Cell to cell hijacking: Role of membrane nanotubes

Cell to Cell communications is the pivot for life processes. Any event that disrupts this leads to the loss of physiological function, eventually leading to cell death. Evolutionarily, cells developed an elaborate mechanism to undertake this paramount responsibility through cell surface glycocalyx, receptors, integrins, and other recognition molecules. Cells also exchange through local acting soluble mediators as well as through vesicles and exosomes. Recent development in this field led to the identification of a spectacular network of membrane process that seems to be the supremo of all that was known about cellular communications. These are called membrane nanotubes or tunneling nanotubes (TNT). Cellular communication can be subdivided into contact and contactless. The former provides more rapid and molecule transfer as compared to the latter. Tunneling nanotubes (TNTs) are a novel type of contact-based communication. TNTs are straight, thin membrane extensions connecting cells over long distances first reported in PC12 cells in 2004. TNT is believed to form from actin-based membrane protrusion. There are three different models of TNT formation. a>Protrusions from one cell grow and extend until it reaches the other cell, followed by a membrane fusion. b> Membrane protrusions grow from both cells until they meet and establish a connection c> TNT formation by cell dislodgement when cells migrate further apart from each other, and during this movement, TNT is formed. It is possible that all these three models may be operational depending on cell types and context. Tunneling nanotubes (TNT) are dynamic connections between cells, representing a novel route for cell-to-cell communication. TNT was reported in various cell types, like epithelial cells, neuronal cells, mesenchyma cells, and immune cells engaged in intercellular exchanges of molecules, subcellular organelles, and pathogen and viruses transport routes. TNT can extend up to 200 µm in length and about 50 nm to 1500 nm in diameter in macrophages. TNT can be established between similar cell types (homo-TNT) or between one cell type and another ( hetro TNT) and thus may be involved in the initiation and growth of cancer as well as dissemination of cancer cells. TNTs are also assumed to play a role in treatment resistance, e.g., in chemotherapy treatment of cancer. Recently, TNT has been used to hijack mitochondria from healthy cells by the cancer cells as a source of energy. TNT was also reported to transport miRNA and other RNA to the surrounding stroma creating an environment suitable for cancer growth. More research in this discipline is needed to understand the full function of these wonderful nanostructures.

Melatonin, tunneling nanotubes and anastasis: Cheating cell death

When healthy neurons are exposed to toxins or physiological insults such as ischemia, apoptosis is often initiated. Once underway, this mechanistically-well described process was thought to routinely run its course with the disintegration of the cell and phagocytosis of the debris. Within the last decade, the consistency of this process has been questioned. It is now known that some damaged cells can recover, i.e., they avoid death; this restoration process is referred to as anastasis. The reestablishment of a healthy cell phenotype is highly energy-requiring, so optimally functioning mitochondria are obviously beneficial during the regenerative process. Some healthy mitochondria that end up in regenerating cells are transferred there by adjacent healthier cells through tunneling nanotubes. Tunneling nanotubes generally form under stressful conditions when these micron-size tubules link adjacent cells. These tubules transfer soluble factors and organelles, including mitochondria, between the connected cells. When damaged cells receive high APT-producing mitochondria via this means, they support the ability of the cells to recover. Two recent comprehensive publications show that melatonin aids the transfer of mitochondria through nanotubes that connect neurons thereby likely assisting the recovery of the damaged recipient cell. Thus, melatonin not only protects normal neurons from damage by neutralizing the agents that initiate apoptosis, e.g., free radicals, etc., but also reverses this process once it is underway.

RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell–cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

Global analysis of human-to-mouse intercellular RNA transfer in cell culture

Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer- and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

Intercellular Propagation and Aggregate Seeding of Mutant Ataxin-1

Intercellular propagation of aggregated protein inclusions along actin-based tunneling nanotubes (TNTs) has been reported as a means of pathogenic spread in Alzheimer’s, Parkinson’s, and Huntington’s diseases. Propagation of oligomeric-structured polyglutamine-expanded ataxin-1 (Atxn1[154Q]) has been reported in the cerebellum of a Spinocerebellar ataxia type 1 (SCA1) knock-in mouse to correlate with disease propagation. In this study, we investigated whether a physiologically relevant polyglutamine-expanded ATXN1 protein (ATXN1[82Q]) could propagate intercellularly. Using a cerebellar-derived live cell model, we observed ATXN1 aggregates form in the nucleus, subsequently form in the cytoplasm, and finally, propagate to neighboring cells along actin-based intercellular connections. Additionally, we observed the facilitation of aggregate-resistant proteins into aggregates given the presence of aggregation-prone proteins within cells. Taken together, our results support a pathogenic role of intercellular propagation of polyglutamine-expanded ATXN1 inclusions.

Tunneling nanotubes and related structures: molecular mechanisms of formation and function

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.

Tunneling nanotubes provide a novel route for SARS-CoV-2 spreading between permissive cells and to non-permissive neuronal cells.

SARS-CoV-2 entry into host cells is mediated by the binding of its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor, highly expressed in several organs, but very low in the brain. The mechanism through which SARS-CoV-2 infects neurons is not understood. Tunneling nanotubes (TNTs), actin-based intercellular conduits that connect distant cells, allow the transfer of cargos, including viruses. Here, we explored the neuroinvasive potential of SARS-CoV-2 and whether TNTs are involved in its spreading between cells in vitro. We report that neuronal cells, not permissive to SARS-CoV-2 through an exocytosis/endocytosis dependent pathway, can be infected when co-cultured with permissive infected epithelial cells. SARS-CoV-2 induces TNTs formation between permissive cells and exploits this route to spread to uninfected permissive cells in co-culture. Correlative Cryo-electron tomography reveals that SARS-CoV-2 is associated with the plasma membrane of TNTs formed between permissive cells and virus-like vesicular structures are inside TNTs established both between permissive cells and between permissive and non-permissive cells. Our data highlight a potential novel mechanism of SARS-CoV-2 spreading which could serve as route to invade non-permissive cells and potentiate infection in permissive cells.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: 1)-long tunneling nanotubes that connect two or more host cells over submillimeter distances; 2)-large scale multiple cell fusion events (syncytia); and 3)-abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Our findings may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

Mechanisms of stem cells action: reality and hypotheses

Despite the long history of stem cells studying, the mechanisms of their action are still not fully understood. A classic example is the described non-hematological effect of hematopoietic stem cells (HSC), presumably due to the ability to transdifferentiation and cell fusion. The focus of clinical application in the field of regenerative medicine is mesenchymal stromal cells (MSC), and the understanding of the ways of implementfnion of their regenerative potential has evolved significantly. MSC were discovered and described as a structural unit of the HSC niche responsible for the repair of connective tissue through differentiation. However, it later turned out that they are a regulator of various processes in the body as a whole. These processes include anti-inflammatory, antifibrotic, and immunomodulatory effects, which are realized in various ways. Among these pathways, the paracrine mechanism has already been identified – the release of various growth factors, exosomes and microvesicles, the mechanism of direct transfer of mitochondria and other cellular organelles from cell to cell using tunneling nanotubes, the mechanism of efferocytosis. One of the latest discoveries in this field was the immunomodulatory effect of apoptotic MSC.In general, the study of the stem cells mechanisms of action is a living, developing field of science in which the last word will not be said soon.