Tunnelling nanotube formation is driven by Eps8/IRSp53-dependent linear actin polymerization.

Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer-based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.

Seeing eye to eye: photoreceptors employ nanotube-like connections for material transfer.

Cytoskeletal protrusions are emerging as key elements in the development of cellular networks through which material is readily exchanged. In parallel studies, Ortin-Martinez et al (2021) and Kalargyrou et al (2021) report for the first time a direct transfer of cytoplasmic and membrane-bound material between photoreceptors through nanotube-like connections, providing further evidence toward the existence of nanotube-mediated material transfer in vivo within the central nervous system.

Peering into tunneling nanotubes-The path forward.

The identification of Tunneling Nanotubes (TNTs) and TNT-like structures signified a critical turning point in the field of cell-cell communication. With hypothesized roles in development and disease progression, TNTs' ability to transport biological cargo between distant cells has elevated these structures to a unique and privileged position among other mechanisms of intercellular communication. However, the field faces numerous challenges-some of the most pressing issues being the demonstration of TNTs in vivo and understanding how they form and function. Another stumbling block is represented by the vast disparity in structures classified as TNTs. In order to address this ambiguity, we propose a clear nomenclature and provide a comprehensive overview of the existing knowledge concerning TNTs. We also discuss their structure, formation-related pathways, biological function, as well as their proposed role in disease. Furthermore, we pinpoint gaps and dichotomies found across the field and highlight unexplored research avenues. Lastly, we review the methods employed to date and suggest the application of new technologies to better understand these elusive biological structures.

Actin-based protrusions at a glance.

Actin-based protrusions are at the base of many fundamental cellular processes, such as cell adhesion, migration and intercellular communication. In recent decades, the discovery of new types of actin-based protrusions with unique functions has enriched our comprehension of cellular processes. However, as the repertoire of protrusions continues to expand, the rationale behind the classification of newly identified and previously known structures becomes unclear. Although current nomenclature allows good categorization of protrusions based on their functions, it struggles to distinguish them when it comes to structure, composition or formation mechanisms. In this Cell Science at a Glance article, we discuss the different types of actin-based protrusions, focusing on filopodia, cytonemes and tunneling nanotubes, to help better distinguish and categorize them based on their structural and functional differences and similarities.

Tunneling nanotubes and tumor microtubes-Emerging data on their roles in intercellular communication and pathophysiology: Summary of an International FASEB Catalyst Conference October 2023.

In the past decade, there has been a steady rise in interest in studying novel cellular extensions and their potential roles in facilitating human diseases, including neurologic diseases, viral infectious diseases, cancer, and others. One of the exciting new aspects of this field is improved characterization and understanding of the functions and potential mechanisms of tunneling nanotubes (TNTs), which are actin-based filamentous protrusions that are structurally distinct from filopodia. TNTs form and connect cells at long distance and serve as direct conduits for intercellular communication in a wide range of cell types in vitro and in vivo. More researchers are entering this field and investigating the role of TNTs in mediating cancer cell invasion and drug resistance, cellular transfer of proteins, RNA or organelles, and intercellular spread of infectious agents, such as viruses, bacteria, and prions. Even further, the elucidation of highly functional membrane tubes called "tumor microtubes" (TMs) in incurable gliomas has further paved a new path for understanding how and why the tumor type is highly invasive at the cellular level and also resistant to standard therapies. Due to the wide-ranging and rapidly growing applicability of TNTs and TMs in pathophysiology across the spectrum of biology, it has become vital to bring researchers in the field together to discuss advances and the future of research in this important niche of protrusion biology.

CellWalker: a user-friendly and modular computational pipeline for morphological analysis of microscopy images.

SUMMARY: The implementation of computational tools for analysis of microscopy images has been one of the most important technological innovations in biology, providing researchers unmatched capabilities to comprehend cell shape and connectivity. While numerous tools exist for image annotation and segmentation, there is a noticeable gap when it comes to morphometric analysis of microscopy images. Most existing tools often measure features solely on 2D serial images, which can be difficult to extrapolate to 3D. For this reason, we introduce CellWalker, a computational toolbox that runs inside Blender, an open-source computer graphics software. This add-on improves the morphological analysis by seamlessly integrating analysis tools into the Blender workflow, providing visual feedback through a powerful 3D visualization, and leveraging the resources of Blender's community. CellWalker provides several morphometric analysis tools that can be used to calculate distances, volume, surface areas and to determine cross-sectional properties. It also includes tools to build skeletons, calculate distributions of subcellular organelles. In addition, this python-based tool contains 'visible-source' IPython notebooks accessories for segmentation of 2D/3D microscopy images using deep learning and visualization of the segmented images that are required as input to CellWalker. Overall, CellWalker provides practical tools for segmentation and morphological analysis of microscopy images in the form of an open-source and modular pipeline which allows a complete access to fine-tuning of algorithms through visible-source code while still retaining a result-oriented interface. AVAILABILITY AND IMPLEMENTATION: CellWalker source code is available on GitHub (https://github.com/utraf-pasteur-institute/Cellwalker-blender and https://github.com/utraf-pasteur-institute/Cellwalker-notebooks) under a GPL-3 license.

α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes.

The accumulation of α-synuclein (α-syn) aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson disease (PD). α-Syn aggregates propagate in a "prion-like" manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs). However, how lysosomes participate in the spreading of α-syn aggregates is unclear. Here, by using super-resolution (SR) and electron microscopy (EM), we find that α-syn fibrils affect the morphology of lysosomes and impair their function in neuronal cells. In addition, we demonstrate that α-syn fibrils induce peripheral redistribution of lysosomes, likely mediated by transcription factor EB (TFEB), increasing the efficiency of α-syn fibrils' transfer to neighboring cells. We also show that lysosomal membrane permeabilization (LMP) allows the seeding of soluble α-syn in cells that have taken up α-syn fibrils from the culture medium, and, more importantly, in healthy cells in coculture, following lysosome-mediated transfer of the fibrils. Moreover, we demonstrate that seeding occurs mainly at lysosomes in both donor and acceptor cells, after uptake of α-syn fibrils from the medium and following their transfer, respectively. Finally, by using a heterotypic coculture system, we determine the origin and nature of the lysosomes transferred between cells, and we show that donor cells bearing α-syn fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them. These findings thus contribute to the elucidation of the mechanism by which α-syn fibrils spread through TNTs, while also revealing the crucial role of lysosomes, working as a Trojan horse for both seeding and propagation of disease pathology.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

The Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes.

Tunneling nanotubes (TNTs) are actin-based transient tubular connections that allow direct communication between distant cells. TNTs play an important role in several physiological (development, immunity, and tissue regeneration) and pathological (cancer, neurodegeneration, and pathogens transmission) processes. Here, we report that the Wnt/Ca2+ pathway, an intracellular cascade that is involved in actin cytoskeleton remodeling, has a role in TNT formation and TNT-mediated transfer of cargoes. Specifically, we found that Ca2+ /calmodulin-dependent protein kinase II (CaMKII), a transducer of the Wnt/Ca2+ pathway, regulates TNTs in a neuronal cell line and in primary neurons. We identified the ß isoform of CaMKII as a key molecule in modulating TNT formation and transfer, showing that this depends on the actin-binding activity of the protein. Finally, we found that the transfer of vesicles and aggregated α-synuclein between primary neurons can be regulated by the activation of the Wnt/Ca2+ pathway. Our findings suggest that Wnt/Ca2+ pathway could be a novel promising target for therapies designed to impair TNT-mediated propagation of pathogens.

Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.

Organization of GPI-anchored proteins at the cell surface and its physiopathological relevance.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrPC, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.

Rab11a-Rab8a cascade regulates the formation of tunneling nanotubes through vesicle recycling.

Tunneling nanotubes (TNTs) are actin-enriched membranous channels enabling cells to communicate over long distances. TNT-like structures form between various cell types and mediate the exchange of different cargos, such as ions, vesicles, organelles and pathogens; thus, they may play a role in physiological conditions and diseases (e.g. cancer and infection). TNTs also allow the intercellular passage of protein aggregates related to neurodegenerative diseases, thus propagating protein misfolding. Understanding the mechanism of TNT formation is mandatory in order to reveal the mechanism of disease propagation and to uncover their physiological function. Vesicular transport controlled by the small GTPases Rab11a and Rab8a can promote the formation of different plasma membrane protrusions (filopodia, cilia and neurites). Here, we report that inhibiting membrane recycling reduces the number of TNT-connected cells and that overexpression of Rab11a and Rab8a increases the number of TNT-connected cells and the propagation of vesicles between cells in co-culture. We demonstrate that these two Rab GTPases act in a cascade in which Rab11a activation of Rab8a is independent of Rabin8. We also show that VAMP3 acts downstream of Rab8a to regulate TNT formation.

The best of both worlds-bringing together cell biology and infection at the Institut Pasteur.

Only a profound understanding of the structure and function of cells-either as single units or in the context of tissues and whole organisms-will allow a comprehension of what happens in pathological conditions and provides the means to fight disease. The Cell Biology and Infection (BCI for Biologie Cellulaire et Infection) department was created in 2002 at the Institut Pasteur in Paris to develop a research program under the umbrella of cell biology, infection biology, and microbiology. Its visionary ambition was to shape a common framework for cellular microbiology, and to interface the latter with hard sciences like physics and mathematics and cutting-edge technology. This concept, ahead of time, has given high visibility to the field of cellular microbiology and quantitative cell biology, and it has allowed the successful execution of highly interdisciplinary research programs linking a molecular understanding of cellular events with disease. Now, the BCI department embraces additional pathologies, namely cancer and neurodegenerative diseases. Here, we will portray how the integrative research approach of BCI has led to major scientific breakthroughs during the last 10 years, and where we see scientific opportunities for the near future.

Human NPCs can degrade α-syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures.

Parkinson's disease (PD) is the second most common neurodegenerative disorder whereby loss of midbrain dopaminergic neurons results in motor dysfunction. Transplantation of human induced pluripotent stem cells (iPSCs) into the brain of patients affected by PD is one of the therapeutic approaches that has gained interest to compensate for the degeneration of neurons and improve disease symptoms. However, only a part of transplanted cells can differentiate into mature neurons while the majority remains in undifferentiated state. Here we investigated whether human neuronal precursor cells (hNPCs) derived from iPSCs have an active role in α-synuclein (α-syn) pathology. Our findings demonstrate that α-syn fibrils are taken up by hNPCs and are preferentially localized in lysosomes where they can be degraded. However, α-syn fibrils are also transferred between hNPCs in a cell-to-cell contact dependent manner, and are found in tunneling nanotube (TNT)-like structures. Thus, NPCs can have a dual role in the progression of α-syn pathology, which should be considered in human transplants.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.

Glycosylphosphatidylinositol-anchored proteins: Membrane organization and transport.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of membrane proteins containing a soluble protein attached by a conserved glycolipid anchor to the external leaflet of the plasma membrane. In polarized epithelial cells, GPI-APs are predominantly sorted to the apical surface in the trans-Golgi network (TGN) by clustering in sphingolipid- and cholesterol-dependent microdomains (or rafts), which have been proposed to act as apical sorting platforms. Recent data indicate that the mechanisms of GPI-AP sorting, occurring in the Golgi, control both the membrane transport of GPI-APs and their specific activity at the apical surface of fully polarized epithelial cells. Here, we discuss the most recent findings and the factors regulating apical sorting of GPI-APs at the Golgi in polarized epithelial cells. We also underline the differences in the plasma membrane organization of GPI-APs between polarized and non-polarized cells supporting the existence of various mechanisms that control GPI-AP organization in different cell types.

α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading.

Recent evidence suggests that disease progression in Parkinson's disease (PD) could occur by the spreading of α-synuclein (α-syn) aggregates between neurons. Here we studied the role of astrocytes in the intercellular transfer and fate of α-syn fibrils, using in vitro and ex vivo models. α-Syn fibrils can be transferred to neighboring cells; however, the transfer efficiency changes depending on the cell types. We found that α-syn is efficiently transferred from astrocytes to astrocytes and from neurons to astrocytes, but less efficiently from astrocytes to neurons. Interestingly, α-syn puncta are mainly found inside the lysosomal compartments of the recipient cells. However, differently from neurons, astrocytes are able to efficiently degrade fibrillar α-syn, suggesting an active role for these cells in clearing α-syn deposits. Astrocytes co-cultured with organotypic brain slices are able to take up α-syn fibrils from the slices. Altogether our data support a role for astrocytes in trapping and clearing α-syn pathological deposits in PD.