Analysis of cytoskeletal proteins and Ca ²⁺ -dependent regulation of structure in intestinal brush borders from rachitic chicks

Abstract

We have investigated several structural aspects of-the intestinal epithelial brush border from rachitic chicks. At both the light and electron microscope levels, rachitic brush borders are indistinguishable from controls. Although several of the prominent periodic acid-Schiff-positive proteins ofthe brush border membrane have slightly slower mobilities on sodium dodecyl sulfate/polyacrylamide gels than do corresponding proteins from control brush borders, the major components of the microvillus core, including subunits of 105, 95, and 68 roid, and these molecular events appear to involve both protein synthetic and non-protein-synthetic events.Wilson and Lawson (20) reported effects of vitamin D that may specifically involve the cytoskeletal structure of the brush border. They showed that an early response of rachitic chicks to an acute dose of 1,25-dihydroxyvitamin D3 is the increased rate of synthesis of a 42-to 45-kilodalton (kDal) microvillar protein. This protein was later tentatively identified as f3-and yactin (21). Whether there is an increased turnover or de novo synthesis of the actin-like protein had not been clarified. With the assumption that the 42-to 45-kDal protein is bonafide actin, the results of Wilson and Lawson (20,21) are of considerable interest from the standpoint of both the action of 1,25-dihydroxyvitamin D3 on calcium transport and the potential effect of the vitamin D hormone on the structure and behavior of the microvillar cytoskeleton. The role of calcium in the regulation of brush border contractility (22), brush border myosin phosphorylation (23), and the length and bundling of microvillar actin filaments (24-29) has been established. Also, the microvillus contains considerable amounts of calmodulin (30), for which only one function, activation of the' brush border myosin light chain kinase (23), has as yet been defined. Thus, the significance of calcium in the modulation and regulation of the transmural flow of calcium through the intestinal epithelial cell constituted the basis for the present investigation. The main objective was to determine whether vitamin D alters some of the known molecular components of the microvillus cytoskeleton, and thus to provide the basis for further studies on the relationships among the cytoskeleton ofthe brush border, vitamin D, and calcium transport. Other observations on the protein components of the brush border are reported herein. A review of brush border structure and function has recently appeared (31).MATERIALS AND METHODS Animals. White Leghorn cockerels were maintained on a rachitogenic diet (32) in a room with incandescent lighting for 4-5 weeks. Some chicks were injected with 500 international units of vitamin D3 at least 72 hr before the experiments were performed and fed a vitamin D-complete 'diet; these chicks served as vitamin D replete controls. To verify the vitamin D status of animals, radial immunoassays for vitamin D-dependent calcium-binding protein were performed as described (33) on supernates saved from the first...

Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations.

“…Third, fodrin has been shown to be associated with the cap of lymphocytes during capping (22), indicating that fodrin may anchor cell surface molecules via its attachment to cytoskeletal actin. In addition, two laboratories (15)(16)(17)20) have found, using a iodinated calmodulin overlay technique similar to ours, that calmodulin binds to a protein of Mr 250,000 in intestinal brush borders, but not to microvilli. However, this protein, labeled TW 260/240 (15,16), which is morphologically and immunologically similar to fodrin (15,16), has a different subunit structure.…”

Organization, Chemistry, and Assembly of the Cytoskeletal Apparatus of the Intestinal Brush Border

“…Gel filtration analyses of purified 1l0-kDa-CM complex suggest that it consists of a dimer of 110 kDa (Howe & Mooseker 1983) with variable amounts of associated calmodulin (0.2-2.0 molecules) per 110-kDa subunit (Howe & Mooseker 1983 ;Collins & Borysenko 1984). This was first demonstrated using gel overlay procedures Howe et al 1982). This was first demonstrated using gel overlay procedures Howe et al 1982).…”

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.

“…It has been shown by acrylamide gel overlay techniques that the 240-kdalton subunit binds to calmodulin in the presence but not absence ofcalcium (11,12,14) . Little else is known about this interaction, however .…”

“…They have the potential, at least, to bind specifically to membranes, as determined by binding to inside-out membrane vesicles from erythrocytes (8,9). Finally, they bind to, in a calcium-dependent fashion, the ubiquitous regulatory protein, calmodulin (7,(9)(10)(11)(12)(13)(14).…”