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Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

Signalling endosomes represent a general mechanism for modulating and compartmentalizing cell signalling, which is achieved by delineating specific spatial environments and connecting the plasma membrane with intracellular events. The molecular composition of vesicles, together with their targeting mechanisms and endocytic routes, contributes to the outcome of signalling pathways that are initiated either at the plasma membrane or within endosomes themselves. In T cell signalling, it is now accepted that the spatial distribution of signalling proteins is central to T cell activation not only at the immunological synapse, but also in endosomes travelling to and from the plasma membrane. In addition, there is a global rearrangement of the endosome machinery upon T cell activation, and emerging experimental evidence suggests that vesicles in T cells contain key T cell signalling proteins. We review the various mechanisms by which endosomes contribute to signalling pathways and consider whether signalling endosomes play a role in T cell signalling., (© 2013 FEBS.)

Engagement of the T cell antigen receptor (TCR) triggers signaling pathways that lead to T cell selection, differentiation and clonal expansion. Superimposed onto the biochemical network is a spatial organization that describes individual receptor molecules, dimers, oligomers and higher order structures. Here we discuss recent findings and new concepts that may regulate TCR organization in naïve and memory T cells. A key question that has emerged is how antigen-TCR interactions encode spatial information to direct T cell activation and differentiation. Single molecule super-resolution microscopy may become an important tool in decoding receptor organization at the molecular level.

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis., (© 2011 John Wiley & Sons A/S.)

The exon-exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.

Pluripotency and the capability for unlimited self-renewal make human embryonic stem cells a promising tool for studying development and new cell replacement strategies. Here, we present a simple differentiation protocol, which permits the direct conversion of human embryonic stem cells into neurogenic precursors without formation of embryoid bodies or coculture with other cell types. In this protocol, human embryonic stem cells propagated as adherent cultures are induced to differentiate into the neural lineage in media containing fibroblast growth factor-2. The adherent cells are proliferated to form detaching neurospheres. Upon plating, these neurospheres give rise to a homogenous population of neural precursors capable of generating neurons, astrocytes and oligodendrocytes. Our findings suggest that fibroblast growth factor-2 exposure alone suffices to promote neural conversion of adherently growing human embryonic stem cell cultures.

Using an in vitro assay instead of an approach in vivo greatly facilitates the analysis of complex intracellular mechanisms. This becomes particularly important for studying vesicular trafficking in both the endocytic and exocytic pathways with multiple transport routes connecting cellular organelles. Our chapter describes a novel cell-free assay that reconstitutes endosomal transport to recycling endosomes. The method measures transport of transferrin, a marker for endocytosis/recycling, from an endosome-enriched donor fraction to immunoisolated Rab11-positive acceptor recycling endosomes. Transfer of acridinium-labeled transferrin is detected by a highly sensitive chemiluminescence reaction using a luminometer.

Rab GTPases are key regulators of vesicular protein transport in both the endocytic and exocytic pathways. In endocytosis and recycling, Rab11 plays a role in receptor recycling to plasma membrane via the pericentriolar recycling compartment. However, little is known about the molecular requirements and partners that promote transport through Rab11-positive recycling endosomes. Here, we report a novel approach to reconstitute transport to immunoabsorbed recycling endosomes in vitro. We show that transport is temperature-, energy-, and time-dependent and requires the presence of Rab proteins, as it is inhibited by the Rab-interacting protein Rab GDP-dissociation inhibitor that removes Rab proteins from the membrane. Cytochalasin D, a drug that blocks actin polymerization, inhibits the in vitro assay, suggesting that transport to recycling endosomes depends on an intact actin cytoskeleton. Using an affinity chromatography approach we show the identification of Rab11-interacting proteins including actin that stimulate transport to recycling endosomes in vitro.

The insulin-stimulated glucose transporter in rat adipocytes was inhibited by two protein kinase inhibitors, staurosporine (SSP) and H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). However, whereas SSP (10 microM) blocked the insulin-dependent translocation of glucose transporter, H-7 (3 mM) did not. The latter inhibited glucose transporter activity not only in cells, but also in reconstituted liposomes. On the other hand, SSP blocked both the action of insulin and the insulinomimetic action of GTP gamma S (Guanosine 5'-O-(3-thiotriphosphate)). GTP gamma S had distinct effects on the glucose transport and cAMP phosphodiesterase (PDE) activities. It is suggest that H-7 may inhibit glucose transport activity per se; a SSP sensitive protein kinases (protein kinase C isoforms?) may be involved in cascade of the insulin action on glucose transporter as modulated by GTP gamma S; and glucose transport and PDE activities may be regulated by distinct GTP gamma S-sensitive factors.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.