Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion

A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.     

Prohibitins Are Required for Cancer Cell Proliferation and Adhesion

Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.     

Mechanisms and Functions of Vinculin Interactions with Phospholipids at Cell Adhesion Sites

The cytoskeletal protein vinculin is a major regulator of cell adhesion and attaches to the cell surface by binding to specific phospholipids. Structural, biochemical, and biological studies provided much insight into how vinculin binds to membranes, what components it recognizes, and how lipid binding is regulated. Here we discuss the roles and mechanisms of phospholipids in regulating the structure and function of vinculin and of its muscle-specific metavinculin splice variant. A full appreciation of these processes is necessary for understanding how vinculin regulates cell motility, migration, and wound healing, and for understanding of its role in cancer and cardiovascular diseases.     

NUAK1增加乳腺癌细胞的趋化和粘附能力

本课题组先前已证明NUAK1/ARK5可通过影响F-actin的聚合从而促进乳腺癌细胞的侵袭和转移. 但是NUAK1是否还通过其它机制影响乳腺癌的侵袭和转移尚有待于探讨.本文证明NUAK1还可以影响乳腺癌细胞趋化、粘附能力从而在乳腺癌细胞侵袭转移中起重要作用. 应用化学合成的小RNA干扰质粒转染到乳腺癌细胞系MDA MB 231中,用免疫印迹技术检测NUAK1蛋白的表达情况. 结果显示,在敲除NUAK1的细胞（siNUAK1/MDA231）中, NUAK1蛋白表达水平明显降低;趋化运动实验结果显示, siNUAK1/MDA231细胞的趋化运动能力比未处理组（Scr/MDA231）细胞明显降低;细胞粘附实验结果显示, EGF刺激5 min、15 min后,siNUAK1/MDA231细胞比Scr/MDA231细胞粘附细胞数量均明显减少;免疫印迹技术检测integrin β1磷酸化验证NUAK1影响乳腺癌细胞粘附的机制. 结果显示,siNUAK1/MDA231细胞内integrin β1磷酸化比Scr/MDA231细胞不同程度降低. 上述结果表明, NUAK1通过磷酸化integrin β1促进乳腺癌细胞与纤维粘连蛋白的粘附,从而促进乳腺癌的侵袭和转移.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

High-Throughput Flow Cytometry Screening Reveals a Role for Junctional Adhesion Molecule A as a Cancer Stem Cell Maintenance Factor

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Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane-attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell-intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane-integrated phosphorylated L1–30. Phase separation experiments with Triton X-114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane-anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1-containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.     

Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Cell Adhesion Molecules and the Migration of LHRH Neurons during Development

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.     

Interactions of the Cytoplasmic Domain of P-Selectin with Clathrin-coated Pits Enhance Leukocyte Adhesion under Flow

Flowing leukocytes tether to and roll on P-selectin, a receptor on endothelial cells that is rapidly internalized in clathrin-coated pits. We asked whether the association of P-selectin with clathrin-coated pits contributes to its adhesive function. Under flow, rolling neutrophils accumulated efficiently on CHO cells expressing wild-type P-selectin or a P-selectin construct with a substitution in the cytoplasmic domain that caused even faster internalization than that of the wild-type protein. By contrast, far fewer rolling neutrophils accumulated on CHO cells expressing P-selectin constructs with a deletion or a substitution in the cytoplasmic domain that impaired internalization. Neutrophils rolled on the internalization-competent constructs with greater adhesive strength, slower velocity, and more uniform motion. Flowing neutrophils tethered equivalently to internalization-competent or internalization-defective P-selectin, but after tethering, they rolled further on internalization-competent P-selectin. Confocal microscopy demonstrated colocalization of α-adaptin, a component of clathrin-coated pits, with wild-type P-selectin, but not with P-selectin lacking the cytoplasmic domain. Treatment of CHO cells or endothelial cells with hypertonic medium reversibly impaired the clathrin-mediated internalization of P-selectin and its ability to support neutrophil rolling. Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits provide a novel mechanism to enhance leukocyte adhesion under flow.     

Biochemical Specialization of Photosynthetic Cell Layers and Carbon Flow Paths in Suaeda monoica

Suaeda monoica Frossk. ex J. F. Gmel is a C₄ plant with three different photosynthesizing cell layers. The outer chlorenchymatous layer shows a high activity of phosphoenolpyruvate (PEP) carboxylase but none of ribulose bisphosphate (RuBP) carboxylase. The electrophoretic protein band of RuBP carboxylase was missing in this layer. The second chlorenchymatous cells layer shows a very high activity of RuBP carboxylase and NAD malic enzyme and only traces of activity of PEP carboxylase. The third photosynthesizing cell type is comprised of the water tissue. It has moderate activities of RuBP carboxylase and PEP carboxylase. A model for carbon flow in Suaeda monoica leaves is proposed.     

SHC–α5β1 Integrin Interactions Regulate Breast Cancer Cell Adhesion and Motility

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with α5β1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.     

Nuclear Localized Phosphorylated FADD Induces Cell Proliferation and is Associated with Aggressive Lung Cancer

Fas-associated death domain (FADD)/Mort1 was initially reported as a pro-apoptotic adaptor molecule that recruits the initiator caspases 8 and 10 to promote formation of the death-inducing signal complex (DISC) and mediates receptor induced apoptosis. Recent studies have brought to light ancillary death receptor induced apoptosis-independent activities of FADD that include cell cycle regulation, NF-κB activation, cell proliferation and role during embryonic development. We have recently shown that in lung adenocarcinomas increased FADD mRNA and protein are significantly associated with poor survival and that FADD overexpression was not due to gene amplification and/or mutation. In this study we showed that the nuclear localization of FADD and elevated expression of the phosphorylated form of FADD (p-FADD) correlated most closely with an increase in NF-κB activity and poor clinical outcome. These results suggest that levels of p-FADD may be used as a prognostic biomarker for predicting survival of lung cancer patients.     

Co-Occurring Gland Angularity in Localized Subgraphs: Predicting Biochemical Recurrence in Intermediate-Risk Prostate Cancer Patients

Quantitative histomorphometry (QH) refers to the application of advanced computational image analysis to reproducibly describe disease appearance on digitized histopathology images. QH thus could serve as an important complementary tool for pathologists in interrogating and interpreting cancer morphology and malignancy. In the US, annually, over 60,000 prostate cancer patients undergo radical prostatectomy treatment. Around 10,000 of these men experience biochemical recurrence within 5 years of surgery, a marker for local or distant disease recurrence. The ability to predict the risk of biochemical recurrence soon after surgery could allow for adjuvant therapies to be prescribed as necessary to improve long term treatment outcomes. The underlying hypothesis with our approach, co-occurring gland angularity (CGA), is that in benign or less aggressive prostate cancer, gland orientations within local neighborhoods are similar to each other but are more chaotically arranged in aggressive disease. By modeling the extent of the disorder, we can differentiate surgically removed prostate tissue sections from (a) benign and malignant regions and (b) more and less aggressive prostate cancer. For a cohort of 40 intermediate-risk (mostly Gleason sum 7) surgically cured prostate cancer patients where half suffered biochemical recurrence, the CGA features were able to predict biochemical recurrence with 73% accuracy. Additionally, for 80 regions of interest chosen from the 40 studies, corresponding to both normal and cancerous cases, the CGA features yielded a 99% accuracy. CGAs were shown to be statistically signicantly () better at predicting BCR compared to state-of-the-art QH methods and postoperative prostate cancer nomograms.     

蛋白酶活化受体2促进结肠癌细胞与基质粘附

目的:蛋白酶活化受体2激活对结肠癌细胞HT29粘附能力的影响。方法:通过激活多肽活化细胞膜表面的蛋白酶活化受体2,检测对结肠癌细胞与FN和Matrigel粘附能力的影响。建立蛋白酶活化受2敲降的结肠癌细胞,检测对结肠癌细胞与基质粘附能力的影响。结果:蛋白酶活化受体2激活,能够促进结肠癌细胞与基质的粘附能力;而蛋白酶活化受体敲降则抑制粘附。结论:蛋白酶活化受体2参与对结肠癌细胞粘附能力的调控,其激活能够促进结肠癌细胞与基质的粘附能力。     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Spatio-Temporally Restricted Expression of Cell Adhesion Molecules during Chicken Embryonic Development

Differential cell adhesive properties are known to regulate important developmental events like cell sorting and cell migration. Cadherins and protocadherins are known to mediate these cellular properties. Though a large number of such molecules have been predicted, their characterization in terms of interactive properties and cellular roles is far from being comprehensive. To narrow down the tissue context and collect correlative evidence for tissue specific roles of these molecules, we have carried out whole-mount in situ hybridization based RNA expression study for seven cadherins and four protocadherins. In developing chicken embryos (HH stages 18, 22, 26 and 28) cadherins and protocadherins are expressed in tissue restricted manner. This expression study elucidates precise expression domains of cell adhesion molecules in the context of developing embryos. These expression domains provide spatio-temporal context in which the function of these genes can be further explored.