metastatic niche. These prometastatic roles of tumor-derived EVs would pass through their interaction with the extracellular matrix (ECM) and its remodeling, in turn providing a substrate favoring persistent tumor cell migration. Nevertheless, the capacity of EVs to directly interact with ECM components is still questionable. In this study, we use electron microscopy and a pull-down assay to test the capacity of sEVs, derived from different melanoma cell lines, to physically interact with collagen I. We were able to generate collagen fibrils coated with sEVs and to show that melanoma cells release subpopulations of sEVs that can differentially interact with collagen.

Exosomes are small extracellular vesicles that are enriched in specific proteins and lipids during their generation as intraluminal vesicles in multivesicular endosomes. Contrary to proteins, the endosomal sorting mechanisms of lipids on intraluminal vesicles are still ill-defined. Here, we find that the tetraspanin CD63, highly enriched in ILVs and exosomes, regulates cholesterol sorting on ILVs and exosomes. This process generates a pool of cholesterol that affects exosomes physical properties and can be mobilized by the NPC1/2 complex from ILVs and exosomes. Absence of CD63 redirects cholesterol from endosomes to the Trans-Golgi Network by actin dependent vesicular transport, placing CD63 and cholesterol at the center of a balance between inward and outward budding of endomembranes. These results establish CD63 as cholesterol sorting mechanism within endosomes and place ILVs and exosomes as alternative providers of cholesterol.

Animals precisely control the morphology and assembly of highly reflective guanine crystals to produce diverse optical phenomena used in coloration and vision. However, little is known about how organisms regulate crystallization to produce optically useful crystal morphologies which express highly reflective crystal faces. Guanine crystals form inside iridosome vesicles within specialized chromatophore cells called iridophores. By following the formation of iridosomes in developing scallop eyes we show that, despite their different physical properties, the morphogenesis of iridosomes bears a striking resemblance to melanosome morphogenesis in vertebrates. A key finding is that pre-assembled intravesicular fibrillar sheets template crystal nucleation, growth, and orientation, dictating the formation of optically functional, but thermodynamically disfavored plate-like habits. The common control mechanisms for melanin and guanine formation inspire new approaches for manipulating the morphologies and properties of molecular materials.

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 (PI4KIIα and PI4KIIβ) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIβ 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 PI4KIIα and PI4KIIβ function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation toward 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.

SynopsisMelanins, the main pigments of the skin and hair in mammals, are synthesized within membrane-bound organelles of melanocytes called melanosomes. Melanosome structure and function are determined by a cohort of resident transmembrane proteins, many of which are expressed only in pigment cells and localize specifically to melanosomes. Defects in the genes that encode melanosome-specific proteins or components of the machinery required for their transport in and out of melanosomes underlie various forms of ocular or oculocutaneous albinism, characterized by hypopigmentation of the hair, skin, and eyes and by visual impairment. We review major components of melanosomes, including the enzymes that catalyze steps in melanin synthesis from tyrosine precursors, solute transporters that allow these enzymes to function, and structural proteins that underlie melanosome shape and melanin deposition. We then review the molecular mechanisms by which these components are biosynthetically delivered to newly forming melanosomes—many of which are shared by other cell types that generate cell type-specific lysosome-related organelles. We also highlight unanswered questions that need to be addressed by future investigation.

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, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. 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 β-catenin activity, revealing a function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer., Aktary et al. identify novel roles for the dynein light chain Dynlt3 in melanosome transport, maturation, and transfer to keratinocytes. They also find that the Wnt/βcatenin signalling pathway controls Dynlt3 levels and thus also contributes to the regulation of melanocyte transport and skin pigmentation.

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 μ 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.

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.

International audience; The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (“reverse topology”), but they can also be directed toward the cytosol (“normal topology”). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA + ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B–IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B–IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B–IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

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 anex vivomodel 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.

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 (PI4KIIα and PI4KIIβ) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIβ 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 PI4KIIα and PI4KIIβ 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, PI4KIIα and PI4KIIβ, sequentially generate phosphatidylinositol-4-phosphate during tubule initiation and elongation for ultimate melanosome content delivery.

International audience; 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.

Unique functions of specialised cells such as those of the immune and haemostasis systems, skin, blood vessels, lung, and bone require specialised compartments, collectively referred to as lysosome-related organelles (LROs), that share features of endosomes and lysosomes. LROs harbour unique morphological features and cell type-specific contents, and most if not all undergo regulated secretion for diverse functions. Ongoing research, largely driven by analyses of inherited diseases and their model systems, is unravelling the mechanisms involved in LRO generation, maturation, transport and secretion. A molecular understanding of these features will provide targets and markers that can be exploited for diagnosis and therapy of a myriad of diseases.

Extracellular vesicles (EVs), cell-derived membrane structures, are secreted after fusion of endosomes with the plasma membrane (exosomes) or shed from the plasma membrane (microvesicles). EVs play a key role both in physiological balance and homeostasis and in disease processes by their ability to participate in intercellular signaling and communication.

Bowman et al. show that in melanocytes, the vSNARE VAMP7 is sorted from endosomes into tubular membrane transport carriers bound for maturing melanosomes in a complex with the tSNARE syntaxin 13 via redundant recognition of each SNARE by an AP-3–BLOC-1 super-complex., Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1–dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3–BLOC-1 super-complex.

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.

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 β-catenin activity, revealing a novel function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.

The single critical output of a blockchain is creating trust where previously impossible. While this feature delivers compelling value for many use cases (bitcoin for money, standards setting and data sharing for permissioned blockchains, audit trails and protection against liability in supply chains), the most novel use case has been something unexpected: the birth of a new type of social structure to provide goods and services. The early examples of this new type of social structure have shown themselves to be incredibly effective at providing those services to their users.

Clinical research evolved side-by-side with technology, leading to exponential data generation contributing to social and economic development. Nevertheless, data storage, integrity, and privacy concerns have emerged, raising trust issues regarding data sharing. This chapter will demonstrate how blockchain technology (BT) can address these problems and help optimizing processes, minimize costs, and monetize data. It will explain why these models are not fully explored and how cryptocurrencies are advantageous compared to traditional currency. Worldwide examples of companies developing network infrastructures that rely on private players will be provided, and European cases, where consortium models that count with different partners to build health blockchain infrastructures are being developed, will be discussed. Considering the business models to be addressed under the European Union (EU) jurisdiction, a hypothetical BT-based healthcare model with potential application in the EU scenario will also be highlighted.

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 artifacts 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 minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during 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.