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 ?-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.This study was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2011-25095 and BFU2015- 63684-P, MINECO, FEDER, UE) to LMG

Carta de Salvador Viniegra a Miguel de Unamuno. [S.l.], 24 de abril de 1917

Carta de Salvador Viniegra, representante en España de la revista “Caras y Caretas”, a Miguel de Unamuno reiterándole la solicitud de que le mande otra correspondencia para enviarla a la redacción de la revista en Buenos Aires

Tarjeta postal de Salvador Viniegra a Miguel de Unamuno. [Madrid], [23 de septiembre de 19--?]

Tarjeta postal de Salvador Viniegra, representante en España de la revista “Caras y Caretas”, a Miguel de Unamuno informándole que ya ha remitido a la revista “Caras y caretas” su artículo y en cuanto reciba el dinero por su colaboración se lo enviará

Carta de Salvador Viniegra a Miguel de Unamuno. Madrid, 26 de enero de 1914

Carta de Salvador Viniegra, representante en España de la revista “Caras y Caretas”, a Miguel de Unamuno informándole que ha recibido su narración “Un grito de muerte en las tinieblas”, la cual ha remitido a la dirección de la revista “Caras y Caretas”. También le adjunta un cheque por el pago de dicha correspondencia

Carta de Salvador Viniegra a Miguel de Unamuno. [S.l.], 2 de abril de [1917]

Carta de Salvador Viniegra, representante en España de la revista “Caras y Caretas”, a Miguel de Unamuno informándole que se ha publicado en la revista “Caras y Caretas” del día 24 de febrero su artículo “Batracófilos y batracófobos”, rogándole además que mande otro original

A single amino acid near the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells

Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome-associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a construct containing complete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu203, the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP-25. Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.This work was supported by grants from the Spanish Dirección General de Investigación Científica y Técnica to S.V. and L.M.G. (DGICYT, PM 95-0110) and M.C. (DGICYT PB95–0690) and Generalitat Valenciana (GV-D-vs.-20-158-96) to M.C. and S.V.Peer reviewe

Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells

Truncation of the C-terminal domain of the synaptosomal associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin A (BoNT A) has been shown to block neuroendocrine cell secretion. It is unclear, however, if toxin mechanism involved the affection of a single or more events of the exocytotic cascade. BoNT A induced changes in both the degree of inhibition and the kinetics of catecholamine secretion from populations of cultured bovine chromaffin cells. Ca2+-dependent secretion from digitonin-permeabilized cells showed partial inhibition associated with the alteration of a slow secretory phase at different toxin concentrations. In contrast, in intact cells stimulated by depolarization, cell treatment with low concentrations (1 nm) of the toxin affected the late phase of secretion, whereas 100 nm BoNT A-poisoned cells showed an alteration even of fast components. The high degree of inhibition associated with fast secretory component alteration was dependent on Ca2+ entry through the Ca2+ channels, as it was absent from cells permeated with the A23187 Ca2+ ionophore. Vesicle pools implicated in the effect of BoNT A on the secretory response from single cells were identified using amperometry. These studies supported the macroscopic view by showing that secretion from BoNT A-treated permeabilized cells presented specific inhibition of late vesicle fusions. Intact cells showed alterations in the late vesicle pool (t = 39 s) recruited during prolonged or repetitive KCl depolarizations using 1 nm BoNT A-treated cells as well as in an intermediate kinetic pool (t = 18 s) at higher toxin concentrations (100 nm). The faster resolved component (t = 8 s) or the membrane fusion event itself were not affected. Our results demonstrate that removal of the last nine C-terminal amino acids of SNAP-25 by BoNT A has a specific effect on two different and distal secretory stages in chromaffin cells.This work was supported by a Grant from the Spanish Dirección General de Investigación Científica y Técnica (DGICYT, PM 95–0110). A.G. was a recipient of a research aid from Instituto Juan Gil-Albert.Peer reviewe

Temperature and PMA affect different phases of exocytosis in bovine chromaffin cells

Amperometry was used to study secretory kinetics of single bovine chromaffin cells stimulated by transient depolarizations at different temperatures. The initial rate of release was moderately enhanced when the temperature was raised from 18 to 22 and 37 degrees C. Secretion increased drastically at a later period, 5-10 s after the initiation of stimulus. Interestingly, incubation of the cells with phorbol 12-myristate 13-acetate (PMA) clearly enhanced fast secretory components. In addition, the rate of secretion of the slower component recruited by prolonged depolarizations (t > 30 s) was unaffected at the range of temperatures normally used in secretory experiments (22-37 degrees C). A 'counting events' analysis of secretion, which avoids the influence of event charge changes, showed specific increases in a population of vesicles fusing between 7 and 12 s over the same range of temperatures, and a marked increase in vesicles fusing during the initial phase (1-5 s), of PMA-treated cell secretion. An analysis of temperature influence on transient components released by high sucrose, the secretion elicited by cell permeabilization with digitonin, and studies of the individual characteristics of amperometric events, allow us to conclude that an increase in the size of a secondary-released vesicle population is the main factor contributing to temperature-dependent enhancement of secretion, in clear contrast to the enhancement of fast releasable pools caused by phorbol esters.This work was supported by a Grants from the Spanish Dirección General de Investigación Científica y Técnica to S.V (PM98-0101) and Dirección General de Enseñanzas Universitarias e Investigación de la Generalitat Valenciana to L.M.G. (GV99-155-1-05). A.G. is a recipient of a Glaxo-Wellcome-CSIC fellowship.Peer reviewe

The F-actin cytoskeleton modulates slow secretory components rather than readily releasable vesicle pools in bovine chromaffin cells

Adrenal chromaffin cells were used to test the role of the peripheral cytoskeleton of F-actin in controlling different vesicle pools. Phorbol 12-myristate 13-acetate and calyculin A, two substances affecting phosphorylation–dephosphorylation cycles, produced different degrees of F-actin reorganization, inducing the partial and the almost total disassembly of this structure, respectively, as visualized using rhodamine–phalloidin staining. Consequently, electron microscopy studies revealed the higher efficiency of calyculin-A over phorbol 12-myristate 13-acetate in promoting vesicle access to the plasmalemma boundary. Surprisingly, only the phorbol ester enhanced fast kinetics and the population of rapidly releasable vesicle pools as studied by single-cell amperometry, whereas both agents, as well as the F-actin severing compound, Latrunculin A, promoted an increase in the population of vesicles recruited in response to prolonged or repetitive stimulations. Taken together, our data support the notion that the F-actin peripheral barrier controls primary granule recruitment from reserve vesicle pools, whereas the phorbol ester effect on the rapidly releasable pools might be related to the alteration of late secretory stage through protein kinase C-dependent phosphorylation of an unidentified target.This work was supported by a Grants from the Spanish Dirección General de Investigación Cientı́fica y Técnica to S.V. (PM98-0101) and Dirección General de Enseñanzas Universitarias e Investigación de la Generalitat Valenciana to L.M.G. (GV99-155-1-05). A.G. is the recipient of a Glaxo-Wellcome–CSIC fellowship.Peer reviewe