Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin.

Abstract

Abstract. Two extremely high molecular weight proteins were found to be components of the intestinal epithelial cell brush border cytoskeleton. The largest brush border protein, designated T-protein, migrated on SDS gels as a doublet of polypeptides with molecular weights similar to muscle titin T I and T II. The other large brush border protein, designated N-protein, was found to have a polypeptide molecular weight similar to muscle nebulin. In Western analysis, a polyclonal antibody raised against brush border T-protein reacted specifically with T-protein in isolated brush borders and cross-reacted with titin in pectoralis and cardiac muscle samples. T-protein was distinguished from the muscle titins by an anti-cardiac titin mAb. A polyclonal antibody raised against N-protein was specific for N-protein in brush borders and crossreacted with nothing in pectoralis muscle. Immunolocalization in cryosections of intestinal epithelia and SDS-PAGE analysis of fractionated brush borders revealed that both T-protein and N-protein are concentrated distinctly in the brush border terminal web region subjacent to the microvilli, but absent from the microvilli. EM of rotary-replicated T-protein samples revealed many of the molecules to be long (912 + 40 nm) and fibrous with a globular head on one end. In some of the molecules, the head domain appeared to be extended in a fibrous conformation yielding T-protein up to 1,700-nm long. The brush border N-protein was found as long polymers with a repeating structural unit of ~450 nm. Our findings indicate that brush border T-protein is a cellular isoform of titin and suggest that both T-protein and N-protein play structural roles in the brush border terminal web.T n~ brush border array of microvilli at the apical end of intestinal epithelial cells is supported by one of the most extensively organized actin-based cytoskeletons found in nonmuscle cells (for review of brush border structure, see references 1, 12, 18). This cytoskeleton is a useful model system with which to identify structural components, because it can be isolated intact and in amounts sufficient for biochemical analysis. Moreover, the multiple domain structure of the brush border cytoskeleton aids in determining the functional and structural contributions of each component.In the brush border, each of the numerous microvilli is supported by a core bundle of parallel actin filaments that is rooted in the underlying terminal web. Numerous filaments of myosin II and fodrin cross-link the microvillar bundle rootlets. This interrootlet domain is encompassed by a circumferential ring of anti-parallel actin filaments. Several major brush border proteins have been characterized and their localization in these brush border domains has yielded insight into their possible functions. Many of the proteins in this integrated system are related to components of various cytoskeletal organizations in other cells (12).We report here the discovery of two huge proteins in the brush border terminal web. These cytoskeletal proteins ten...

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber

“…More recently, evidence for expression of bona fide titin isoforms encoded by the titin gene in smooth muscle has been presented [Labeit et al, 2006]. Previously, our lab has identified a titin-like protein (cellular titin, c-titin) in nonmuscle cells, first in the terminal web domain of the intestinal cell brush border cytoskeleton and then in human blood platelets [Eilertsen and Keller, III, 1992;Keller, III et al, 2000]. Characterization of c-titin isolated from brush borders and platelets revealed that it interacts with a-actinin and arranges nonmuscle myosin II bipolar filaments in a side-by-side and end-to-end ordered array similar to that of myosin II organization in the stress fibers of nonmuscle cells [Eilertsen and Keller, III, 1992;Eilertsen et al, 1994].…”

Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery

“…One non-muscle site of titin expression has been reported in the terminal web region of the brush border array of microvilli of intestinal epithelial cells, where it is thought to mediate the association of myosin II and alpha-actinin with the cytoskeleton [Eilertsen and Keller, 1992]. Titin's intracellular interactions are thought to organize and stabilize actin-myosin II associated cytoskeletal structures [Eilertsen et al, 1994].…”

Smooth Muscle Titin Zq Domain Interaction with the Smooth Muscle α-Actinin Central Rod

“…The powder was extracted using 1ϫ SDS-sample buffer. The SDS extract was fractionated on highly porous 4 -20% gradient SDS-polyacrylamide, as described previously (29). The gel was electroblotted onto nitrocellulose using semidry blot transfer system (Bio-Rad Laboratories) and stained using Ponceau S. The blots were blocked in 5.0% nonfat dry milk for 1-2 h at room temperature.…”