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Journal: Journal of cell science


Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein LST1 induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found LST1 to induce nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined LST1 to recruit the actin-crosslinking protein filamin to the plasma membrane and to interact with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes.

Concepts: Membrane protein, Developmental biology, Stromule, Membrane biology, Protein, Cell membrane, Signal transduction, Cell


Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell-cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell-cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiologic morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Concepts: Claudin, Electron microscope, Electron, Fluorescence, Protein, Occludin, Tight junction, Cell membrane


Store-Operated Calcium Entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T-cell activation and mast cell degranulation. SOCE is activated through intricate coordination between the Ca(2+) sensor on the ER membrane (STIM1) and the plasma membrane channel Orai1. When Ca(2+) stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain resulting in Orai1 gating and Ca(2+) influx. Here we engineer novel inter- and intra-molecular FRET sensors in the context of the full-length membrane anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched ‘open’ conformation that exposes SOAR/CAD allowing it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted 1(st) and 3(rd) coiled-coil domains of STIM1 are not involved in maintaining the ‘closed’ inactive conformation. In addition, they argue that an amphipathic α-helix between residues 317-336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1.

Concepts: ORAI1, ER, Immune system, Cell membrane, Degranulation, Mast cell


Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules (kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.

Concepts: Eukaryote, Cell nucleus, Motor protein, Spindle apparatus, Metaphase, Cell cycle, Kinetochore, Mitosis


Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent endocytic routes exist but the molecular mechanism of each pathway remains to be uncovered. This study focuses on a clathrin-independent, dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Rac1 and its targets the p21-activated kinases (Pak) are specific regulators of this pathway, acting on cortactin and actin polymerization. Here, our study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2R endocytosis. Firstly, the inhibition of the catalytic activity of PI 3-kinase strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI 3-kinase activation, is specifically involved in IL-2R entry. The second action of PI 3-kinase is via its regulatory subunit, p85α, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85α mutant missing the Rac1 binding motif, leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85α mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85α acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85α, Rac1 and IL-2R that is enhanced by IL-2. In summary our results point out a key role of class I PI 3-kinase in IL-2R endocytosis that creates a link with IL-2 signalling.

Concepts: Eukaryote, Wortmannin, Actin, Interleukin 2, Microscopy, Endocytosis, Immune system, Signal transduction


Although the Golgi apparatus has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organization in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here we identify the Golgi apparatus inNaegleria, the first description of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species ofNaegleriaand show that nearly all are expressed in mouse-passagedN. fowlericells. We then study distribution of the Golgi markerNgCOPB by fluorescence, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immuno-electron microscopy reveals thatNgCOPB localizes to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.

Concepts: Organism, Species, Lysosome, Eukaryote, Golgi apparatus, Organelle, Endoplasmic reticulum, Cell


Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes and glycans show characteristic cell type dependent distributions. Using an immortalized human mesenchymal stromal cell (MSC) line model we show that both N- and O-glycan processing in the Golgi functionally modulate early steps of osteogenic differentiation. Inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts three weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway through reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first two days of osteogenesis we were able to phenocopy the effect of inhibiting N-glycan processing. Thus modulating the functional outcome of differentiation through glycan processing provides another layer of regulation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes.

Concepts: Cell type, Cell, DNA, Developmental biology, Mesenchymal stem cell, Cellular differentiation, Stem cell, Bone marrow


Recent advances in microscope automation provide new opportunities for high-throughput cell biology, such as image-based screening. High-complex image analysis tasks often make the implementation of static and predefined processing rules a cumbersome effort. Machine-learning methods, instead, seek to use intrinsic data structure, as well as the expert annotations of biologists to infer models that can be used to solve versatile data analysis tasks. Here, we explain how machine-learning methods work and what needs to be considered for their successful application in cell biology. We outline how microscopy images can be converted into a data representation suitable for machine learning, and then introduce various state-of-the-art machine-learning algorithms, highlighting recent applications in image-based screening. Our Commentary aims to provide the biologist with a guide to the application of machine learning to microscopy assays and we therefore include extensive discussion on how to optimize experimental workflow as well as the data analysis pipeline.

Concepts: Microscope, Cell biology, Microscopy, Gene, Computer, Scientific method, Machine learning, Biology


Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to CNS injury by a process known as astrogliosis, which involves morphological changes including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity, and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown.Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK/RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. On the other hand, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression.Our findings identify a novel morphological outcome for Arp2/3 activation in restricting rather than promoting outward movement of the plasma membrane in astrocytes. Arp2/3 regulators PICK1 and N-WASP/WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

Concepts: Gap junction, Glial scar, Actin, Myosin, Synapse, Gene expression, Nervous system, Neuron


The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks. The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions. In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs. The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease.

Concepts: Collagen, Cytoplasm, Extracellular matrix, Cell biology, Signal transduction, Integrin, Cell membrane, Protein