The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.
The harmonious orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions, called tunneling nanotubes, that allow for the intercellular transport of various cargoes, including viruses, organelles, and proteins between the cytoplasm of distant cells in vitro and in vivo. Over the last decade, studies have focused on their functional role but information regarding their structure and the differences with other cellular protrusions such as filopodia, is still lacking.Here, we report the structural characterization of tunneling nanotubes using correlative light-and cryo-electron microscopy approaches. We demonstrate their structural identity compared to filopodia by showing that they are comprised of a bundle of functional individual Tunneling Nanotubes containing membrane-bound compartments and allowing organelle transfer. effectively improved our understanding of these novel structures and underscored their role in cellto-cell communication, facilitating the bi-and uni-directional transfer of compounds between cells, including: organelles, pathogens, ions, genetic material, and misfolded proteins 5 . Altogether, in vitro and in vivo evidence has shown that TNTs can be involved in many different processes such as stem cell differentiation, tissue regeneration, neurodegenerative disorders, immune response, and cancer 2,6-11 .Although these in vitro and in vivo studies have been informative, the structural complexity of TNTs remains largely unknown. As a result, TNTs have been regarded with skepticism by one part of the scientific community 5,12 . Two major issues that remain to be clarified are whether these protrusions are different from other previously studied cellular processes such as filopodia 13 and whether their function in allowing the exchange of cargos between distant cells is due to direct communication between the cytoplasm of distant cells or to a classic exo-endocytosis process or a trogocytosis event 14 .Addressing these questions has been difficult due to considerable technical challenges in preserving the ultrastructure of TNTs for electron microscopy (EM) studies. To date, the ultrastructure of TNTs using Scanning and Transmission EM (SEM and TEM, respectively) has only been analyzed in a handful of articles 1,15-18 .Although very similar under fluorescence microscopy (FM), TNT formation appears to be oppositely regulated by the same actin modifiers that act on filopodia 19 . Furthermore, filopodia have not been shown to allow cargo transfer 13,20,21 . Thus, we hypothesize that TNTs might be different organelles from filopodia and display structural differences in morphology and actin architecture.In order to compare the ultrastructure and actin architecture of TNTs and filopodia at high resolution and ensure that the structures identified by TEM/SEM represent the functional units observed by FM, we employed a combination of live imaging, correlative light-and cryo-electron tomog...
Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique. IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of “Molliviridae,” which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.
Although vaccinia virus (VACV) is the best studied poxvirus, the structure of the mature virus (MV) remains poorly understood. Its asymmetric shape, size and compactness poses a major challenge for electron microscopy (EM) analysis, including cryoEM. Sub-viral particles, in particular membrane-free viral cores, may overcome these limitations. We compare cores obtained by detergent-stripping MVs with cores in the cellular cytoplasm, early in infection. By combining cryo-electron tomography (cryoET), subtomogram averaging (STA) and AlphaFold2 (AF2), abundant core-structures are analyzed, focusing on the prominent palisade layer located on the core surface. On detergent-stripped cores, the palisade is composed of densely packed trimers of the major core protein A10. They display a random order on the core surface and classification of the trimers indicate structural flexibility. On cytoplasmic cores A10 is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. CryoET and STA also uncover unexpected details of the layers beneath the palisade both on in vitro and in situ cores, that are compared to AF2 structure predictions of known VACV core-associated proteins. Altogether, our data identify for the first time the structure and molecular composition of the palisade units. The results are discussed in the context of the VACV replicative cycle, the assembly and disassembly of the infectious MV.
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