Brush Border Assembly Research Articles

The cytoskeleton in development of epithelial cell polarity.

The polarization of intestinal epithelial cells and the stereotypic arrangement of their actin-based cytoskeleton have made these epithelia an excellent system to explore the organization and formation of a cortical actin-based cytoskeleton. Through a combined morphological and biochemical analysis, the molecular arrangement of many of the components of the brush border has been elucidated. Study of brush border assembly in the Crypts of Lieberkühn suggests that cytoskeletal mRNA and protein expression, as well as morphological development, occur rapidly following cell differentiation. Protein kinases appear to be important regulators of intestinal cell growth, for differentiating cells in the crypts possess 15-fold higher levels of tyrosine phosphorylated proteins than differentiated cells of the villus. One of these kinases, pp60c-src, has a 4- to 7-fold higher activity in crypts and increased association with the cytoskeleton than it has in villus cells. The development and maintenance of polarization in epithelial cells require the targeting and transport of specific proteins to the apical and basolateral plasma membrane. It has been proposed that a dynein-like, microtubule-based motor is involved in the transport of apically directed materials from the trans-Golgi to the apical plasma membrane. However, microtubules do not reach the plasma membrane, but terminate below the actin-rich network of filaments comprising the terminal web. We propose that vesicles translocate from the Golgi to the apical cytoplasm along microtubules using dynein, and then move through the terminal web to reach the apical plasma membrane using the actin-based motor myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells.

Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were 'depolarized' by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Intercellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.

Familial Microvillous Atrophy

Twenty-three cases of microvillous atrophy were reviewed to determine clinical and morphological characteristics of the disease. Congenital and late-onset forms of presentation were clearly identified in which the late-onset cases appeared to have a better prognosis. Three different, and distinctive, appearances of the proximal small intestinal mucosa were found. Careful orientation of mucosal samples allowed a temporal sequence of events to be delineated in which the first morphological abnormality to be detected in the epithelium was the accumulation of "secretory granules"; microvillous inclusions were seen in older cells in the upper villous region. It is suggested that, in familial microvillous atrophy, diarrhoea and disorganisation of the brush border assembly occur as a consequence of a more fundamental defect that affects the intracellular traffic of certain cell components, as indicated by the accumulation of "secretory granules."

Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion

Selected strains of the human immunodeficiency virus (HIV) types 1 and 2 are able to infect human colon epithelial cells in vitro, suggesting a mechanism for the anal route of HIV transmission. In some cases, HIV is not produced by infected colon cells but can be rescued after coculture with T-lymphoid cells. One of the HIV strains (HIV1-NDK) replicated well in colonic cells. A transmission electron microscope study demonstrated two major structural perturbations in producer colon cells: an unusual number of secretion bodies and the appearance of intracellular lumina with disorganized microvilli, indicating a defect in brush border assembly and differentiation. Either abnormality could account for HIV-induced enteropathy consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens. Moreover, HT29 cells infected with HIV provide a unique model for selection of enterotropic HIV strains.

Assembly of distinctive coated pit and microvillar microdomains in the renal brush border

The plasma membrane of the kidney brush border is composed of two compositionally distinct microdomains, microvilli and clathrin-coated pits. To study their assembly we have immunolocalized brush border marker proteins in the developing proximal tubule epithelium of the neonatal rat and compared their time and site of appearance with those of basolateral markers, Na-K-ATPase and fodrin. The proteins studied were dipeptidyl peptidase IV (DPPIV) (microvilli), actin and villin (microvillar cytoskeletal proteins), glycoprotein 330 (gp330) (coated pits), and clathrin (coated pit cytoskeleton). Although apical microvilli and coated pits were first seen in the stage III nephron, many brush border markers including DPPIV, actin, and clathrin appeared earlier in the development and initially were not polarized. Only during stage III did they become concentrated at the apical membrane. Villin first appeared in the stage III proximal tubule where it was located diffusely in the cytoplasm and in lysosomes as well as along the apical membrane. It did not completely colocalize with actin until stage IV. Gp330 first appeared during stage III and from the beginning was restricted to the apical clathrin-coated membrane domains and endosomes. The results demonstrate that 1) the expression of renal brush border proteins during development is asynchronous, and 2) unlike the basolateral plasmalemmal domain, which is established early in nephrogenesis, brush border assembly occurs later, approximately coinciding with the onset of glomerular filtration.

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).

Structural and compositional analysis of early stages in microvillus assembly in the enterocyte of the chick embryo

Abstract Morphological and immunocytochemical techniques were used to examine the distribution of villin, with respect to actin, during the early events of brush border morphogenesis in the embryonic chicken intestine. Immunolocalization studies indicate that actin and villin exist as a cortical array in the apical domain of embryonic enterocytes at a time when few surface microvilli are visible by scanning and transmission electron microscopic techniques. A population of villin is also localized at the level of the junctional complex. With time, the density of microvilli increases and the cells begin to flatten. In these cells, villin is detected in the newly formed microvilli and also in the subjacent cortex, where microvillar rootlets are beginning to appear. The significance of actin-villin associations in the process of brush border assembly is discussed in the light of the functional properties of villin.

Assembly of the brush border cytoskeleton: Changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine

The assembly of the intestinal microvillus cytoskeleton was examined during the differentiation of enterocytes along the crypt-villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (hc) of brush border (BB) myosin I (110K-calmodulin complex) and rhodamine-conjugated phalloidin as a probe for F-actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (hc) did not assume this localization until the crypt-villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt-villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane cytoskeleton as well. The pattern of expression of the microvillar core proteins along the crypt-villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.