Biomedical subjects

Jim J-C Lin

Publications and source records attributed to Jim J-C Lin.

13 recordsLinked to original sources

PubMed2006

Tropomyosin and caldesmon regulate cytokinesis speed and membrane stability during cell division.

The contractile ring and the cell cortex generate force to divide the cell while maintaining symmetrical shape. This requires temporal and spatial regulation of the actin cytoskeleton at these areas. We force-expressed misregulated versions of actin-binding proteins, tropomyosin and caldesmon, into cells and analyzed their effects on cell division. Cells expressing proteins that increase actomyosin ATPase, such as human tropomyosin chimera (hTM5/3), significantly speed up division, whereas cells expressing proteins that inhibit actomyosin, such as caldesmon mutants defective in Ca(2+)/calmodulin binding (CaD39-AB) and in cdk1 phosphorylation sites (CaD39-6F), divide slowly. hTM5 and hTM5/3-expressing cells lift one daughter cell off the substrate and twist. Furthermore, CaD39-AB- and CaD39-6F-expressing cells are sensitive to hypotonic swelling and show severe blebbing during division, whereas hTM5/3-expressing cells are resistant to hypotonic swelling and produce membrane bulges. These results support a model where Ca(2+)/calmodulin and cdk1 dynamically control caldesmon inhibition of tropomyosin-activated actomyosin to regulate division speed and to suppress membrane blebs.

Animals↗

PubMed2006

Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration.

Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.

Alanine↗

PubMed2006

Autoimmunity against human tropomyosin isoforms in ulcerative colitis: localization of specific human tropomyosin isoforms in the intestine and extraintestinal organs.

BACKGROUND: Tropomyosins (TMs) are microfilament cytoskeletal proteins, and 5 major human TM isoforms (hTM1-5) are described. hTMs, particularly isoform 5 (hTM5), is capable of inducing autoantibodies and T-cell response in ulcerative colitis (UC). However, cellular localization of hTM isoforms in the colon and in extraintestinal organs commonly involved in UC is unknown. METHODS: Using isoform-specific monoclonal antibodies, we localized hTMs through immunoperoxidase assay in normal colon (n = 12), small intestine (n = 14), esophagus (n = 10), skin (n = 19), eye (n = 12), gallbladder (n = 16), liver, including bile duct at the porta hepatis (n = 4), lungs (n = 4), and pancreas (n = 4). RESULTS: There is intense expression of hTM5, but not other isoforms, in the epithelium of the colon, gallbladder, and skin. In the eye, hTM5 is expressed only in the nonpigmented ciliary epithelium. Although extrahepatic and interlobar large ductal biliary epithelium was positive, bile canaliculi at the portal tract are negative. The immunoreactivity in epithelial cells from these organs is diffuse cytoplasmic and along the periphery. In colon epithelium, there is intense expression along basolateral areas and luminal (apical) surface. In the small intestinal epithelium, however, hTM5 expression is weak and distinctly different than in the colon. hTM5 was not detected in the squamous epithelium of the esophagus, although it was strongly positive in the skin. hTM1, hTM2, and hTM3 are localized predominantly in smooth muscle of the intestine and blood vessel wall but not the epithelium. HTM4 is localized in the endothelial cells and basement membrane of the colonic epithelium. CONCLUSIONS: hTM5 is the predominant isoform in the epithelium of colon and extraintestinal organs commonly involved in UC. The unique expression of hTM5 may allow its interaction with effector immune cells involved in the immunopathogenesis of UC and its extraintestinal manifestations.

Antibodies, Monoclonal↗

PubMed2005

h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.

Actins↗

PubMed2005

Specific features of neuronal size and shape are regulated by tropomyosin isoforms.

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.

Actin Cytoskeleton↗

PubMed2004

Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Animals↗

PubMed2004

High-throughput gene discovery in the rat.

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3' ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to approximately 50,000 rat UniGene clusters. We have identified, arrayed, and derived 5' ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI.

Animals↗

PubMed2004

1274 full-open reading frames of transcripts expressed in the developing mouse nervous system.

As part of the trans-National Institutes of Health (NIH) Mouse Brain Molecular Anatomy Project (BMAP), and in close coordination with the NIH Mammalian Gene Collection Program (MGC), we initiated a large-scale project to clone, identify, and sequence the complete open reading frame (ORF) of transcripts expressed in the developing mouse nervous system. Here we report the analysis of the ORF sequence of 1274 cDNAs, obtained from 47 full-length-enriched cDNA libraries, constructed by using a novel approach, herein described. cDNA libraries were derived from size-fractionated cytoplasmic mRNA isolated from brain and eye tissues obtained at several embryonic stages and postnatal days. Altogether, including the full-ORF MGC sequences derived from these libraries by the MGC sequencing team, NIH_BMAP full-ORF sequences correspond to approximately 20% of all transcripts currently represented in mouse MGC. We show that NIH_BMAP clones comprise 68% of mouse MGC cDNAs > or =5 kb, and 54% of those > or =4 kb, as of March 15, 2004. Importantly, we identified transcripts, among the 1274 full-ORF sequences, that are exclusively or predominantly expressed in brain and eye tissues, many of which encode yet uncharacterized proteins.

Animals↗

PubMed2003

Specification of actin filament function and molecular composition by tropomyosin isoforms.

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.

Actins↗

PubMed2002

Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling.

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses.

Animals↗

PubMed2002

Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle-specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281-1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole-mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both beta-catenin and N-cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with beta-catenin within the N-cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell-cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell-cell contacts and possibly in myofibrillogenesis.

Animals↗

PubMed2002

Role of VEGF family members and receptors in coronary vessel formation.

The specific roles of vascular endothelial growth factor (VEGF) family members and their receptors (VEGFRs) in coronary vessel formation were studied. By using the quail heart explant model, we found that neutralizing antibodies to VEGF-B or VEGF-C inhibited tube formation on the collagen gel more than anti-VEGF-A. Soluble VEGFR-1, a receptor for VEGF-A and -B, inhibited tube formation by 87%, a finding consistent with that of VEGF-B inhibition. In contrast, addition of soluble VEGFR-2, a receptor for VEGF family members A, C, D, and E, inhibited tube formation by only 43%. Acidic FGF-induced tube formation dependency on VEGF was demonstrated by the attenuating effect of a soluble VEGFR-1 and -2 chimera. The localization of VEGF R-2 and R-3 was demonstrated by in situ hybridization of serial sections, which documented marked accumulations of transcripts for both receptors at the base of the truncus arteriosus coinciding with the temporal and spatial formation of the coronary arteries by means of ingrowth of capillary plexuses. This finding suggests that both VEGFR-2 and R-3 may play a role in the formation of the coronary artery roots. In summary, these experiments document a role for multiple members of the VEGF family and their receptors in formation of the coronary vascular bed.

Animals↗

PubMed2002

Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer.

BACKGROUND & AIMS: Nonmuscle human tropomyosin (hTM) isoforms have distinct functions and may play important roles in various disease processes. METHODS: In an attempt to identify colon epithelial tropomyosin isoform, a complementary DNA library prepared from a human colon cancer cell line T84 was screened by an oligonucleotide probe complementary to messages of all known hTM isoforms. A novel clone called TC22 was obtained. The amino acid sequence of TC22 isoform is identical to isoform 5 (hTM5) apart from the C terminal domain, amino acids 222-247 coding the exon 9. RESULTS: Northern blot analysis showed that TC22 message is expressed in transformed epithelial cell lines and tumor tissues but not in normal epithelial cells. We developed a monoclonal antibody specific to TC22 isoform (TC22-4). By Western blot and immunoperoxidase assays, we analyzed 105 colonic specimens (fresh frozen and formalin fixed) from 96 patients with colon polyps (hyperplastic) or adenomas with or without dysplasia and cancer. Twenty-one of 22 (95%) of colon cancer specimens showed the presence of TC22, compared with only 1 of the 17 normal colon specimens and none of the 13 hyperplastic polyps (P < 0.0001). As assayed by immunoperoxidase staining, TC22 expression progressively increased in benign adenomatous polyps (35%) and polyps with mild and severe dysplasia (57% and 100%, respectively). CONCLUSIONS: We cloned and sequenced a novel hTM isoform, TC22, which is strongly associated with colonic neoplasia and carcinoma. TC22 may provide a useful biomarker for surveillance of colon cancer.

Adenoma↗