Multiple Mechanisms Driving F-actin-Dependent Transport of Organelles to and From Secretory Sites in Bovine Chromaffin Cells.

Neuroendocrine chromaffin cells represent an excellent model to study the molecular mechanisms associated with the exo-endocytotic cycle of neurotransmitter release. In this study, EGFP-Lifeact and confocal microscopy has been used to analyze the re-organization of the cortical F-actin cytoskeleton associated to organelle transport during secretion with unprecedented detail. In these cells secretory events accumulate in temperature-sensitive and myosin II-dependent F-actin expansions and retractions affecting specific regions of the sub-membrane space. Interestingly, not only vesicles but also mitochondria are transported toward the plasmalemma during these expansions. Simultaneously, we found F-actin cytoskeletal retraction withdraws vesicles from the sub-plasmalemmal space, forming novel empty internal spaces into which organelles can be transported. In addition to these well-coordinated, F-actin-myosin II dependent processes that drive the transport of the majority of vesicles, fast transport of chromaffin vesicles was observed, albeit less frequently, which used F-actin comet tails nucleated from the granular membrane. Thus, upon cell stimulation F-actin structures use diverse mechanisms to transport organelles to and from the membrane during the exo-endocytotic cycle taking place in specific areas of cell periphery.

The Differential Organization of F-Actin Alters the Distribution of Organelles in Cultured When Compared to Native Chromaffin Cells.

Cultured bovine chromaffin cells have been used extensively as a neuroendocrine model to study regulated secretion. In order to extend such experimental findings to the physiological situation, it is necessary to study mayor cellular structures affecting secretion in cultured cells with their counterparts present in the adrenomedullary tissue. F-actin concentrates in a peripheral ring in cultured cells, as witnessed by phalloidin-rodhamine labeling, while extends throughout the cytoplasm in native cells. This result is also confirmed when studying the localization of α-fodrin, a F-actin-associated protein. Furthermore, as a consequence of this redistribution of F-actin, we observed that chromaffin granules and mitochondria located into two different cortical and internal populations in cultured cells, whereas they are homogeneously distributed throughout the cytoplasm in the adrenomedullary tissue. Nevertheless, secretion from isolated cells and adrenal gland pieces is remarkably similar when measured by amperometry. Finally, we generate mathematical models to consider how the distribution of organelles affects the secretory kinetics of intact and cultured cells. Our results imply that we have to consider F-actin structural changes to interpret functional data obtained in cultured neuroendocrine cells.

F-actin cytoskeleton and the fate of organelles in chromaffin cells.

In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss the role of transport systems and the existence of an F-actin cortical structure as the main factors behind the formation of organelle subpopulations in this neuroendocrine cell model. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015). Cover image for this issue: doi: 10.1111/jnc.13322.

The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

Role of protease-activated receptor 2 in lung injury development during acute pancreatitis in rats.

OBJECTIVE: The objective of this study was to evaluate whether an uncontrolled activation of mast cells and macrophages through protease-activated receptor-2 (PAR-2) during acute pancreatitis could develop lung injury. METHODS: Pancreatitis was induced in rats by intraductal infusion of sodium taurocholate. In a group of animals, PAR-2 antagonist or trypsin (TRP) inhibitor was intravenously administered before the pancreatitis induction. In additional groups, the animals were treated with PAR-2-activating peptide or pancreatic TRP. The myeloperoxidase (MPO) activity was measured to evaluate the progression of inflammation. RESULTS: Plasma from the animals with pancreatitis and pancreatic TRP induced the secretion of mast cells and alveolar macrophages as well as increased the density of PAR-2 in the plasma membrane. The treatment of alveolar macrophages with TRP, tryptase, as well as PAR-1- and PAR-2-activating peptide led to an increase in calcium-triggered exocytosis. Similar results were obtained in acinar cells. The intravenous injection of PAR-2-activating peptide and TRP induced an increase in MPO activity in the lung. The intravenous injection of PAR-2 antagonist or TRP inhibitor before the pancreatitis induction could prevent the increase in MPO activity in the pancreas and the lung. CONCLUSIONS: The TRP generated during acute pancreatitis could be involved in the progression of lung injury through the activation of PAR-2 in alveolar macrophages.

F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.

The F-actin cortex in chromaffin granule dynamics and fusion: a minireview.

Chromaffin granules are restrained in a dense cortical cytoskeleton before releasing their complex mix of active substances in response to cell stimulation. In recent years, the complex organization and dynamics of the chromaffin cell cortex has been unveiled through its analysis with a range of techniques to visualize this structure, including confocal fluorescence, transmitted light, and evanescent field microscopy. Accordingly, it has become apparent that the cortex is a dense F-actin mesh that contains open polygonal spaces through which vesicles can access the submembrane space. In addition to its retentive role, this structure also influences vesicle motion in both the resting state and during cell stimulation with secretagogues. During secretion, the chromaffin cell cortex undergoes a complex reorganization, helping to replenish the empty fast releasable pool of vesicles. Such changes in the cortical cytoskeleton and in the vesicle motion are governed by the activity of molecular motors, such as myosins II and Va. Interestingly, the F-actin/myosin II network also affects the final stages of exocytosis, which involve the opening and expansion of the fusion pore, and the extrusion of the vesicles contents.

The F-actin cortical network is a major factor influencing the organization of the secretory machinery in chromaffin cells.

We have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca(2+) levels ([Ca(2+)](i)) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca(2+)](i) and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca(2+)-sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca(2+)-insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L- and P-Q-type Ca(2+) channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca(2+) entry. The influence of this cortical organization on the propagation of [Ca(2+)](i) can be modelled, illustrating how it serves to define rapid exocytosis.

Sulfur-containing antioxidants increase in vitro several functions of lymphocytes from mice.

The in vitro effects of several sulfur-containing antioxidants, such as glutathione (GSH), N-acetylcysteine (NAC), thioproline (TP) and taurine (TAU), at different concentrations, on key functions of lymphocytes from axillary nodes, spleen, thymus and peritoneum from young-adult BALB/c mice have been investigated. The functions studied have been proliferation, both spontaneous and in response to the mitogen Concanavalin A, mobility both spontaneous and directed to a chemical attractant (chemotaxis) and adherence to substrate. The effect of these antioxidants on the viability of leukocytes was also investigated. The results show an antioxidant-induced stimulation of all the functions studied. The highest concentrations used of each antioxidant were the most effective in proliferation (5mM for GSH, 1mM for TP and NAC and 40 mM for TAU). These concentrations increase mobility significantly. The presence of TP+NAC enhances the chemotaxis of peritoneal lymphocytes more than each antioxidant separately. The adherence capacity of peritoneal lymphocytes also increased at 10 min of incubation with GSH, TP and NAC. All these antioxidants increase the viability of leukocytes in culture, especially in cells from spleen. In conclusion, the sulfur-containing antioxidants studied in vitro improve the functional capacity of lymphocytes from young-adult mice and these results showing that the improvement of the immune response, and specifically of the lymphocyte functions, found after ingesting diet supplemented with the antioxidants studied, are due to a direct action of these compounds in the immune cells.

Association of SNAREs and calcium channels with the borders of cytoskeletal cages organizes the secretory machinery in chromaffin cells.

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500-600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.

The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis.

The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage.

Vesicle motion and fusion are altered in chromaffin cells with increased SNARE cluster dynamics.

The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a Delta9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 Delta9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 Delta9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population.

Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells.

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.

Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells.

Transmitted light images showed an intricate and dynamic cytoplasmic structural network in cultured bovine chromaffin cells observed under high magnification. These structures were sensitive to chemicals altering F-actin-myosin and colocalised with peripheral F-actin, beta-actin and myosin II. Interestingly, secretagogues induced a Ca2+-dependent, rapid (>10 second) and transitory (60-second cycle) disassembling of these cortical structures. The simultaneous formation of channel-like structures perpendicular to the plasmalemma conducting vesicles to the cell limits and open spaces devoid of F-actin in the cytoplasm were also observed. Vesicles moved using F-actin pathways and avoided diffusion in open, empty zones. These reorganisations representing F-actin transfer from the cortical barrier to the adjacent cytoplasmic area have been also confirmed by studying fluorescence changes in cells expressing GFP-beta-actin. Thus, these data support the function of F-actin-myosin II network acting simultaneously as a barrier and carrier system during secretion, and that transmitted light images could be used as an alternative to fluorescence in the study of cytoskeleton dynamics in neuroendocrine cells.

New roles of myosin II during vesicle transport and fusion in chromaffin cells.

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.

Small peptides patterned after the N-terminus domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis.

Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.

Modifications in the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) and in the complementary region of synaptobrevin affect the final steps of exocytosis.

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis.