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


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


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


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


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


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


A hallmark of eukaryotic cells is the spatial separation of molecular and biochemical processes into membrane-bound organelles, such as mitochondria, endoplasmic reticulum and Golgi. At the ‘Cell dynamics: organelle-cytoskeleton interface’ meeting held in Lisbon, researchers from around the world discussed their findings of how the cytoskeleton regulates dynamics, interaction, and function of organelles in health and disease. Organised by Edgar Gomes, Heidi McBride, Sharon Tooze and Michael Way, the meeting created an open, stimulating and collaborative environment for scientific exchange and an opportunity to highlight the newest trends in the field.


Eukaryotic cells respond to an overload of unfolded proteins in the endoplasmic reticulum (ER) by activating signaling pathways that are referred to as the unfolded protein response (UPR). Much UPR research has been conducted in cultured cells that exhibit no baseline UPR activity until they are challenged by ER stress initiated by chemicals or mutant proteins. At the same time, many genes that mediate UPR signaling are essential for the development of organisms ranging from Drosophila and fish to mice and humans, indicating that there is physiological ER stress that requires UPR in normally developing animal tissues. Recent studies have elucidated the tissue-specific roles of all three branches of UPR in distinct developing tissues of Drosophila, fish and mammals. As discussed in this Review, these studies not only reveal the physiological functions of the UPR pathways but also highlight a surprising degree of specificity associated with each UPR branch in development.


Recycling endosomes are stations that sort endocytic cargoes to their appropriate destinations. Tubular endosomes have been characterized as a recycling endosomal compartment for clathrin-independent cargoes. However, the molecular mechanism by which tubular endosome formation is regulated is poorly understood. In this study we identified Rab10 as a novel protein localized at tubular endosomes by using a comprehensive localization screen of EGFP-tagged Rab small GTPases. Knockout of Rab10 completely abolished tubular endosomal structures in HeLaM cells. We also identified kinesin motors KIF13A/B as novel Rab10-interacting proteins by means of in silico screening. The results of this study demonstrated that both the Rab10-binding homology domain and the motor domain of KIF13A are required for Rab10-positive tubular endosome formation. Our findings provide insight into the mechanism by which the Rab10-KIF13A/B complex regulates tubular endosome formation.


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