Differential expression of sialic acid and N-acetylgalactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential.

In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions of the cells. Fluorescence and electron microscopic analyses revealed differences in the expression profile of carbohydrates recognized by the lectins used on the cell surface of IEC-6, Caco-2, and HCT-116 cells. Lectin blotting identified a range of eight HPA-binding glycoprotein bands with molecular weights of 16-66 kD in Caco-2 cells, six glycoproteins of 16-36 kD, and three protein bands of 35, 24, and 21 kD in IEC-6 cells. A minor band of 66 kD and a major one of 50 kD for WGA-binding glycoproteins were observed in IEC-6 cells and seven glycoproteins of 18-97 kD in Caco-2 and HCT-116 cells but with a visible higher expression of these glycoproteins in the latter. Furthermore, significant quantitative difference in levels of expression of WGA- but not of HPA-binding glycoconjugates was noted, as analyzed by high-resolution scanning electron microscopy using backscattered electron images of cells incubated with gold-labeled lectins.     

Plasma membrane proteins associated with undifferentiated and embryonicDaucus carota tissue

Summary Membranes and membrane proteins from undifferentiated cells and torpedo-stage embryos were compared. A comparison of marker enzyme profiles on linear sucrose gradients showed that the membrane vesicles obtained from 14-day-old embryos were consistently less dense than those obtained from undifferentiated carrot cells. The density of the endoplasmic reticulum, for instance, was 1.10g/cm³ in embryos and 1.12g/cm² in undifferentiated cells. Proteins and glycoproteins from endoplasmic reticulum-, Golgi apparatus-, and plasma membrane-enriched fractions were compared from undifferentiated carrot cells with 14-day-old embryos by 2D SDS-PAGE. When these two tissues were compared, extensive qualitative and quantitative changes in the steady-state endomembrane and plasma membrane proteins were observed. The plasma membrane was examined further by labeling the plasma membrane proteins with sulfosuccinimidylbiotin. Using this specific label, plasma membrane proteins of 54 kD, 41 kD, 16 kD, and 15 kD were found to be uniquely associated with the embryonic state. Conversely, a 70 kD protein and a 45 kD glycoprotein were found to be associated with only undifferentiated cells. These results demonstrate that proteins of the plasma membrane exhibit distinct changes as a result of somatic embryogenesis in carrot.Abbreviations conA concanavilin A - 2,4-D 2,4-dichlorophenoxyacetic acid - p protein - gp glycoprotein - kD kilodalton - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Intracellular localization of the P21rho proteins

We have surveyed the proteins expressed at the surface of different primary neurons as a first step in elucidating how axons regulate their ensheathment by glial cells. We characterized the surface proteins of dorsal root ganglion neurons, superior cervical ganglion neurons, and cerebellar granule cells which are myelinated, ensheathed but unmyelinated, and unensheathed, respectively. We found that the most abundant proteins are common to all three types of neurons. Reproducible differences in the composition of the integral membrane proteins (enriched by partitioning into a Triton X-114 detergent phase) were detected. These differences were most striking when the expression of glycosylphosphatidyl-inositol (GPI)-anchored membrane proteins by these different neurons was compared. Variations in the relative abundance and degree of glycosylation of several well known GPI-anchored proteins, including Thy-1, F3/F11, and the 120-kD form of the neural cell adhesion molecule (N-CAM), and an abundant 60-kD GPI-linked protein were observed. In addition, we have identified several potentially novel GPI-anchored glycoproteins on each class of neurons. These include a protein that is present only on superior cervical ganglion neurons and is 90 kD; an abundant protein of 69 kD that is essentially restricted in its expression to dorsal root ganglion neurons; and proteins of 38 and 31 kD that are expressed only on granule cell neurons. Finally, the relative abundance of the three major isoforms of N-CAM was found to vary significantly between these different primary neurons. These results are the first demonstration that nerve fibers with diverse ensheathment fates differ significantly in the composition of their surface proteins and suggest an important role for GPI-anchored proteins in generating diversity of the neuronal cell surface.     

神经细胞粘附分子结构特征和生理功能

神经细胞粘附分子是一类调节细胞与细胞、细胞与细胞外基质间粘附作用的膜表面糖蛋白,主要有NCAM-180、NCAM-140、NCAM-120三种形式,多与PSA结合在一起。在神经系统中,NCAM的表达具有时间和空间特异性,最主要的作用为调节神经系统的可塑性,这种作用可能是通过PSA-NCAM对AMPA的调节作用,主要是通过调节蛋白激酶的表达和细胞内Ca^2 浓度来实现的。     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.     

GALECTIN-3 expression in differentiating human myeloid cells

Galectin-3 is an endogenous mammalian carbohydrate-binding protein with affinity for ABH group carbohydrate epitopes and polylactosamine glycans present on cell surface and extracellular matrix glycoproteins. It has been shown to play a role in cell differentiation, morphogenesis, adhesion and cell proliferation regulation. Progenitor cell proliferation in bone marrow depends on stem cell factors including those modulating their adhesion to the bone marrow stroma. The present study shows that the 32 kD galectin-3 is developmentally expressed in human myeloid cells and is strongly upregulated on the cell surface of late mature myeloid cells. Despite the fact that the expression of the galectin-3 is very low in CD34+ early myeloid cell, a 70 kD protein is found by Western blotting using antibodies specific to galectin-3, exclusively in those cells. Finally, exogenous human recombinant galectin-3 binds strongly to CD34+ early myeloid cells and emphasizes granulocyte-colony stimulating factor (G-CSF) driven proliferation in vitro.     

Gap junction protein tissue distribution and abundance in the adult brain in Drosophila.

The distribution of gap junction (GJ) protein in Drosophila tissues and developmental stages was determined by probing immuno-blots with an anti-Drosophila GJ protein antibody (R2AP18). All tissues and developmental stages examined contained 18, 24 or 72 kD GJ protein. GJ protein was notably abundant in immuno-blots of homogenates of adult brain tissue. This was confirmed by the direct visualization of GJs in thin sections of adult brain by electron microscopy. GJs were particularly large and numerous between glial cells in the optic lobes and peripheral glial sheath. R2AP18 reactivity was used to identify GJ protein in immunoblots of cell fractions from isolated adult heads. The final GJ-enriched pellets, derived by extracting crude membrane fractions with urea and N-lauroyl sarcosine, contained GJs with reduced profile widths (13-15 nm vs 16-18 nm for native GJs) and which, unlike native GJs in the crude membrane fractions, were immuno-labelled by R2AP18. Immuno-blots of the urea-sarcosine extracted GJ pellets and supernatant contained higher molecular weight R2AP18 immuno-reactive proteins in addition to the 18 kD form which was present in the tissue homogenate and crude membrane fractions. The results confirm previous observations that urea-sarcosine causes alterations in GJ structure and suggest that urea-sarcosine treatment exposes antigenic determinant(s) which are unavailable for R2AP18 binding in non-extracted native GJs. The abundance of GJs in the adult brain and the relatively simple R2AP18 staining patterns in immuno-blots of GJ-enriched fractions from isolated adult heads suggest that this tissue will be useful for further biochemical and molecular studies of GJs in Drosophila.     

高及低转移潜能小鼠肝癌细胞株糖蛋白凝集素结合特征的研究

为探讨肿瘤转移与细胞表面的糖结构的关系,对小鼠肝癌细胞的高、低淋巴道转移株Ｈｃａ－Ｆ和Ｈｃａ－Ｐ进行了蛋白质电泳及经蛋白质印迹术后的５种凝集素（ＣｏｎＡ、ＷＧＡ、ＵＥＡ、ＳＢＡ、ＰＮＡ）结合糖蛋白谱的对比分析．结果表明：高、低转移两株细胞的ＳＤＳ－ＰＡＧＥ谱基本相同;ＣｏｎＡ特异结合糖蛋白共有５种（～７２,８０～９０,～１０４,～１５０,～２００ｋＤ）;其中较明显的差异为～７２ｋＤＣｏｎＡ特异结合糖蛋白,它在Ｈｃａ－Ｐ细胞的表达明显高于Ｈｃａ－Ｆ细胞．ＷＧＡ特异结合糖蛋白１种（～１５０ｋＤ）,在Ｈｃａ－Ｐ细胞的表达略高于Ｈｃａ－Ｆ细胞．此外,实验发现两种性质未明的蛋白质（～７９,～１３０ｋＤ）,后者在Ｈｃａ－Ｐ细胞的含量明显高于Ｈｃａ－Ｐ细胞．结果提示Ｈｃａ－Ｆ和Ｈｃａ－Ｐ细胞不同的转移表型可能与其糖蛋白的表达有一定的关联．     

Sulfoglycolipids bind to adhesive protein amphoterin (P30) in the nervous system.

HNK-1 antibody reactive carbohydrate epitope carried by glycolipids and glycoproteins has been shown to be involved in cell to cell interactions. It has been proposed that the HNK-1 reactive 3-sulfoglucuronyl carbohydrate epitope in glycolipids may interact with a cell surface receptor to promote the biological response in the developing nervous system. The possible occurrence of such a receptor was examined in rat nervous system. A specific binding of sulfoglycolipids to a 30 kD protein from adult rat cerebellum is described. Little binding was found with neutral glycolipids and gangliosides. The 30 kD protein from cerebellum was partially purified on a sulfatide-octyl-Sepharose affinity column. Binding of sulfoglucuronyl glycolipids to a similar 30 kD protein from forebrain previously identified as amphoterin is also shown. Amphoterin is developmentally regulated and is involved in neural cell adhesion and neurite extension.     

Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.     

Preferential turnover of membrane proteins in the intact Chlamydomonas flagellum

Radioactive labeling studies demonstrate a continuous incorporation of newly synthesized proteins and glycoproteins into the intact flagella of Chlamydomonas. This apparent turnover is preferentially occurring for membrane components. In particular, two classes of flagellar membrane components, one a high molecular weight (HMW) group of closely migrating glycoproteins and the other a protein with a MW around 65 kD, are continuously turning over in the vegetative cell. This selective protein turnover may explain the ability of Chlamydomonas to rapidly recover from proteolytic modification of the flagellar surface and to change its flagellar surface properties during the early events in mating.     

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.     

Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma

mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.