Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration.
ABSTRACT: The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.
Project description:BACKGROUND:Among gynecological cancers, ovarian carcinoma has the highest mortality rate, and chemoresistance is highly prevalent in this cancer. Therefore, novel strategies are required to improve its poor prognosis. Formation and disassembly of focal adhesions are regulated dynamically during cell migration, which plays an essential role in cancer metastasis. Metastasis is intricately linked with resistance to chemotherapy, but the molecular basis for this link is unknown. METHODS:Transwell migration and wound healing migration assays were used to analyze the migration ability of ovarian cancer cells. Real-time recordings by total internal reflection fluorescence microscope (TIRFM) were performed to assess the turnover of focal adhesions with fluorescence protein-tagged focal adhesion molecules. SOCE inhibitors were used to verify the effects of SOCE on focal adhesion dynamics, cell migration, and chemoresistance in chemoresistant cells. RESULTS:We found that mesenchymal-like chemoresistant IGROV1 ovarian cancer cells have higher migration properties because of their rapid regulation of focal adhesion dynamics through FAK, paxillin, vinculin, and talin. Focal adhesions in chemoresistant cells, they were smaller and exhibited strong adhesive force, which caused the cells to migrate rapidly. Store-operated Ca2+ entry (SOCE) regulates focal adhesion turnover, and cell polarization and migration. Herein, we compared SOCE upregulation in chemoresistant ovarian cancer cells to its parental cells. SOCE inhibitors attenuated the assembly and disassembly of focal adhesions significantly. Results of wound healing and transwell assays revealed that SOCE inhibitors decreased chemoresistant cell migration. Additionally, SOCE inhibitors combined with chemotherapeutic drugs could reverse ovarian cancer drug resistance. CONCLUSION:Our findings describe the role of SOCE in chemoresistance-mediated focal adhesion turnover, cell migration, and viability. Consequently, SOCE might be a promising therapeutic target in epithelial ovarian cancer.
Project description:Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP-CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.
Project description:Total RNA was extracted from skin biopsies before and after imatinib (Gleevec) treatment using Qiagen RNeasy fibrous tissue kit. RNA was amplified using the Ambion Amino Allyl MessageAmp II aRNA kit. Amplified skin RNA (labeled with Cy5) and amplified Stratagene Human Universal Reference RNA (labeled with Cy3) were competitively hybridized to HEEBO microarrays in duplicate as described (http://www.microarray.org/sfgf/heebo.do). 161a/b: Patient 1, pre-Gleevec treatment 170a/b: Patient 1, post-Gleevec treatment 215a/b: Patient 2, pre-Gleevec treatment 232a/b: Patient 2, post-Gleevec treatment A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed. Compound Based Treatment: Treated with 100 mg Gleevec twice daily for 3 months or 200 mg Gleevec daily for 6 months Keywords: compound_treatment_design Overall design: Computed
Project description:Total RNA was extracted from skin biopsies before and after imatinib (Gleevec) treatment using Qiagen RNeasy fibrous tissue kit. RNA was amplified using the Ambion Amino Allyl MessageAmp II aRNA kit. Amplified skin RNA (labeled with Cy5) and amplified Stratagene Human Universal Reference RNA (labeled with Cy3) were competitively hybridized to HEEBO microarrays in duplicate as described (http://www.microarray.org/sfgf/heebo.do). 161a/b: Patient 1, pre-Gleevec treatment 170a/b: Patient 1, post-Gleevec treatment 215a/b: Patient 2, pre-Gleevec treatment 232a/b: Patient 2, post-Gleevec treatment A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed. Compound Based Treatment: Treated with 100 mg Gleevec twice daily for 3 months or 200 mg Gleevec daily for 6 months Keywords: compound_treatment_design Computed
Project description:Leukocytes circulating in the blood stream leave out of blood vessels and infiltrate into inflamed tissues to perform immune responses. Endothelial cells (ECs) lining interior of the post-capillary venules regulate various steps of leukocyte extravasation. In response to inflammatory signals, ECs upregulate adhesion molecules and produce/present chemokines to support firm adhesion and intraluminal crawling of leukocytes. They also remodel junctions to facilitate leukocyte transendothelial migration (TEM). While roles of apical/lateral components of EC layers in regulating leukocyte extravasation have been extensively investigated, relatively little attention has been paid to the basal part of EC layers comprising subendothelial spaces. In this study, we employed interference reflection microscopy (IRM), a microscopy technique specialized for label-free visualization of cell-substrate contact, to study detailed dynamic interactions between basal part of ECs and T cells underneath EC monolayer. For TEM, T cells on EC monolayer extended protrusions through junctions to explore subendothelial spaces, and EC focal adhesions (EC-FAs) acted as physical barrier for the protrusion. Therefore, preferential TEM occurred through junctions where near-junction focal adhesion (NJ-FA) density of ECs was low. After TEM, T cells performed subendothelial crawling (SEC) with flattened morphology and reduced migration velocity due to tight confinement. T cell SEC mostly occurred through gaps formed in between EC-FAs with minimally breaking EC-FAs. Tumor necrosis factor-? (TNF-?) treatment significantly loosened confinement in subendothelial spaces and reduced NJ-FA density of ECs, thus remodeled basal part of EC layer to facilitate leukocyte extravasation.
Project description:Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools.
Project description:Blocking phosphorylation of peroxisome proliferator-activated receptor (PPAR)? at Ser(273) is one of the key mechanisms for antidiabetes drugs to target PPAR?. Using high-throughput phosphorylation screening, we here describe that Gleevec blocks cyclin-dependent kinase 5-mediated PPAR? phosphorylation devoid of classical agonism as a PPAR? antagonist ligand. In high fat-fed mice, Gleevec improved insulin sensitivity without causing severe side effects associated with other PPAR?-targeting drugs. Furthermore, Gleevec reduces lipogenic and gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues. Interestingly, Gleevec increases browning of white adipose tissue and energy expenditure. Taken together, the results indicate that Gleevec exhibits greater beneficial effects on both glucose/lipid metabolism and energy homeostasis by blocking PPAR? phosphorylation. These data illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type 2 diabetes.
Project description:Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.
Project description:Tyrosine kinases present attractive drug targets for specific types of cancers. Gleevec, a well-known therapeutic agent against chronic myelogenous leukemia, is an effective inhibitor of Abl tyrosine kinase. However, Gleevec fails to inhibit closely homologous tyrosine kinases, such as c-Src. Because many structural features of the binding site are conserved, the molecular determinants responsible for binding specificity are not immediately apparent. Some have attributed the difference in binding specificity of Gleevec to subtle variations in ligand-protein interactions (binding affinity control), whereas others have proposed that it is the conformation of the DFG motif, in which ligand binding is only accessible to Abl and not to c-Src (conformational selection control). To address this issue, the absolute binding free energy was computed using all-atom molecular dynamics simulations with explicit solvent. The results of the free energy simulations are in good agreement with experiments, thereby enabling a meaningful decomposition of the binding free energy to elucidate the factors controlling Gleevec's binding specificity. The latter is shown to be controlled by a conformational selection mechanism and also by differences in key van der Waals interactions responsible for the stabilization of Gleevec in the binding pocket of Abl.
Project description:Mechanical linkage between cell-cell and cell-extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell-cell and cell-ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell-cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell-cell and cell-ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell-cell pairs resulted in shorter junction lengths and constant cell-cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell-cell forces and was evenly distributed along cell-cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.