Observations of calcium dynamics in cortical secretory vesicles

Abstract

SUMMARY Calcium (Ca2+) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca2+ concentration in the secretory vesicles was estimated to be in the range of ~10 – 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca2+ dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca2+ signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Cav2.1) Ca2+ channels. Blocking the p-type Ca2+ channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca2+ dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca2+ indicator fluorescence. The results suggest that the free Ca2+ content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process.

Combined Targeted Omic and Functional Assays Identify Phospholipases A2 that Regulate Docking/Priming in Calcium-Triggered Exocytosis

“…In this regard, localization of the endogenous catalytically active PLA 2 isozymes that supply LPC and FFA near the docking and/or fusion site is of critical importance. The internal CV microenvironment has a pH of ~5.5 and contains ~100 mM calcium, mostly in a bound state, as has also been found in other (mammalian) secretory vesicles [30,86,99,100,101]; however, the estimated global luminal [Ca 2+ ] free is ~1–10 µM, occurring as transients, linked in part to the opening of p-type Ca 2+ channels [30]. As these are global measures, the local [Ca 2+ ] free at the CV membrane would be much higher.…”

“…In transfected CHO-K1 and HEK293 cells that lack regulated secretory vesicles, expressed sPLA 2 was localized to the golgi and related intracellular vesicles and released ARA, indicating basal activity [28]. Of note is that adequate concentrations of free calcium ([Ca 2+ ] free ) may be (transiently) present within regulated secretory vesicles to support luminal sPLA 2 activity under basal conditions [30,31,32,33]. In contrast, iPLA 2 are intracellular enzymes that do not require Ca 2+ beyond basal cytosolic levels for activity, contain serine in the GXSXG active site and possess a nucleotide-binding (GXGXXG) consensus sequence.…”

Imaging approaches to measuring lysosomal calcium

“…For the most part, this has been carried out in purified or enriched granule preparations because the cytosol is also labeled and would contaminate the vesicular signal (Gerasimenko, Gerasimenko, Belan & Petersen 1996;Nguyen, Chin, & Verdugo, 1998;Quesada et al, 2003;Raveh et al, 2012). The inference is that secretory vesicles contain esterases and retain the dyes.…”

Study on PREP localization in mouse seminal vesicles and its possible involvement during regulated exocytosis

“…, no reports have directly associated PREP with the epithelial cells of seminal vesicles (SV), exocrine glands that, together with the prostate and Cowper’s gland, constitute the male sex accessory system. During the exocytosis of molecules such as citric acid, prostaglandin, fructose and proteins, many cellular elements have an active role, such as microtubules for vesicle trafficking (Müsch, 2004; Park & Loh, 2008; Noordstra & Akhmanova, 2017) and Ca 2+ for active exocytosis (Petersen & Tepikin, 2008; Raveh et al ., 2012; Petersen, 2014). In a previous study, we reported the hypothesis that the formin DAAM1 may participate in SV physiology, suggesting its possible involvement as an actor in morphofunctional remodelling and organization of the epithelium and smooth muscle of these exocrine glands (Venditti et al ., 2018).…”