The cytoskeleton as a barrier to exocytosis in secretory cells

Chromaffin cells of the adrenal medulla synthesize, store and secrete catecholamines. These cells contain numerous electron-dense secretory granules which discharge their contents into the extracellular space by exocytosis. The subplasmalemmal area of the chromaffin cell is characterized by the presence of a highly organized cytoskeletal network. F-Actin seems to be exclusively localized in this area and together with specific actin-binding proteins forms a dense viscoelastic gel; fodrin, vinculin and caldesmon, three actin cross-linking proteins, and gelsolin, an actin-severing protein, are found in this subplasmalemmal region. Since fodrin-, caldesmon- and alpha-actinin-binding sites exist on secretory granule membranes, actin filaments can also link secretory granules. Chromaffin granules can be entrapped in this subplasmalemmal lattice and thus the cytoskeleton acts as a barrier preventing exocytosis. When cells are stimulated, molecular rearrangements of the subplasmalemmal cytoskeleton take place: F-actin depolymerizes and fodrin reorganizes into patches. In addition, introduction of monospecific antifodrin immunoglobulins into digitonin-permeabilized cells blocks exocytosis, demonstrating the crucial role of this actin-binding protein. In bacterial toxin-permeabilized chromaffin cells, experiments using actin-perturbing agents such as cytochalasin D and DNAase I suggest that exocytosis is in part controlled by the cytoskeleton. The intracellular signal governing the cytoskeletal reorganization (associated with exocytosis) is calcium. Calcium inhibits some and activates other actin-binding proteins and consequently causes dissolution of the subplasmalemmal cytoskeleton. This dissolution of cytoskeletal filaments should result in granule detachment and permit granules free access to exocytotic sites on the plasma membrane.

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Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells.

The Journal of Cell Biology ◽

10.1083/jcb.102.2.636 ◽

1986 ◽

Vol 102 (2) ◽

pp. 636-646 ◽

Cited By ~ 53

Author(s):

M F Bader ◽

J M Trifaró ◽

O K Langley ◽

D Thiersé ◽

D Aunis

Keyword(s):

Adrenal Medulla ◽

Chromaffin Cells ◽

Binding Proteins ◽

Binding Protein ◽

Actin Binding ◽

Secretory Process ◽

Secretory Cells ◽

Breakdown Product ◽

Actin Binding Proteins ◽

Actin Binding Protein

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.

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Tubulin- and Actin-binding Proteins in Chromaffin Cells

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1987.tb27229.x ◽

1987 ◽

Vol 493 (1 Cellular and) ◽

pp. 435-447 ◽

Cited By ~ 5

Author(s):

DOMINIQUE AUNIS ◽

MARIE-FRANCE BADER ◽

O. KEITH LANGLEY ◽

DOMINIQUE PERRIN

Keyword(s):

Chromaffin Cells ◽

Binding Proteins ◽

Actin Binding ◽

Actin Binding Proteins

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Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Molecular Biology of the Cell ◽

10.1091/mbc.6.3.247 ◽

1995 ◽

Vol 6 (3) ◽

pp. 247-259 ◽

Cited By ~ 111

Author(s):

K Pestonjamasp ◽

M R Amieva ◽

C P Strassel ◽

W M Nauseef ◽

H Furthmayr ◽

...

Keyword(s):

Plasma Membranes ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Actin Binding ◽

Actin Binding Proteins ◽

Permeabilized Cells ◽

Plasma Gelsolin ◽

Conserved Sequence ◽

Myosin Subfragment 1

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

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