Insulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these... Show moreInsulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5 beta (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5 beta or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchangewere stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release. Show less
Verkoeijen, S.; Ma, Y.F.; Roosmalen, W. van; Lalai, R.; Miltenburg, M.H.A.M. van; Graauw, M. de; ... ; Le Dévédec, S.E. 2019
Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesiondynamics, a process required for the tumor cell migration and invasion.... Show morePaxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesiondynamics, a process required for the tumor cell migration and invasion. Phosphorylation of the serine residue 178requires c-Jun NH2-terminal kinase (JNK) activation, which occurs downstream of epidermal growth factor receptor (EGFR)-mediated signaling and drives cell migration. In this study, we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs. With those, we next performed several in vitro assays including cell proliferation, migration and focal adhesion dynamics. Finally, we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration, which was correlated with impaired focal adhesion dynamics. Moreover, paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model, demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression. Expression of paxillinS178A caused a decrease in EGFR expression while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics. Furthermore, re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby, in EGF-driven cell migration and metastasis formation. Show less
Le Dévédec, S.E.; Geverts, B.; Bont, H.J.G.M. de; Yan, K.; Verbeek, F.J.; Houtsmuller, A.B.; Water, B. van de 2012
Understanding the molecular mechanisms of survival and migratory pathways in cancer cells is essential to better comprehending cancer progression, metastasis formation and drug resistance, thereby... Show moreUnderstanding the molecular mechanisms of survival and migratory pathways in cancer cells is essential to better comprehending cancer progression, metastasis formation and drug resistance, thereby benefiting the development of novel anticancer treatments. The overall goal of the work is to better understand the role and mechanism of focal adhesion mediated signaling in the control of anticancer drug-related survival signaling of breast tumor cells in vivo as well as the regulation of cell migration of breast tumor cells in vitro. Mainly, we focused on the role of the focal adhesion associated proteins (focal adhesion kinase and paxillin) in cell survival, cytoskeleton reorganization and migration. Moreover, we would like to identify the pattern of C-X-C chemokines and corresponding receptors that mediate the downstream signaling events in tumor cell migration and invasion. Show less
The elucidation of the molecular and cellular mechanisms of ischemic ARF very important in finding new strategies to reduce or prevent renal injury. FAK is an important FA protein with tyrosine... Show moreThe elucidation of the molecular and cellular mechanisms of ischemic ARF very important in finding new strategies to reduce or prevent renal injury. FAK is an important FA protein with tyrosine kinase and scaffolding function. The general goal of this thesis was to investigate the role of FAK during I/R. Using a unilateral renal I/R rat model, we show the presence of tyrosine phosphorylated FAs in vivo and disruption of FAs and the F-actin network after ischemia and rebuild during reperfusion. FAK phosphorylation occured on different tyrosine residues during the reperfusion implicating a role of FAK. ERK is known to be involved in FA signaling. We studied the role of ERK signaling pathway during I/R in vivo using the inhibitor U0126. Inhibition prevented the changes in FA protein phosphorylation after ischemia and diminished injury. We used an inducible proximal tubule cell specific FAK knockout model to investigate the role of FAK in I/R. We show that FAK knockout mice are less susceptible to I/R injury compared to their wildtype littermates. Furthermore we studied FAK signaling under normal and ATP depletion in vitro. FAK deleted renal cells show no differences in morphology. However FAK knockout cells have increased FAs, aberrant stress fibers and impaired spreading. During recovery from ATP depletion, FAK deleted cells show impaired recovery of FAs and stress fibers. Show less
