Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

Structure and function of the membrane microdomains in osteoclasts.

The cell membrane structure is closely related to the occurrence and progression of many metabolic bone diseases observed in the clinic and is an important target to the development of therapeutic strategies for these diseases. Strong experimental evidence supports the existence of membrane microdomains in osteoclasts (OCs). However, the potential membrane microdomains and the crucial mechanisms underlying their roles in OCs have not been fully characterized. Membrane microdomain components, such as scaffolding proteins and the actin cytoskeleton, as well as the roles of individual membrane proteins, need to be elucidated. In this review, we discuss the compositions and critical functions of membrane microdomains that determine the biological behavior of OCs through the three main stages of the OC life cycle.

Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread.

IMPORTANCE: Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.

The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila.

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. We show in Drosophila that the zona pellucida (ZP) domain protein Piopio interacts and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. We also show conserved ectodomain shedding of the human TGFß type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gßγ/PKCα/FARP1/Cdc42 axis.

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

CYRI proteins: controllers of actin dynamics in the cellular 'eat vs walk' decision.

Cells use actin-based protrusions not only to migrate, but also to sample their environment and take up liquids and particles, including nutrients, antigens and pathogens. Lamellipodia are sheet-like actin-based protrusions involved in sensing the substratum and directing cell migration. Related structures, macropinocytic cups, arise from lamellipodia ruffles and can take in large gulps of the surrounding medium. How cells regulate the balance between using lamellipodia for migration and macropinocytosis is not yet well understood. We recently identified CYRI proteins as RAC1-binding regulators of the dynamics of lamellipodia and macropinocytic events. This review discusses recent advances in our understanding of how cells regulate the balance between eating and walking by repurposing their actin cytoskeletons in response to environmental cues.

Actin cytoskeleton and complex cell architecture in an Asgard archaeon.

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell1-3. A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated4-6, but never visualized. Here we describe a highly enriched culture of 'Candidatus Lokiarchaeum ossiferum', a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7-14 days, reaches cell densities of up to 5 × 107 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain7. ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying 'Ca. L. ossiferum' cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin-one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures.

Melatonin maintained higher contents of unsaturated fatty acid and cell membrane structure integrity in banana peel and alleviated postharvest chilling injury.

Exogenous melatonin confers the chilling tolerance of banana fruit by promoting reactive oxygen species (ROS) scavenging system and inducing the unsaturated fatty acid synthesis. The results showed that melatonin treatment increased the contents of phospholipids, promoted the ROS scavenging enzyme, and restrained the activities of lipoxygenase (LOX), and thus reduced the lipid peroxidation of banana peel. In addition, melatonin treatment increased the flavonoids and proline contents, which was conducive to antioxidant capacity. Interestingly, the enhanced antioxidant capacity is conducive to the stability of unsaturated fatty acids and reduce the enzymatic browning reaction. Moreover, melatonin treatment induced the expression of omega-3/6 fatty acid desaturase and triggered the fatty acid metabolism activity, by which maintained higher contents of unsaturated fatty acid in banana peel. Moreover, melatonin treatment stimulated the accumulation of fatty acids in banana peel, and was involved in alleviating fruit chilling injury.

Quantification of ruffle area and dynamics in live or fixed lung adenocarcinoma cells.

Ruffles are actin-rich membrane protrusions implicated in actin reorganization and initiation of cell motility. Here, we describe methods for measuring and analyzing ruffle dynamics in live cells and average ruffle area per cell in fixed samples. The specific steps described are for the analysis of A549 lung adenocarcinoma cells, but the protocol can be applied to other cell types. The protocol has applications for dissecting the signaling events linked to ruffling. For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021).

p97/UBXD1 Generate Ubiquitylated Proteins That Are Sequestered into Nuclear Envelope Herniations in Torsin-Deficient Cells.

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.

Tunneling Nanotube-Mediated Mitochondrial Transfer Rescues Nucleus Pulposus Cells from Mitochondrial Dysfunction and Apoptosis.

Stem cell-based therapy has been indicated to be beneficial for intervertebral disc regeneration. However, the underlying mechanisms have not been fully identified. The present study showed that bone marrow mesenchymal stem cells (BMSCs) donated mitochondria to adjacent nucleus pulposus cells (NPCs) in a coculture system. The mode of mitochondrial transfer between these cells was intercellular tunneling nanotube (TNT), which acted as a transportation expressway for mitochondria. NPCs acquired additional mitochondria from BMSCs in a concentration-dependent manner after rotenone-induced mitochondrial dysfunction in NPCs. Further research demonstrated that TNT-mediated mitochondrial transfer rescued NPCs from mitochondrial dysfunction and apoptosis, which was indicated by the recovery of the mitochondrial respiratory chain, the increase in mitochondrial membrane potential, and the decreases in reactive oxygen species (ROS) levels and apoptosis rates. Furthermore, Miro1, a critical protein that regulates mitochondrial movement, was knocked down in BMSCs and significantly reduced mitochondrial transfer from BMSCs to NPCs. These results suggested that Miro1 depletion inhibited the rescue of NPCs with mitochondrial dysfunction. Taken together, our data shed light on a novel mechanism by which BMSCs rescue impaired NPCs, providing a concrete foundation to study the critical role of intercellular interactions in disc regeneration.

Tunneling Nanotubes: A New Target for Nanomedicine?

Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.

Stem cells from human exfoliated deciduous teeth (SHED) have mitochondrial transfer ability in stromal-derived inducing activity (SDIA) co-culture system.

Stem cells from human exfoliated deciduous teeth (SHED) have stromal-derived inducing activity (SDIA): which means these stromal cells induce neural differentiation where they are used as a substratum for embryonic stem cell (ESCs) culture. Recent studies show that mitochondria or mitochondrial products, as paracrine factors, can be released and transferred from one cell to another. With this information, we were curious to know whether in the SDIA co-culture system, SHED release or donate their mitochondria to ESCs. For this purpose, before co-culture, SHED s' mitochondria and ESCs s' cell membranes were separately labeled with specific fluorescent probes. After co-culture, SHED s' mitochondria were tracked by fluorescent microscope and flow cytometry analysis. Co-culture also performed in the presence of inhibitors that block probable transfer pathways suchlike tunneling nanotubes, gap junctions or vesicles. Results showed that mitochondrial transfer takes place from SHED to ESCs. This transfer partly occurs by tunneling nanotubes and not through gap junctions or vesicles; also was not dependent on intracellular calcium level. This kind of horizontal gene transfer may open a new prospect for further research on probable role of mitochondria on fate choice and neural induction processes.

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.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton-Supported Intercellular Bridges Requires mTOR and CDC42 Signalling.

Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of senescent cells with increasing age is thought to contribute adversely to the development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell-autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of proinflammatory cytokines, chemokines, and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges-or tunnelling nanotubes (TNTs)-containing the cytoskeletal components actin and tubulin, which form direct physical connections between cells. We observe the presence of mitochondria in these TNTs and show organelle transfer through the TNTs to adjacent cells. While transport of individual mitochondria along single TNTs appears by time-lapse studies to be unidirectional, we show by differentially labelled co-culture experiments that organelle transfer through TNTs can occur between different cells of equivalent cell age, but that senescent cells, rather than proliferating cells, appear to be predominant mitochondrial donors. Using small molecule inhibitors, we demonstrate that senescent cell TNTs are dependent on signalling through the mTOR pathway, which we further show is mediated at least in part through the downstream actin-cytoskeleton regulatory factor CDC42. These findings have significant implications for the development of senomodifying therapies, as they highlight the need to account for local direct cell-cell contacts as well as the SASP in order to treat cancer and diseases of ageing in which senescence is a key factor.

Membranous Structures Directly Come in Contact With p62/SQSTM1 Bodies.

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.

Tunneling nanotubes: A novel pharmacological target for neurodegenerative diseases?

Diversiform ways of intercellular communication are vital links in maintaining homeostasis and disseminating physiological states. Among intercellular bridges, tunneling nanotubes (TNTs) discovered in 2004 were recognized as potential pharmacology targets related to the pathogenesis of common or infrequent neurodegenerative disorders. The neurotoxic aggregates in neurodegenerative diseases including scrapie prion protein (PrPSc), mutant tau protein, amyloid-beta (Aß) protein, alpha-synuclein (α-syn) as well as mutant Huntington (mHTT) protein could promote TNT formation via certain physiological mechanisms, in turn, mediating the intercellular transmission of neurotoxicity. In this review, we described in detail the skeleton, the formation, the physicochemical properties, and the functions of TNTs, while paying particular attention to the key role of TNTs in the transport of pathological proteins during neurodegeneration.

RNA transfer through tunneling nanotubes.

It was already suggested in the early '70's that RNA molecules might transfer between mammalian cells in culture. Yet, more direct evidence for RNA transfer in animal and plant cells was only provided decades later, as this field became established. In this mini-review, we will describe evidence for the transfer of different types of RNA between cells through tunneling nanotubes (TNTs). TNTs are long, yet thin, open-ended cellular protrusions that are structurally distinct from filopodia. TNTs connect cells and can transfer many types of cargo, including small molecules, proteins, vesicles, pathogens, and organelles. Recent work has shown that TNTs can also transfer mRNAs, viral RNAs and non-coding RNAs. Here, we will review the evidence for TNT-mediated RNA transfer, discuss the technical challenges in this field, and conjecture about the possible significance of this pathway in health and disease.

Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.