Roy Cohen

Highly Aminated Mesoporous Silica Nanoparticles with Cubic Pore Structure

Mesoporous silica with cubic symmetry has attracted interest from researchers for some time. Here, we present the room temperature synthesis of mesoporous silica nanoparticles possessing cubic Pm3n symmetry with very high molar ratios (>50%) of 3-aminopropyl triethoxysilane. The synthesis is robust allowing, for example, co-condensation of organic dyes without loss of structure. By means of pore expander molecules, the pore size can be enlarged from 2.7 to 5 nm, while particle size decreases. Adding pore expander and co-condensing fluorescent dyes in the same synthesis reduces average particle size further down to 100 nm. After PEGylation, such fluorescent aminated mesoporous silica nanoparticles are spontaneously taken up by cells as demonstrated by fluorescence microscopy.

Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis.

The coupling of voltage‐gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization‐evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion‐free solution. Cd2+, nifedipine, or verapamil inhibited depolarization‐evoked secretion in La3+, indicating specific binding of La3+ at the pore of L‐type VGCC, probably at the poly‐glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+ > Ce3+ > Pr3+, consistent with a size‐selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura‐2 imaging assay (∼1 pm) detected no La3+ entry, cytosolic La3+ build‐up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build‐up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build‐up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.

R-type voltage-gated ca2+ channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of xenopus oocytes

It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.

Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation

Summary Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca2+ mobilization, but spatio-temporal relationships between stimulated Ca2+ mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)–dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC–dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca2+ dynamics to demonstrate a spatial relationship between the sites of Ca2+ wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca2+ signaling and secretory events correlate with participation of TRPC1 channels in Ca2+ mobilization.

Ionic dependence of Ca2+ channel modulation by syntaxin 1A

Alteration of the kinetic properties of voltage-gated Ca2+ channels, Cav1.2 (Lc-type), Cav2.2 (N type), and Cav2.3 (R type), by syntaxin 1A (Syn1A) and synaptotagmin could modulate exocytosis. We tested how switching divalent charge carriers from Ca2+ to Sr2+ and Ba2+ affected Syn1A and synaptotagmin modulation of Ca2+-channel activation. Syn1A accelerated Cav1.2 activation if Ca2+ was the charge carrier; and by substituting for Ba2+, Syn1A slowed Cav1.2 activation. Syn1A also significantly accelerated Cav2.3 activation in Ca2+ and marginally in Ba2+. Synaptotagmin, on the other hand, increased the rate of activation of Cav2.3 and Cav2.2 in all permeating ions tested. The Syn1A-channel interaction, unlike the synaptotagmin-channel interaction, proved significantly more sensitive to the type of permeating ion. It is well established that exocytosis is affected by switching the charge carriers. Based on the present results, we suggest that the channel-Syn1A interaction could respond to the conformational changes induced within the channel during membrane depolarization and divalent ion binding. These changes could partially account for the charge specificity of synaptic transmission as well as for the fast signaling between the Ca2+ source and the fusion apparatus of channel-associated-vesicles (CAV). Furthermore, propagation of conformational changes induced by the divalent ions appear to affect the concerted interaction of the channel with the fusion/docking machinery upstream to free Ca2+ buildup and/or binding to a cytosolic Ca2+ sensor. These results raise the intriguing possibility that the channel is the Ca2+ sensor in the process of fast neurotransmitter release.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3s pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1s lipid and sugar components and CaV2.3s α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells

Ca2+ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca2+ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca2+ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca2+ oscillations throughout the cell that usually follow the initiating Ca2+ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca2+ puffs at the region of cell contact. Initiating Ca2+ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca2+ release from endoplasmic reticulum stores and to extracellular Ca2+, but they do not depend on store-operated Ca2+ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response.

Roles for Ca2+ Mobilization and Its Regulation in Mast Cell Functions

Mobilization of Ca2+ in response to IgE receptor-mediated signaling is a key process in many aspects of mast cell function. Here we summarize our current understanding of the molecular bases for this process and the roles that it plays in physiologically relevant mast cell biology. Activation of IgE receptor signaling by antigen that crosslinks these complexes initiates Ca2+ mobilization as a fast wave that is frequently followed by a series of Ca2+ oscillations which are dependent on Ca2+ influx-mediated by coupling of the endoplasmic reticulum luminal Ca2+ sensor STIM1 to the calcium release activated calcium channel protein Orai1. Granule exocytosis depends on this process, together with the activation of protein kinase C isoforms, and specific roles for these signaling steps are beginning to be understood. Ca2+ mobilization also plays important roles in stimulated exocytosis of recycling endosomes and newly synthesized cytokines, as well as in antigen-mediated chemotaxis of rat mucosal mast cells. Phosphoinositide metabolism plays key roles in all of these processes, and we highlight these roles in several cases.

Mitochondrial translocator protein (TSPO) function is not essential for heme biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes.

Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.