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

1988 ◽  
Vol 107 (3) ◽  
pp. 1037-1048 ◽  
Author(s):  
D Drenckhahn ◽  
R Dermietzel
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Close Association ◽  
Zonula Occludens ◽  
Terminal Web ◽  
Ultrathin Sections ◽  
Microfilament System ◽  
Human Intestinal Epithelium ◽  
Insight Into ◽  
Specific Label

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 brush-border 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 110-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 interrootlet 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 actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-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 alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-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 110-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.

scholarly journals Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

2009 ◽  
Vol 20 (10) ◽  
pp. 2549-2562 ◽  
Author(s):  
Eva-Maria S. Grimm-Günter ◽  
Céline Revenu ◽  
Sonia Ramos ◽  
Ilse Hurbain ◽  
Neil Smyth ◽  
...  
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Keratin 19 ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Important Regulator ◽  
Increased Sensitivity ◽  
Filament Network ◽  
Deficient Mice

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.

scholarly journals Mechanism of brush border contractility studied by the quick-freeze, deep-etch method.

1983 ◽  
Vol 96 (5) ◽  
pp. 1325-1336 ◽  
Author(s):  
N Hirokawa ◽  
T C Keller ◽  
R Chasan ◽  
M S Mooseker
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Ultrastructural Changes ◽  
Immunofluorescent Localization ◽  
Terminal Web ◽  
Myosin S1 ◽  
Brush Borders ◽  
Mixed Polarity ◽  
The Individual ◽  
Replication Technique

We have analyzed terminal web contraction in sheets of glycerinated chicken small intestine epithelium and in isolated intestinal brush borders using a quick-freeze, deep-etch, rotary shadow replication technique. In the presence of Mg-ATP at 37 degrees C, the terminal web region of each cell in the glycerinated sheet and of each isolated brush border became severely constricted at the level of its zonula adherens (ZA). Consequently, the individual brush borders rounded up, splaying out their microvilli in fanlike patterns. The most prominent ultrastructural changes that occurred during terminal web contraction were a dramatic decrease in the diameter of the circumferential ring composed of a bundle of 8-9-nm filaments adjacent to the zonula adherens and a decrease in the number of cross-linkers between the microvillus rootlets. Microvilli were not retracted into the terminal web. We have used myosin S1 decoration to demonstrate that most of the circumferential bundle filaments are actin and that the actin filaments are arranged in the bundle with mixed polarity. Some filaments within the bundle did not decorate with myosin S1 and had tiny projections that appeared to be attached to adjacent actin filaments. Because of their morphology and immunofluorescent localization of myosin within this region of the terminal web, we propose that these undecorated filaments are myosin. From these results, we conclude that brush border contraction is caused primarily by an active sliding of actin and myosin filaments within the circumferential bundle of filaments associated with the ZA.

Structure and function of neural networks in the retina

1988 ◽  
Vol 46 ◽  
pp. 26-27
Author(s):  
Peter Sterling
Keyword(s):  
Ganglion Cells ◽  
Three Dimensional ◽  
Structure And Function ◽  
Synaptic Connections ◽  
Visual Axis ◽  
One Dimensional ◽  
Cat Retina ◽  
Ultrathin Sections ◽  
And Function ◽  
Insight Into

The synaptic connections in cat retina that link photoreceptors to ganglion cells have been analyzed quantitatively. Our approach has been to prepare serial, ultrathin sections and photograph en montage at low magnification (˜2000X) in the electron microscope. Six series, 100-300 sections long, have been prepared over the last decade. They derive from different cats but always from the same region of retina, about one degree from the center of the visual axis. The material has been analyzed by reconstructing adjacent neurons in each array and then identifying systematically the synaptic connections between arrays. Most reconstructions were done manually by tracing the outlines of processes in successive sections onto acetate sheets aligned on a cartoonist's jig. The tracings were then digitized, stacked by computer, and printed with the hidden lines removed. The results have provided rather than the usual one-dimensional account of pathways, a three-dimensional account of circuits. From this has emerged insight into the functional architecture.

Ultrastructural studies of the relationship between actin filaments and secretory granules

1990 ◽  
Vol 48 (3) ◽  
pp. 458-459
Author(s):  
Ellen Holm Nielsen
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Granule Membrane ◽  
Ultrathin Sections ◽  
Lowicryl K4m

In secretory cells a dense and complex network of actin filaments is seen in the subplasmalemmal space attached to the cell membrane. During exocytosis this network is undergoing a rearrangement facilitating access of granules to plasma membrane in order that fusion of the membranes can take place. A filamentous network related to secretory granules has been reported, but its structural organization and composition have not been examined, although this network may be important for exocytosis.Samples of peritoneal mast cells were frozen at -70°C and thawed at 4°C in order to rupture the cells in such a gentle way that the granule membrane is still intact. Unruptured and ruptured cells were fixed in 2% paraformaldehyde and 0.075% glutaraldehyde, dehydrated in ethanol. For TEM (transmission electron microscopy) cells were embedded in Lowicryl K4M at -35°C and for SEM (scanning electron microscopy) they were placed on copper blocks, critical point dried and coated. For immunoelectron microscopy ultrathin sections were incubated with monoclonal anti-actin and colloidal gold labelled IgM. Ruptured cells were also placed on cover glasses, prefixed, and incubated with anti-actin and colloidal gold labelled IgM.

Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Non-muscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

2020 ◽  
Author(s):  
Colbie R. Chinowsky ◽  
Julia A. Pinette ◽  
Leslie M. Meenderink ◽  
Matthew J. Tyska
Keyword(s):  
Brush Border ◽  
Isotropic Layer ◽  
Intestinal Epithelial ◽  
Actin Bundles ◽  
Terminal Web ◽  
Actin Turnover ◽  
Genetic Perturbations ◽  
And Function ◽  
Pointed End ◽  
Muscle Myosin

ABSTRACTEpithelial brush borders are large arrays of microvilli that enable efficient solute uptake from luminal spaces. In the context of the intestinal tract, brush border microvilli drive functions that are critical for physiological homeostasis, including nutrient uptake and host defense. However, cytoskeletal mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a highly crosslinked filamentous meshwork referred to as the “terminal web”. Although classic EM studies revealed complex ultrastructure, the composition, organization, and function of the terminal web remains unclear. Here, we identify non-muscle myosin-2C (NM2C) as a major component of the brush border terminal web. NM2C is found in a dense, isotropic layer of puncta across the sub-apical domain, which transects the rootlets of microvillar actin bundles. Puncta in this network are separated by ∼210 nm, dimensions that are comparable to the expected size of filaments formed by NM2C. In primary intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations to disrupt NM2C activity in cultured intestinal epithelial cells, we found that this motor controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Development of macrociliary cells in Beroe. I. Actin bundles and centriole migration

1988 ◽  
Vol 89 (1) ◽  
pp. 67-80
Author(s):  
S. Tamm ◽  
S.L. Tamm
Keyword(s):  
Cell Membrane ◽  
Basal Body ◽  
Actin Filaments ◽  
Close Association ◽  
Directed Assembly ◽  
Double Layers ◽  
Basal Bodies ◽  
Apical Surface ◽  
Actin Bundle ◽  
Microfilament Bundle

Differentiation of macrociliary cells on regenerating lips of the ctenophore Beroe was studied by transmission electron microscopy. In this study of early development, we found that basal bodies for macrocilia arise by an acentriolar pathway near the nucleus and Golgi apparatus, in close association with plaques of dense fibrogranular bodies. Procentrioles are often aligned side-by-side in double layers with the cartwheel ends facing outward toward the surrounding plaques of dense granules. Newly formed basal bodies then disband from groups and develop a long striated rootlet at one end. At the same time, an array of microfilaments arises in the basal cytoplasm. The microfilaments are arranged in parallel strands oriented toward the cell surface. The basal body-rootlet units are transported to the apical surface in close association with the assembling actin filament bundle. Microfilaments run parallel to and alongside the striated rootlets, to which they often appear attached. Basal body-rootlet units migrate at the heads of trails of microfilaments, as if they are pushed upwards by elongation of their attached actin filaments. Near the apical surface the actin bundle curves and runs below the cell membrane. Newly arrived basal body-rootlets tilt upwards out of the microfilament bundle to contact the cell membrane and initiate ciliogenesis. The basal bodies tilt parallel to the flat sides of the rootlets, and away from the direction in which the basal feet point. The actin bundle continues to enlarge during ciliogenesis. These results suggest that basal body migration may be driven by the directed assembly of attached actin filaments.

The Fine-Structural Organization of the Brush Border of Intestinal Epithelial Cells

1971 ◽  
Vol 8 (3) ◽  
pp. 573-599
Author(s):  
T. M. MUKHERJEE ◽  
L. A. STAEHELIN
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Membrane Surface ◽  
Intestinal Epithelial ◽  
The Core ◽  
Unit Structure ◽  
Terminal Web ◽  
Freeze Etching

The fine structure of the brush border of intestinal epithelial cells of the mouse has been studied with both normal sectioning and freeze-etching techniques. Freeze-etching reveals the plasma membrane of the microvilli as consisting of a continuous layer, that is split during the cleaving process, in which numerous particles, 5-9 nm in diameter, are embedded, while other particle-like structures, with diameters of 7-10 nm, appear attached to the true outer membrane surface. The mucopolysaccharide surface coats of the microvilli show up more clearly in sectioned material than in freeze-etched specimens. Inside each microvillus 2 different filament systems can be demonstrated: (1) bundles of fairly closely packed and straight core microfilaments, which lead into the tip of the microvillus, and (2) short cross-filaments. Under suitable conditions the core microfilaments display a sub-unit structure with a repeating distance of approximately 6 nm. The diameter of a microfilament can vary along its length from 6 to 11 nm. Two strands of globular particles wound helically around each other seem to make up each microfilament. These and other data support the idea that the core microfilaments are actin-like. No substructure has been found on the cross-filaments, which have an orientation approximately radial to the axis of the microvilli and seem to be attached at one end to the core microfilaments and at the other to the inner surface of the microvillous membrane. The interwoven terminal web filaments also show no substructure. They form a continuous flexible platform-like structure into which the bundles of core microfilaments extend. Some terminal web filaments appear attached to the plasma membrane between the microvilli. It is suggested that the core microfilaments represent mechanical supporting elements and that the terminal web and cross-filaments are tensile elements of the brush border. In addition all 3 filament systems may also be involved in possible contractile movements of the microvilli.

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