Release Of Vesicle Cargo Research Articles

The Arabidopsis SNARE complex genes regulate the early stages of pollen-stigma interactions.

The VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigma's pollen responses. In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen-stigma interactions.

Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy.

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

Near‐Infrared Light Triggered‐Release in Deep Brain Regions Using Ultra‐photosensitive Nanovesicles

AbstractRemote and minimally‐invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold‐coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad‐PC‐Rad as a tool for the delivery of bioactive molecules into brain tissue. Near‐infrared picosecond laser pulses activated the gold‐coating on the surface of nanovesicles, creating nanomechanical stress and leading to near‐complete vesicle cargo release in sub‐seconds. Compared to natural phospholipid liposomes, the photo‐release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Imaging the Rapid Recruitment of Dynamins at the Exocytic Fusion Pore

The size and shape of the exocytic fusion pore directly modulates the release of vesicle cargoes like insulin, hormones, and neurotransmitters. Though a number of proteins have been implicated in fusion pore regulation, the molecular details of this process and how these proteins work together to shape the membrane are largely unknown. Using TIRF microscopy in living INS-1 cells we directly image the coincident and transient recruitment of dynamin proteins, the regulatory lipid PIP2, and BAR domain proteins amphiphysin, syndapin, and endophilin to exocytic sites. Recruitment occurs at the moment of membrane fusion. Evidence from imaging and siRNA depletion of endogenous dynamins suggests that dynamin-1 and dynamin-2 have distinct functional roles at sites of exocytosis. We show that perturbing dynamin-1 interaction with PIP2 or amphiphysin blocks its recruitment to exocytic sites and has functional effects on vesicle cargo release. We turn to stimulated emission depletion (STED) super-resolution fluorescence microscopy to directly visualize the organization of dynamins at sites of exocytosis in living cells. The spatiotemporal coincident recruitment of dynamins, PIP2, and BAR domain proteins, and their known interactions, strongly suggest these molecules form a complex at the nascent exocytic fusion pore. Such a protein and lipid assembly represents a novel paradigm for controlling fusion pore size and shape and directly modulating endocrine cargo release.

Amyotrophic Lateral Sclerosis IgGs Enhance the Mobility of Lysotracker-Labelled Vesicles in Cultured Rat Astrocytes

We examined the effect of purified immunoglobulins G (IgG) from amyotrophic lateral sclerosis (ALS) patients on the mobility and exocytotic release from Lysotracker-stained vesicles in cultured rat astrocytes. Time-lapse confocal images were acquired and vesicle mobility analyzed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. At rest, when mobility was monitored for 2 minutes in bath with Ca2+, two vesicle populations were discovered: i) non-mobile vesicles (6.1%) with total track length (TL) 1 μm, averaging at 3.03±0.01 μm (n=20200). ALS IgG (0.1 mg/ml) from 12 out of 13 patients increased the TL of mobile vesicles by ∼24% and maximal displacement (MD) by ∼26% within 4 minutes, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca2+, indicating that ALS-IgG vesicle mobility-enhancement involves changes in Ca2+ homeostasis. To examine, if enhanced mobility relates to elevated Ca2+ activity, cells were stimulated by 1 mM ATP, a cytosolic Ca2+ increasing agent, in the presence (2 mM) and in the absence of extracellular Ca2+. ATP stimulation triggered an increase in TL by ∼7% and ∼12%, and a decrease in MD by ∼11% and ∼1%, within 4 minutes respectively. Interestingly none of the stimuli triggered the release of vesicle cargo. It is concluded that ALS IgG-enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.

Heat Profiling of Three-Dimensionally Optically Trapped Gold Nanoparticles using Vesicle Cargo Release

Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature sensitive permeability. The surface temperature can increase by hundreds of degrees Celsius even at moderate laser powers. Also, there are significant differences of the heat profiles in two-dimensional and three-dimensional trapping assays.

Cell Secretion: Current Structural and Biochemical Insights

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

Fusion Pore Regulation of Peptidergic Vesicles

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus. This process begins with the formation of a fusion pore, an aqueous channel between a spherical vesicle and the nearly “flat” plasma membrane, through which cargo molecules diffuse into the cell exterior. After formation the fusion pore can expand, leading to the complete merger of the vesicle membrane and releasing all of the vesicle cargo (full fusion exocytosis). On the other hand, fusion pore can relatively swiftly close, allowing only partial release of vesicle cargo and retaining vesicle physical integrity (transient exocytosis). We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. Perifused cells spontaneously released prolactin at room temperature, and hypotonicity evoked a transient increase in prolactin release, followed by a sustained depression, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently-labelled atrial natriuretic peptide cargo from ∼2% of vesicles/cell, while KCl-induced depolarization resulted in a response of ∼10% of vesicles/cell, with different unloading/loading time-course of the two fluorescent probes. High resolution changes of membrane capacitance were recorded in both, unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, since over 95% of fusion events were transient. However, stimulation prolonged the average pore dwell-time (hypotonicity and KCl depolarization for 25%, respectively), increased the frequency of occurrence (hypotonicity for 35%, KCl depolarization tenfold) and the fusion pore conductance (hypotonicity and KCl depolarization for 50%, respectively). Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that transient exocytosis appears to be the dominant mode of exocytosis at spontaneous as well as at stimulated conditions.

Insulinoma-Associated Protein IA-2, a Vesicle Transmembrane Protein, Genetically Interacts with UNC-31/CAPS and Affects Neurosecretion in Caenorhabditis elegans.

IA-2 (insulinoma-associated protein 2), a major autoantigen in type 1 diabetes, is a receptor-tyrosine phosphatase-like protein associated with the membrane of secretory granules of neural and endocrine-specific cells. Loss of IA-2 activity in the mouse results in reduced insulin release and additional phenotypes, consistent with a general effect on neurosecretion and hormone release. To gain further insight into the cellular mechanisms of IA-2 function, we have studied the Caenorhabditis elegans homolog, CeIA-2 encoded by the ida-1 gene. Using two independent putative null alleles of ida-1, we demonstrate that animals lacking CeIA-2 activity are viable and exhibit subtle defects. Genetic studies of mutants in ida-1 and several genes involved in neurosecretory vesicle cargo release and signaling highlight two roles for CeIA-2. First, CeIA-2 has a specific and novel genetic interaction with UNC-31/CAPS, a protein that has been shown in other systems to regulate dense-core vesicle cargo release. Second, loss of CeIA-2 activity enhances weak alleles in the insulin-like signaling pathway. These results suggest that CeIA-2 may be an important factor in dense-core vesicle cargo release with parallels to insulin signaling in mammals.