Mesoporous silica with cubic symmetry has attracted interest from researchers for some times. 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, e.g. for 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 uptaken by cells as demonstrated by fluorescence microscopy.Significant research efforts in recent years have been devoted to the development of nanoparticles for applications in biomedical imaging, sensing and drug delivery.1 -4 Nanoparticle architecture and composition are key to the achievable property profiles. Silica is one of the most studied nanoparticle matrix materials due to low toxicity, versatile bulk and surface chemistry and biocompatibility.5 -9 Ordered mesoporous silica in particular has attracted considerable interest due to its ability to reversibly load other compounds. It provides high surface area and large pore volume, necessary in sorption and catalysis applications, while maintaining the intrinsic properties of silica.10 , 11 Mesoporous siliceous materials with three-dimensional pore systems, such as MCM-48, provide advantages in diffusion and transport over one-dimensional channel systems such as in MCM-41-type materials.6 , 11 , 12Among several three-dimensional mesoporous structures reported, mesoporous silica with cubic Pm3n symmetry and possessing a cage-type structure that is three-dimensionally interconnected with small open windows is a promising material, e.g. as carrier for biologically active molecules.12 , 13 Compared to cubic MCM-48 materials only a few studies report on Pm3n -type silica.14 -18 Recently, amine functionalized Pm3n mesoporous ubw1@cornell.edu. Supporting Information Available: Detailed experimental methods, characterizations, and fluorescent imaging procedure. This method is available free of charge via the Internet at http://pubs.acs.org. Herein, we report the room temperature synthesis of discrete, faceted Pm3n highly aminated mesoporous silica nanoparticles (NH 2 -MSNs), from 54 mol % APTES. To our surprise, the synthesis protocol is quite robust allowing the co-condensation of other functional moieties in the same synthesis, e.g. organic dyes, without appreciable loss of structure control. We further demonstrate that the addition of pore expander 1,3,5-trimethylbenzene (TMB) to the synthesis increases pore size from 2.7 to 5 nm while decreasing overall particle size. Rendering these highly aminated, pore-expanded particles fluorescent by co-condensing organic dyes into the particles reduces particle size even further, down to about 100 nm, th...

Abstract

Summary 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 Ca2+ influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3’s pore-forming α1E subunit showed altered Ca2+ responses, reduced AE, and a strong sub-fertility phenotype. Surprisingly, AE depended on spatio-temporal information encoded by flux through CaV2.3—not merely the presence/ amplitude of Ca2+ waves. Using both studies in sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1’s lipid and sugar components and CaV2.3’s α1E and α2δ subunits. Our results provide mechanistic understanding of membrane lipid regulation of Ca2+ flux and therefore Ca2+-dependent cellular and developmental processes such as exocytosis and fertilization.

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.

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.