Bradykinin Induces Vinculin-Stained Focal Adhesions (FA) Dissipation by Vesicles Internalization.

MARÍA G MÁRQUEZ; MARÍA C. FERNÁNDEZ ; NICOLÁS O. FAVALE; NORMA B. STERIN-SPEZIALE

Mar del Plata

Congreso; Reunion anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2007

SAIB

CB-P18.RAFT-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE(PIP2) IS ESSENTIAL FOR TUBULAR ORGANIZATION OFRENAL PAPILLAMárquez G, Leocata Nieto F, Fernández-Tome M, Sterin-SpezialeN.Biología Celular, Facultad de Farmacia y Bioquímica, UBA.IQUIFIB-CONICET. E-mail: g_marquez@uolsinectis.com.arTubular organization of renal papilla depends on cell-cell and cellmatrix attachment, which are principally mediated by adherensjunctions (AJ) and focal adhesions (FA), respectively. Vinculin (V)is present in both type of adhesions, and talin (T) only in FA. Toinvestigate whether PIP2 constitutes a molecular platform for FAand AJ assembly, we treated collecting duct cells with the raftdisruptionagent cyclodextrin (CD), and cell adhesion structureswere observed by confocal microscopy. CD caused importantchanges in cell morphology with loss of cell-cell adhesions and cellretraction, dissipation of V, T and PIP2 from FA accompanied by adistribution in certain zones of the plasma membrane suggesting adelocalization of these proteins from raft, where FA were locatedbefore treatment. We also treated cultured cells with the specificPIP2 sequestering agent neomycin, and with the PIP2 synthesisinhibitor LiCl. Neomycin induced an almost complete loss of Vfrom FA and AJ, and T from FA, and intensity of fluorescence ofPIP2 staining appeared to be increased. LiCl induced a dissipationof V from FA and AJ, whereas T - immunostaining FA remainedintact, and PIP2 immunoreactivity was substantially reduced.Westernblot analysis correlated with the above observations Theseresults demonstrate that the existence of PIP2-enriched raft is arequirement for maintenance of FA and AJ which ensures tubularorganization of renal tissue.CB-P19.TNF-α PRODUCTION BY HUMAN LYMPHOCYTES ISREDUCED BY LACTOBACILLUS REUTERI CRL 1098:EFFECT OF LOW CELLULAR MEMBRANE CHOLESTEROLSoria MA, Font de Valdez G, Rodriguez AV.CERELA. Chacabuco 145. 4000- Tucumán. Argentina. E-mail:anavirr@cerela.org.arCholesterol-rich microdomains, named lipid rafts, present in themembrane of eukaryotic cells, play a critical role in several biologicalprocesses such us production of cytokines. We investigated theeffect of probiotic Lactobacillus reuteri CRL 1098 on theproinflamatory cytokine TNF-α production by humanlymphocytes with low membrane cholesterol content. Humanlymphocytes were isolated with a density gradient centrifugationand treated with 10 mM methyl-beta-cyclodextrin (MßCD) for 12min at 37°C to reduce cholesterol levels. Under these experimentalconditions cell viability was higher than 90%, and cholesterol wasreduced by 55%. TNF-α was detected by chemisluminescent assay.The lymphocytes treated with and without MßCD were thenincubated with L. reuteri at different MOI and times of incubation.The results show that TNF-α production of 149pg/ml/ 1.00x 106by normal lymphocytes, was reduced by 23% when the cells wereincubated with L. reuteri at 20 MOI for 4 h. When low-cholesterollymphocytes were incubated with L. reuteri under the sameconditions, a substantial reduction of 51% of TNF-α amount wasobserved. These results show for the first time that a probioticbacteria reduced TNF-α production by human lymphocytes andsuggest that lipid rafts could be involved in this cellular response.CB-P20.MICROFILAMENTS ARE REQUIRED FOR THEBIOGENESIS OF THE COXIELLA-REPLICATIVECOMPARTMENTAguilera M, Carminati S, Beron W.IHEM-CONICET, Facultad de Ciencias Medicas, UniversidadNacional de Cuyo, Mendoza, Argentina. E-mail:moaguile@fcm.uncu.edu.arQ fiber is a disease caused by the intracellular pathogen Coxiellaburnetii. This bacterium generates a large replicative compartmentin the host cell, known as parasitophorous vacuole (PV). We haveinvestigated the role of the actin cytoeskeleton in the biogenesis ofthe PV by using different agents. CHO cells were infected with C.burnetii Phase II for 16 hours and actin was visualized by fluorescencemicroscopy using phalloidin, at different times post infection. Atearly times after infection some small Coxiella-containing vacuoleswere decorated by actin. However by 24-48 hs post infection therewas a marked increase in the size of the PV and the colocalizationwith actin increased to 68-95%. Treatment of infected cells withthe actin polymerization inhibitor latrunculin completely blockedPV formation. Interestingly, this inhibitory effect was reversed bywashing out the drug and the large PV reformed after few hours.Similarly, PV generation was hampered by the miosin inhibitorbutanodionemonoxime (BDM) although in an irreversible way. Theseresults indicate that actin and miosin motors play a critical role inthe biogenesis of the C. burnetii replicative niche.Posters