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Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease. Brain tumours are difficult to treat because of their propensity to infiltrate brain tissue; here long processes, or tumour microtubes, extended by astrocytomas are shown to promote brain infiltration and to create an interconnected network that enables multicellular communication and that protects the tumours from radiotherapy-induced cell death, suggesting that disruption of the network could be a new therapeutic approach. Microtube network protects tumours from therapeutics One of the factors making astrocyte-derived brain tumors difficult to treat is their tendency to infiltrate brain tissue. Frank Winkler and colleagues show that the long processes, or tumour microtubes, extended by astrocytomas promote brain infiltration and create an interconnected network that enables multicellular communication and protects the tumours from radiotherapy-induced cell death. The neuronal growth-associated protein 43 is identified as an important factor in this process. Disruption of the network of astrocytoma cell by targeting their tumour microtubes could be a new therapeutic approach.

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 architecture of functional TNTs is still under debate. Here, the authors combine correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structure of TNTs in neuronal cells, showing that they form structures that are distinct form other membrane protrusions.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1 + cells) regulate immune responses 1 – 5 . CX3CR1 + cells take up luminal antigens by protruding their dendrites into the lumen 1 – 4 , 6 . However, it remains unclear how dendrite protrusion by CX3CR1 + cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1 + cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1 + cells, showed defective dendrite protrusions of CX3CR1 + cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1 + cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1 + cells of wild-type mice, but not of Gpr31b −/− mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1 + cells in the small intestine of wild-type mice, but not in that of Gpr31b −/− mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1 + cells. In the mouse intestine, pyruvate and lactate produced from bacterial metabolites enhance immune responses through inducing dendrite protrusion, mediated by GPR31, of small intestinal mononuclear cells that express CX3CR1.

Most human cells require anchorage for survival. Cell–substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis—a form of programmed cell death 1 . Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue 2 , 3 . In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs 4 – 11 . Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios 12 . Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K—well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance. A study demonstrates that sustained membrane blebs in cancer cells recruit curvature-sensing septins that form plasma membrane-proximal signalling hubs that promote cancer cell survival.

Key Points Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane. They act as sites of attachment to — and degradation of — the extracellular matrix. These structures contain actin regulators such as cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), adaptor proteins such as Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and several pericellular proteases. Podosomes are found in vascular smooth muscle and endothelial cells, as well as in cells derived from monocyte lineages. Their presence correlates with migratory ability. Invadopodia are found in invasive human cancer cells. In two-dimensional culture, their presence correlates with invasive behaviour. However, in three-dimensional culture and in vivo , invadopodium-associated proteins are also required for cell growth. Podosome-associated proteins have been implicated in human developmental and immune disorders, and dysregulation of podosome formation is associated with atherosclerosis. Small-molecule regulation of podosomes and invadopodia might represent a new therapeutic strategy to treat several diseases. Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. Progress has been made in our understanding of the regulation and function of these structures, and their role in human disease. Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.

“Frustrated” coated pits associate with collagen fibers and appear to promote cell adhesion in three-dimensional collagen networks. Clathrin-coated pits are well known to be involved in receptor-mediated endocytosis. Independent of their role in endocytosis, Elkhatib et al. observed that clathrin-coated structures strongly accumulated along collagen fibers in migrating cells. Clathrin-coated structures assembled on and then partially wrapped around and pinched the fibers. In a three-dimensional (3D) network, this mechanism provided multiple anchoring points along cellular protrusions. In the absence of clathrin-coated structures, protrusions were shorter and migration was impaired. This mode of adhesion may cooperate with classical focal adhesions to help cancer cells move in a 3D environment. Science , this issue p. eaal4713 Migrating cells often use focal adhesions in order to move. Focal adhesions are less prominent in cells migrating in three-dimensional (3D) as compared with 2D environments. We looked for alternative adhesion structures supporting cell migration. We analyzed the dynamics of clathrin-coated pits in cells migrating in a 3D environment of collagen fibers. Both topological cues and local engagement of integrins triggered the accumulation of clathrin-coated structures on fibers. Clathrin/adaptor protein 2 (AP-2) lattices pinched collagen fibers by adopting a tube-like morphology and regulated adhesion to fibers in an endocytosis-independent manner. During migration, tubular clathrin/AP-2 lattices stabilized cellular protrusions by providing anchoring points to collagen fibers. Thus, tubular clathrin/AP-2 lattices promote cell adhesion that, in coordination with focal adhesions, supports cell migration in 3D.

The eukaryotic pathway of galactolipid synthesis involves fatty acid synthesis in the chloroplast, followed by assembly of phosphatidylcholine (PC) in the endoplasmic reticulum (ER), and then turnover of PC to provide a substrate for chloroplast galactolipid synthesis. However, the mechanisms and classes of lipids transported between the chloroplast and the ER are unclear. PC, PC-derived diacylglycerol, phosphatidic acid, and lyso-phosphatidylcholine (LPC) have all been implicated in ER-to-chloroplast lipid transfer. LPC transport requires lysophosphatidylcholine acyltransferase (LPCAT) activity at the chloroplast to form PC before conversion to galactolipids. However, LPCAT has also been implicated in the opposite chloroplast-to-ER trafficking of newly synthesized fatty acids through PC acyl editing. To understand the role of LPC and LPCAT in acyl trafficking we produced and analyzed the Arabidopsis (Arabidopsis thaliana) act1 lpcat1 lpcat2 triple mutant. LPCAT1 and LPCAT2 encode the major lysophospholipid acyltransferase activity of the chloroplast, and it is predominantly for incorporation of nascent fatty acids exported form the chloroplast into PC by acyl editing. In vivo acyl flux analysis revealed eukaryotic galactolipid synthesis is not impaired in act1 lpcat1 lpcat2 and uses a PC pool distinct from that of PC acyl editing. We present a model for the eukaryotic pathway with metabolically distinct pools of PC, suggesting an underlying spatial organization of PC metabolism as part of the ER–chloroplast metabolic interactions.

The intracellular bacterial pathogen Listeria monocytogenes is shown to exploit efferocytosis—the process by which dead or dying cells are removed by phagocytosis—to promote cell-to-cell spread during infection. Phagocytosis coopted by bacterial pathogens This study of the intracellular bacterial pathogen Listeria monocytogenes , a significant cause of foodborne illness, shows that it exploits the host's efferocytosis system to promote cell-to-cell spread during infection. Efferocytosis is the process by which dead or dying cells are removed by phagocytosis and it relies in part on the receptors that bind to exofacial phosphatidylserine on the surface of cells or cellular debris following the loss of plasma membrane asymmetry. Listerial actin-based motility leads to the formation of protrusions at the cell surface of infected cells, eventually leading to uptake of bacteria by adjacent cells. These findings identify phosphatidylserine as a possible drug target in infections by L. monocytogenes and other bacteria using similar strategies of cell-to-cell spread during infection. Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity 1 . Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes , can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes 2 . Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4 −/− mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.

Key Points Cofilin is an actin-binding protein that can influence actin dynamics to regulate the initiation and shape of cell protrusions. Cofilin severing activity can generate free filament ends that are accessible to G-actin, thus triggering actin polymerization and actin depolymerization without changing the rate constants for actin monomer association and dissociation at either filament end. The cofilin activity cycle includes several activation–inactivation steps that need to be spatially and temporally regulated by different proteins in order to achieve efficient cell motility. Several molecules are involved in the activation of cofilin at protrusions, including Na + –H + exchanger 1 (NHE1), phospholipase C (PLC) and cortactin, which contribute to the first activation step of cofilin. Cofilin can also be activated upon dephosphorylation by phosphatases such as Slingshot (SSH) and chronophin (CIN). Cofilin is inactivated by LIM-domain kinase (LIMK)- and TES kinase (TESK)-mediated phosphorylation at Ser3. Visualization of cofilin activity in live cells is crucial to understand its biological function. Cofilin activity can be studied in vivo using different techniques such as FRAP (fluorescence recovery after photobleaching), FRET (fluorescence resonance energy transfer), FLIP (fluorescence loss in photobleaching), BiFC (bimolecular fluorescence complementation), barbed end assays, PLA (proximity ligation assay) and cofilin uncaging. Cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization. The combination of structural data for filament severing with recently discovered mechanisms for cofilin activation in migrating cells is increasing our understanding of how cofilin activity affects cell behaviour. Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.