Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections.The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with ~12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca 2÷, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.Actin is a major component of the cytoskeletal and contractile elements in nonmuscle cells. It plays an essential role in cell motility and shape determination of the cell (7,16,20,26,31,34). Depending on the physiological state of the cell, actin undergoes assembly and disassembly, and cellular actin levels are also regulated quantitatively in the cell. Actin content becomes a good marker for cell physiology and morphology in some specialized cells (15,32,35) and transformed cells (17,38).In the present studies, the changes in the cytoskeletal components of retinal pigmented epithelial cells were examined during the development of chicken eyes. Relative contents of a 42-kD protein of the cells from a l-d-old chick were found to be much larger than those of an 11-d-old embryo when 590 assayed by SDS gel electrophoresis. The aim of this study is to determine why there is an increase in a 42-kD protein in the cells and how the cells use this additional protein.The increase in 42-kD protein is found to be closely related to the formation and elongation of apical projections that have paracrystalline core bundles in chicken epithelial cells. MATERIALS AND METHODS Preparation of Pigmented Epithelia:Retinal pigmented epithelia were prepared from chicks of various ages, ranging from 11-d-old embryos ...

Section: Discussionmentioning

confidence: 95%

Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye.

Owaribe¹,

Eguchi²

1985

“…The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2, 4, 5, 6, 25, 55). In the presence of Ca ++ (>10 -6 M), villin causes fragmentation of the core bundle whereas the bundling activity of fimbrin appears to be independent of Ca++ (9,17,48,49,55). Villin is also present in the rootlets (23).…”

mentioning

confidence: 99%

“…The microvillus core bundle is attached to the surrounding membrane by periodically arranged lateral bridges that consist of a complex of a rod-shaped ll0-kD protein and calmodulin (13,31,37,46,47,69). The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2,4,5,6,25,55). In the presence of Ca ++ (>10 -6 M), villin causes fragmentation of the core bundle whereas the bundling activity of fimbrin appears to be independent of Ca++ (9,17,48,49,55).…”

mentioning

confidence: 99%

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

Drenckhahn

Dermietzel

1988

229

134

Abstract. In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brushborder cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the ll0-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrooflet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actinactivated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and a-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the ~t-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and ct-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the ll0-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.T HE resorptive surface of the intestinal epithelium, called brush border, is increased 15-30-fold by numerous microvilli that are 1-2 lxm long and 100 nm in diameter (64). Each microvillus is supported by an axial bundle of actin filaments that are in parallel alignment and display identical polarity (51, 53, 59; for review see references 8, 52). The microvillus core bundle is attached to the surrounding membrane by periodically arranged lateral bridges that consist of a complex of a rod-shaped ll0-kD protein and calmodulin (13,31,37,46,47,69). The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2, 4, 5, 6, 25, 55). In the presence of Ca ++ (>10 -6 M), villin causes fragmentation of the core bundle whereas the bundling activity of fimbrin appears to be independent of Ca++ (9,17,48,49,55). Villin is also present in the rootlets (23). In contrast to the core bundle, the rootlets contain tropomyosin (21). There is a report indicating that the muscular Z-line protein, ~t-actinin, may also be present in the rootlets and absent from the microvilli (27).The spac...

“…These observations indicate a role for calcium in maintaining the connection of the cytoskeleton to the membrane. The regulation by calcium of microtubule polymerization, actomyosin-based contraction and cell motility is well documented (11,23,24,26,28,41,43,62) and must also be considered in the context of capping.…”

mentioning

confidence: 99%

Participation of calmodulin in immunoglobulin capping

Nelson¹

1982

When mouse B lymphocytes are incubated with antibodies against their surface immunoglobulin (Ig), patching and capping occur in a process that involves the action of the actomyosin cytoskeleton and the mobilization of cell calcium. Calmodulin (CAM) plays a central role in the Ca ++ regulation of many cellular structures and processes, including the cytoskeleton and membrane-bound enzymes, and therefore was investigated for its role in capping. CaM was isolated from mouse lymphocytes by affinity chromatography on Fluphenazine-Sepharose. Lymphocyte CaM co-migrates with calf brain CaM on SDS polyacrylamide gels, where its rt is Ca++-dependent. It stimulates the activity of the CaM-dependent cyclic AMP phosphodiesterase (PDE) of bovine heart. Several phenothiazine and thioxanthene compounds as well as the drugs W7, WS, and R24571 inhibit CaM in in vitro enzyme assays with ID~o's of from 1 /~M to >1 mM. These were tested for their effects on capping of Ig and were found to inhibit capping in dose-dependent fashions with ID~o'S that corresponded to their anti-CaM potencies. The drugs also disrupted preformed caps and were all reversible. CaM was localized in lymphocytes by staining with a highly fluorescent trifluoperazine derivative (TFP*) produced by photo-oxidation. TFP* staining was diffuse in untreated lymphocytes but stained under caps and in uropods in cells capped with anti-lg antibodies. Staining of cells with antibodies against calf brain and rat testis calmodulin gave similar staining patterns. Staining of patched cells with either antibodies or TFP* showed patched distributions of CaM, but submembranous CaM "patches" did not map one-on-one with respect to Ig patches. These observations suggest that calmodulin participates in the latter stages of ligandinduced Ig redistribution probably by regulating the interaction of the cytoskeleton with the membrane.The capping of surface immunoglobulin on B lymphocytes is a dramatic example of surface receptor redistribution mediated by components of the underlying cytoskeleton (5, 21, 32, 53, 57). The arrangement and regulation of the cell's actomyosinbased motility apparatus and the means by which surface receptors are connected to this machinery through the lipid bilayer are two of the central issues in understanding patching and capping. Cross-linking of receptors by multivalent ligands is required to initiate the energy-independent patching of receptors that must occur before the energy-dependent, cytoskeleton-mediated capping process gathers the patched receptors to one pole of the celi (40,57,58,59). It is during the patching phase of receptor movement that initial connections to the cytoskeleton are made (3,5, 14,18,20,71). The cell's motility machinery then coordinates the movement of receptor aggregates. These two physiologically and structurally distinct steps may be regulated in similar or dissimilar ways.One known regulatory species whose local distribution is altered during cross-linking and patching is the calcium ion. Cross-linking of surface i...