cell migration; chemotaxis; cell adhesion; cell polarization; membrane microdomain; flotillin; neutrophil granulocyte; T-lymphocyte; HT1080 fibrosarcoma cell; Inflammation; metastasis; leukocyte; tumor cell
Martinelli Sibylla, Chen Emily .J.H., Clarke Fiona, Lyck Ruth, Affentranger Sarah, Burkhardt Janis K. (2013), Ezrin/radixin/moesin proteins and flotillins cooperate to promote uropod formation in T cells., in Frontiers in Immunology
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Baumann Tommy, Affentranger Sarah, Niggli Verena (2012), Evidence for chemokine-mediated coalescence of preformed flotillin heterooligomers in human T-cells., in Journal of Biological Chemistry
, 287, 39664-39672.
Lottaz Daniel, Maurer Christoph A., Noel Agnes, Blacher Silvia, Huguenin Maya, Nievergelt Alexandra, Niggli Verena, Kern. Alexander, Müller Stefan, Seibold Frank, Friess Helmut, Becker-Pauly Christoph, Stöcker Walter, Sterchi Erwin E. (2011), Enhanced activity of meprin-alpha, a pro-migratory and pro-angiogenic protease, in colorectal cancer., in PLos One
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Affentranger Sarah, Martinelli Sibylla, Hahn Jonas, Rossy Jérémie, Niggli Verena (2011), Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes., in BMC Cell Biology
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1. Background: Directed cell locomotion is crucial for physiological processes such as embryonal development and host defense against pathogens and plays also an important role in pathological events such as neoplastic invasion. Localized signalling resulting in reorganization of the cytoskeleton, changes in cell shape and cell adhesion have been recognized to be crucial for cell locomotion. Many aspects of localized signalling are incompletely characterized. Membrane microdomains represent a central feature of cellular organization which could be crucial for the local activation of specific signalling cascades and adhesion molecules. Reggie/flotillin-1 and -2 are important microdomain-associated proteins whose functions in cell migration are as yet not well defined. 2. Working hypothesis: Polarized migrating leukocytes contain membrane microdomains of different composition in the leading edge and in the rear. We observed that capping of the microdomain-associated proteins flotillin-1 and -2 is a very early event during neutrophil polarization. Upon stimulation of cells these proteins are concentrated at the site of the later uropod. These novel findings suggest that flotillins play an important role in uropod formation and/or organization of adhesion receptors, cytoskeletal proteins and/or signalling molecules in the uropod. Depletion of flotillins should thus result in defects in migration and adhesion. 3. Specific aims: We plan to study the dynamic reorganization of flotillins in polarizing and migrating leukocytes and tumor cells, and the role of cytoskeletal and signalling proteins in these processes. We also will attempt to interfere with flotillin functions using small interfering RNA (siRNA) or dominant-negative mutants of flotillins. These experiments should lead to information on whether flotillin-1 and -2 are required for adhesion, polarization, migration and chemotaxis of leukocytes and tumor cells. The role of flotillin isoforms in uropod localization of cytoskeletal molecules and adhesion receptors and in regulating chemoattractant-mediated signalling pathways will be addressed. Using expression of tagged flotillins in leukocytes and co-immunoprecipitation we will moreover attempt to identify novel flotillin interaction partners. Expression of mutated flotillins will be used to obtain information on the roles of flotillin domains for scaffold formation, cell motility etc. Fluorescence resonance energy transfer (FRET) will be used to monitor scaffold protein interactions in intact cells.4. Methods: transient transfection of cells with tagged flotillin-2 and/or flotillin-1, and mutants of these proteins; siRNA-mediated depletion of flotillin-1 and -2 in HL-60 cells, lymphocytes, tumor cells; analysis of impact of depletion of flotillin isoforms on signalling, adhesion, polarization and random as well as directed migration of these cells; expression of tagged wild-type and mutated flotillins, co-immunoprecipitation using a tag-directed antibody and mass spectrometry analysis of precipitated proteins for identification of novel flotillin interaction partners; FRET.5. Expected value of the proposed project: We expect that the obtained knowledge provides novel information on the role of flotillin-rich membrane microdomains in regulating signalling, cytoskeletal reorganization, polarization, adhesion and migration in leukocytes and tumor cell lines. This should be important for our understanding of the mechanisms of neutrophil and tumor cell motility, processes involved in inflammation and metastasis.