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Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 μm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular “fruiting bodies” of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium, we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems. [ABSTRACT FROM AUTHOR]

Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MWs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-termIL-4 exposure (1 hour) during phagocytosis of IgGopsonized yeast particles by MWs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomalmembranes provide topological constraints to signalingmolecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell. [ABSTRACT FROM AUTHOR]

Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion.

Signaling by the serine and threonine kinase Akt (also known as protein kinase B), a pathway that is common to all eukaryotic cells, is central to cell survival, proliferation, and gene induction. We sought to elucidate the mechanisms underlying regulation of the kinase activity of Akt in the immune system. We found that the four-transmembrane protein CD37 was essential for B cell survival and long-lived protective immunity. CD37-deficient (Cd37(-/-)) mice had reduced numbers of immunoglobulin G (IgG)-secreting plasma cells in lymphoid organs compared to those in wild-type mice, which we attributed to increased apoptosis of plasma cells in the germinal centers of the spleen, areas in which B cells proliferate and are selected. CD37 was required for the survival of IgG-secreting plasma cells in response to binding of vascular cell adhesion molecule 1 to the α(4)β(1) integrin. Impaired α(4)β(1) integrin-dependent Akt signaling in Cd37(-/-) IgG-secreting plasma cells was the underlying cause responsible for impaired cell survival. CD37 was required for the mobility and clustering of α(4)β(1) integrins in the plasma membrane, thus regulating the membrane distribution of α(4)β(1) integrin necessary for activation of the Akt survival pathway in the immune system.