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T C Keller

Publications and source records attributed to T C Keller.

At least 19 recordsLinked to original sources
PubMed2000

Role of titin in nonmuscle and smooth muscle cells.

Extensive investigation of vertebrate striated muscle titin has yielded significant insight into its structure and function in striated muscle. We have begun to investigate other members of the titin protein family found in vertebrate smooth muscle and nonmuscle cells. Smooth and nonmuscle titins resemble striated muscle titin in molecular size and morphology but differ in their interactions with myosin II filaments and in the structural contexts in which they exist in vivo. Divergence of these titins from the muscle titin paradigm demonstrates the versatility of this remarkable family of giant proteins.

Animals↗
PubMed1999

Differential usage of two 5' splice sites in a complex exon generates additional protein sequence complexity in chicken CLIP-170 isoforms.

Reverse transcription-polymerase chain reaction amplification and cloning of cDNA encoding the chicken CLIP-170(11) isoform of the Cytoplasmic Linker Protein 170 gene revealed an unusual source of protein sequence variation. In addition to differential combinatorial splicing of two cassette exons to yield four CLIP-170 protein isoforms, we found differential usage of alternative 5'-splice junctions in a single exon. Splicing at the downstream site yields message containing 18 bp of nucleotide sequence that is missing from message spliced at the more 5' site. This 18 bp sequence encodes a segment of 6 amino acids that fills a gap in the alignment of chicken and human CLIP-170 homologue sequences. Differential usage of the 5'-splice junctions in this complex exon appears to be tissue- rather than isoform-specific.

Amino Acid Sequence↗
PubMed1998

Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle.

CLIP-170 and Restin, microtubule-binding proteins originally cloned from human cells, are identical except for a stretch of 35 amino acids present in Restin, but missing from CLIP-170. Here we present the discovery of two novel isoforms of the CLIP-170/Restin gene in both chickens and humans. One of the new isoforms, named CLIP-170(11), contains an 11 amino acid insert instead of the 35 amino acid insert found in Restin. Eight of these 11 amino acids, including a helix-breaking proline residue, are perfectly conserved between chickens and humans. The second new isoform, named CLIP-170(11+35), contains both the 11 and 35 amino acid inserts in tandem. PCR analysis of chicken genomic DNA revealed that all four isoforms result from differential splicing of two exons in a region of the CLIP-170 gene that contains approximately 8.6 kb of intervening sequence. We found that the CLIP-170(11) and CLIP-170(11+35) are expressed preferentially in muscle tissues. Chicken and human skeletal muscle express predominantly CLIP-170(11) and to a lesser extent CLIP-170 and CLIP-170(11+35). Adult chicken cardiac and smooth muscles also express CLIP-170(11) and CLIP-170(11+35), but CLIP-170 is the predominant isoform in these muscles as it is in all other tissues except brain. The ratios of CLIP-170 isoform expression found in embryonic and adult chicken cardiac muscles reveal that isoform expression is regulated differentially in different developmental stages as well as in different tissues.

Alternative Splicing↗
PubMed1998

Cloning and expression of chicken CLIP-170 and restin isoforms.

We have cloned cDNA for the chicken homologues of human CLIP-170 and Restin and characterized expression of chicken CLIP-170 and Restin messages in a variety of chicken tissues. Chicken CLIP-170 and Restin, like the human homologues, differ only in a stretch of 35 amino acids present in Restin but missing from CLIP-170. This Restin-specific insert is perfectly conserved between the chicken and human sequences at both the protein and nucleotide level and contributes an additional five heptads to one of the heptad repeat regions in the central alpha-helical coiled-coil rod domain. Other highly conserved chicken and human CLIP-170/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-binding motifs in the N-terminal globular head domain and two CCHC metal-binding motifs in the C-terminal globular tail domain. We have used Southern DNA blot analysis and PCR amplification of exon-intron junctions of chicken genomic DNA to confirm that CLIP-170 and Restin are isoforms encoded by the same gene. Semiquantitative RT-PCR analysis of CLIP-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissues tested, except brain, the message for CLIP-170 was more abundant than that for Restin. Comparison of the levels of CLIP-170 and Restin messages in RNA from chicken and human intestinal epithelial cells revealed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and that the two isoforms have distinct functions in a wide variety of cells.

Alternative Splicing↗
PubMed1997

Interaction of alpha-actinin with cellular titin.

Cellular titin (c-titin) colocalizes with myosin II in cytoskeletal structures containing actin in vivo and organizes highly ordered myosin bipolar filament arrays in the absence of actin in vitro. We report here that the actin-binding protein alpha-actinin associates with coassemblies of c-titin and myosin through direct interaction with c-titin. These results support the possibility that interaction between the myosin-associated protein c-titin and the actin-associated protein alpha-actinin organizes and stabilizes actin-myosin II cytoskeletal structures in vivo.

Actinin↗
PubMed1995

Structure and function of titin and nebulin.

Recent investigations of titin anchorage and elasticity have been supplemented with in vitro expression studies on isolated domains of titin and nebulin. These have yielded new insights into the molecular basis of the functions of these proteins in muscle. The characterization of a cellular (non-muscle) isoform of titin has extended the functional relevance of this family of proteins beyond the realm of muscle.

Animals↗
PubMed1994

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro.

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.

Animals↗
PubMed1992

Functional coupling to brush border creatine kinase imparts a selective energetic advantage to contractile ring myosin in intestinal epithelial cells.

The B-CK isozyme of cytoplasmic creatine kinase is localized distinctly in the terminal web region of the intestinal epithelial cell brush border (Keller and Gordon: Cell Motil. Cytoskeleton 19:169-179, 1991). Experiments were performed to determine whether this CK is energetically coupled to the myosin II that is present in the circumferential ring and interrootlet structural domains of the brush border terminal web. In isolated brush borders, ATP-dependent circumferential ring contraction and interrootlet myosin solubilization were supported either by an exogenous PEP-pyruvate kinase-based ATP-regeneration system (PEP-PK) or by the addition of phosphocreatine to the endogenous B-CK-based ATP-regeneration system (PCr-B-CK). Addition of an exogenous hexokinase-glucose ATP-hydrolysis system (HK-G) effectively blocked both contraction and myosin solubilization in the PEP-PK assay. In contrast, HK-G had no significant effect on PCr-B-CK-supported brush border contraction, although it did inhibit interrootlet myosin solubilization. Thus, when high-energy phosphate is supplied as phosphocreatine, brush border B-CK imparts to the circumferential ring myosin a selective energetic advantage over other ATPases. These results suggest that myosin and B-CK are functionally coupled in the brush border circumferential ring, where they might comprise one end of an energy circuit that supplies energy for contraction, but that colocalization of CK with myosin in the brush border interrootlet domain is insufficient to establish functional coupling.

Adenosine Triphosphatases↗
PubMed1992

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

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 cross-reacted 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 approximately 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.

Animals↗
PubMed1991

Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells.

Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B-CK) and mitochondrial (Mi-CK) creatine kinase isozymes of brain when compared by SDS-PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi-CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B-CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B-CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B-CK was maintained in glycerol-permeabilized cells and in isolated brush borders, indicating that B-CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.

Animals↗
PubMed1991

Multiple polyadenylation signals and 3' untranslated sequences are conserved between chicken and human cellular myosin II transcripts.

We have isolated a chicken cellular myosin II heavy chain isoform cDNA clone that overlaps the published sequence for MHC-A (Shohet et al., 1989, Proc Natl Acad Sci 86, 7726-7730) and contains three canonical AAUAAA-polyadenylation signals in an additional 374 nucleotides at its 3' end. S1 nuclease protection analysis and PCR-amplification of MHC-A cDNA 3' ends have confirmed that all three of the signals are used in vivo. Differential usage of these signals without differential splicing in this region yields three messages that differ at their 3' ends but appear to encode the same protein. Comparison of the new chicken sequence with the homologous human MHC-A cDNA sequence (Saez et al., 1990, Proc Natl Acad Sci 87, 1164-1168) has revealed a number of similarities at this end of their long 3' untranslated regions (3'-UTRs). The three chicken polyadenylation signals reported here are positioned similarly to three signals evident in the human sequence. This region also contains distinct stretches of identity that are interspersed with regions of little homology. Within these regions of identity are a number of conserved sequence motifs, some of which have been demonstrated to be involved in mRNA metabolism in other systems. The pattern of mRNA sequence conservation demonstrated here suggests that the mechanisms for regulating MHC-A mRNA metabolism have been conserved between chickens and humans.

Animals↗
PubMed1985

Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.

Adenosine Triphosphate↗
PubMed1983

Regulation of cytoskeletal structure and contractility in the brush border.

Calcium plays a vital part in the regulation of cytoskeletal structure and contractility in the brush border of intestinal epithelial cells. An increased Ca2+ concentration causes a rapid but reversible solation of microvillar core filaments, which is mediated by an actin-'severing' protein of the core (Mr = 95 000), referred to as MV-95 K or villin. Results of recent experiments on the Ca2+-dependent interaction of MV-95 K with actin are summarized, and various functions for this actin-severing protein in the intestinal epithelial cell are discussed. Calcium also regulates contractility in the brush border, as shown by studies of isolated brush borders in vitro from chicken intestine. In the presence of Ca2+ (greater than 1 microM) and ATP (at 37 degrees C) isolated brush borders dramatically contract, over 1-4 min, via an isometric contraction of the terminal web region, similar to that observed by R. Rodewald on neonatal rat brush borders. This contraction is mediated, at least in part, by contraction of the circumferential bundle of actin filaments that are associated with the zonula adherens and may also involve myosin-mediated contractions between adjacent microvillar rootlets. Analysis of Ca2+-dependent phosphorylation of brush border proteins during terminal web contraction demonstrates a simultaneous phosphorylation of the regulatory light chain of brush border myosin. Like contraction, the brush border myosin kinase is activated by free Ca2+ (greater than 1 microM) and is inhibited by trifluoperazine, an inhibitor of calmodulin function. These results demonstrate that the machinery required for both production and regulation of force are integral components of the brush border cytostructure.

Animals↗
PubMed1983

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

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.

Adenosine Triphosphate↗
PubMed1982

Analysis of cytoskeletal proteins and Ca2+-dependent regulation of structure in intestinal brush borders from rachitic chicks.

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 of the 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 kilodaltons, actin, and calmodulin, are not detectably different. As assayed by a (125)I-labeled calmodulin gel overlay technique, the same calmodulin-binding proteins are present in rachitic and control brush borders. Two proteins, the 105-kilodalton subunit of the microvillus core and an approximately 30-kilodalton membrane protein, bind calmodulin in a calcium-independent manner. Four cytoskeletal proteins (250, 190, 180, and 150 kilodaltons) and one membrane protein (35 kilodaltons) bind calmodulin only in the presence of calcium. Calcium-dependent solation of microvillus core proteins and calcium-dependent phosphorylation of the 20-kilodalton light chain of brush border myosin both occur as in controls. Our results show that rachintic chicks have brush borders that are quite similar to controls with respect to their ultrastructural organization, constituent contractile proteins, and calcium-dependent regulation of contractility and microvillus core structure. Therefore, the decreased absorption of calcium by intestinal epithelial cells in rachitic chicks is probably not due to gross structural or chemical differences in the brush border cytoskeleton.

Actins↗