Sergio D. Rosé

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion

The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine β‐hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time‐dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release‐ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F‐actin networks.

Two pathways control chromaffin cell corticalF-actin dynamics during exocytosis

Neurosecretory cells including chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane. We have proposed that the F-actin network acts as a barrier to the secretory vesicles blocking their access to exocytotic sites at the plasma membrane. Disassembly of cortical F-actin in chromaffin cells in response to stimulation is thought to allow the free movement of secretory vesicles to exocytotic sites. Moreover, experiments by us using morphometric analysis of resting and stimulated chromaffin cells together with membrane capacitance measurements have shown that cortical F-actin controls the traffic of vesicles from the vesicle reserve compartment to the release-ready vesicle compartment. The dynamics of the cortical F-actin is controlled by two pathways: A) stimulation-induced Ca(2+) entry and scinderin activation; and B) protein kinase C (PKC) activation and MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation. When chromaffin cells are stimulated through nicotinic receptors, cortical F-actin disassembly is mainly through the intervention of pathway A, since in the presence of PKC inhibitors, F-actin disassembly in response to cholinergic stimulation is only blocked by 20%. Pathway A involves the activation of scinderin by Ca(2+) with a consequent F-actin severing. Pathway B is fully activated by phorbol esters and in this case PKC blockers inhibit by 100% the disruption of cortical F-actin. This pathway operates through MARCKS. A peptide with amino acid sequence corresponding to the phosphorylation site domain of MARCKS, which also corresponds to its actin binding site, blocks PMA potentiation of Ca(2+)-induced catecholamine release. The results suggest that under physiological conditions (i.e., nicotinic receptor stimulation) pathway A is the principal mechanism for the control of cortical F-actin dynamic changes.

Pathways that control cortical F-actin dynamics during secretion

Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation‐induced chromaffin cell cortical F‐actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+‐dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+‐binding sites. F‐actin severing activity of Sc is Ca2+‐dependent, whereas Sc‐evoked actin nucleation is Ca2+‐independent. Sc domain role in secretion was studied by co‐transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)‐fusion Sc constructs. Cells over‐expressing actin severing Sc1‐6 or Sc1‐2 (first and second actin binding sites) constructs, increased F‐actin disassembly and hGH release upon depolarization. Over‐expression of nucleating Sc5‐6, Sc5 or ScABP3 (third actin site) constructs decreased F‐actin disassembly and hGH release upon stimulation. Over‐expression of ScL5‐6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F‐actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly ⇆ assembly) and release (facilitation ⇆ inhibition). The position of the switch (severing ⇆ nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation‐induced Ca2+ entry would increase Sc‐evoked cortical F‐actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc‐evoked actin nucleation.

Molecular motors involved in chromaffin cell secretion.

Abstract: Neurosecretory cells, including chromaffin cells, possess a mesh of filamentous actin underneath the plasma membrane. It has been proposed that filamentous actin network separates the secretory vesicles into two compartments: the reserve pool and the release‐ready vesicle pool. Disassembly of chromaffin cell cortical filamentous actin in response to stimulation allows the movement of vesicles from the reserve pool into the release‐ready vesicle pool. Electron microscopy of cytoskeletons revealed the presence of polygonal areas almost devoid of actin filaments in stimulated cells. The percentage of stimulated cells showing disrupted cytoskeleton correlates well with the increase in secretion in these cells. Fine filaments also remain in these areas of disassembly, and these reacted with actin antibodies, as demonstrated by immunogold staining. In addition, the movement of vesicles between pools requires Ca2+ and ATP, a condition for activation of a molecular motor. Confocal microscopy images demonstrated colocalization of myosin Va with dopamine‐β‐hydroxylase. Cell depolarization induced the dissociation of myosin Va from chromaffin vesicles. 2,3‐Butadione‐2‐monoxime (BDM), an inhibitor of myosin ATPase, inhibited secretion, suggesting a blockage for chromaffin vesicle transport between the reserve pool and the release‐ready vesicle pool. On the other hand, myosin II subcellular distribution was not affected by cell depolarization. Confocal microscopy images show myosin II to be localized in the cell cortex and in some perinuclear structures. Chromaffin vesicles were not stained by myosin II antibody.

Scinderin, a Ca2+-Dependent Actin Filament Severing Protein that Controls Cortical Actin Network Dynamics During Secretion

Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca2+, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca2+-dependent activity of scinderin together with its Ca2+-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.

Light and Electron Microscopic Study of Changes in the Organization of the Cortical Actin Cytoskeleton During Chromaffin Cell Secretion

Chromaffin cells cultured for 2 days were incubated in the absence or presence of 10 μM nicotine for 40 sec. Resting and stimulated cells were fixed and either prepared for fluorescence microscopy or treated with Triton X-100 to obtain cytoskeletons for ultrastructural studies. Electron microscopy of cytoskeletons revealed the presence of polygonal areas devoid of actin filaments only in nicotinic receptor-stimulated cells. Staining of these cytoskeleton preparations with rhodamine–phalloidin, a probe for filamentous actin, produced fluorescent patterns and three-dimensional images similar to those obtained from resting or stimulated intact cells prepared directly for fluorescence microscopy. Moreover, the percentage of stimulated cells showing disrupted cytoskeleton at the electron microscopic level was similar to the percentage of stimulated cells showing patched rhodamine fluorescence at the fluorescence microscopic level. In addition, cells stimulated with nicotine for 40 sec showed a fivefold increase in amine output and a significant decrease in F-actin levels. These results provide the first ultrastructural evidence for nicotinic receptor-evoked chromaffin cell F-actin disassembly and show that the rhodamine–phalloidin-unstained areas observed in fluorescence microscopy represent the areas devoid of filamentous actin observed at the electron microscopic level.

An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization-induced cortical F-actin disassembly and exocytosis

Chromaffin cell secretion requires cortical F‐actin disassembly and it has been suggested that scinderin, a Ca2+‐dependent F‐actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 µm scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F‐actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization‐evoked cortical F‐actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin‐permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation‐induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (Bmax) secretory response to Ca2+ without modifying the affinity (Km) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine β‐hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle–plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.

Myristoylated alanine‐rich C kinase substrate phosphorylation is involved in thrombin‐induced serotonin release from platelets

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine‐rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin‐permeabilized platelets. MARCKS is known to bind actin and cross‐link actin filaments, and this is inhibited by PKC‐evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala‐MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca2+ and the cortical F-actin network. Cortical F-actin disassembly, a Ca2+-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.