Poster reviderad 091126 copy - PDF

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Poster reviderad 091126 copy - PDF Powered By Docstoc
					Program # 1038




                                                     Influence of Water Fluxes on Cell Morphology and Motility
                                                              Thommie Karlsson , Veronika Borutinskaite , Ruta Navakauskiene , Tommy Sundqvist , Karl-Eric Magnusson , Vesa M Loitto
          1 Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, SE-58185, Linköping, Sweden, 2 Department of Developmental Biology, Institute of Biochemistry, LT-08662, Vilnius, Lithuania



    Abstract                                                                                                                                                                                     AQP9 co-localizes with α-dystrobrevin in di erentiated HL60 cells                                                                                             Conclusion
    Water uxes over the plasma membrane are mainly driven through water                                                                                                                                                                                                                                                                                        This study shows that AQP9 induces lopodia and a ects adhesiveness and migration. Fur-
                                                                                                                                                                                                      α-Dystrobrevin                                             AQP9-GFP                  Merged
    channels known as aquaporins (AQPs). Today thirteen mammalian AQPs are                                                                                                                                                                                                                                                                                     thermore, it augments cellular susceptibility to volume regulation in response to hyper-
    known and they in uence a number of processes including cell motility.                                                                                                                                                                                                                                                                                     osmotic conditions. We believe that interplay between water and ion-channels creates an os-
    AQP9 is present in human leukocytes, which are highly motile cells, and                                                                                                                                                                                                                                                                                    motic gradient that enhances ow of water across the cell membrane and thereby enables
    when it is expressed in broblasts these develop a highly lopodial pheno-
                                                                                                                                              AQP9-GFP
                                                                                                                                                                                                                                                                                                                                                               rapid cellular shape changes such as the formation of membrane protrusions. Such proper-
    type. Using stably transfected AQP9-GFP cell lines and various imaging                                                                                                                                                                                                                                                                                     ties are required for cell movement through narrow pores of transwell inserts or in the extra-
    techniques we show here that AQP9 induces highly dynamic lopodia in                                                                                                                                                                                                                                                                                        cellular matrix. However, the lopodia also contributes by increasing the cell surface area and
    di erent cell types. It also augments their motility, adhesiveness, polariza-                                                                                                                                                                                                                                                                              thereby the area of insertion of adhesion molecules e.g. integrins. Moreover, this process
    tion and sensitivity to hyper-osmotic changes, which is accompanied by an                                                                                                                                                                                                                                                                                  might be strengthened by sca olding proteins such as α-dystrobrevin enabling anchorage of
    increase in β -integrin expression and a co-localization with the sca olding                                                                                                                                                                                                                                                                               AQP9 to specialized membrane micro-domains. A hypothetical working models for AQP9 in-
                                                                                                                                                            GFP
    protein α-dystrobrevin. We suggest that the increased water uxes that                                                                                                                                                                                                                                                                                      duced lopodia is described below.
    occur at the site of AQP insertion enhance the cell ability for shape changes
    and thereby enhance its migratory capacity. Furthermore, the increased                                                                                                                                                                                                                                                                                     Key references:
    surface area that follows induction of lopodia enlarges the area for inser-                                                                                                                                                                                                                                                                                Bolte S and Cordelieres FP, 2006, J Microsc 224: 213                        Hodges BL, et al., 1997, J Cell Sci 110: 2873
                                                                                                                                                            Both α-dystrobrevin and AQP9 are distributed at the cellular membrane where they also co-localize at micro-                                                                                        Cohn RD and Mayer U, 1999, J Neurosci 163: 140                              Hu J and Verkman AS, 2006, Faseb J 20: 1892
    tion of adhesion molecules and thus the attachment. Moreover, this process                                                                              scopic resolution in AQP9 transfected HL60 cells. This localization can further be con rmed by evaluation of                                                                                       Connors NC and Konfuji P, 2006, Glia 53: 124                                Liang HT, et al., 2008, Clin Exp Pharmacol Physiol 35: 7
    is promoted by co-localization with α-dystrobrevin, which could help                                                                                    the Li-graphs where a co-localization yields a C-shaped curve bending towards the upper right corner (B).                                                                                          Cote PD, et al., 2002, J Biol Chem 277: 4672                                Loitto VM, et al., 2007, Exp Cell Res 313: 1295
                                                                                                                                                            Moreover, the Pearson´s Coe cient was calculated to 0.95 for AQP9 and α-dystrobrevin (not shown). Scale bar
    anchor AQP9 to membrane micro-domains through a PDZ-motif interac-                                                                                                                                                                                                                                                                                         Driss A, et al., 2006, Am J Physiol Gastrointest Liver Physiol 290: G1228   Loitto VM, et al., 2009, Cell Motil Cytoskeleton 66: 237
                                                                                                                                                            10um                                                                                                                                                                                               Engel A, et al., 2008, Curr Opin Struct Biol 18:229                         McCoy E and Sontheimer H, 2007, Glia 55: 1034
    tion allowing polarized distribution of the water channels. We also present                                                                                                                                                                                                                                                                                Fort PE, et al., 2008, Glia 56: 597                                         Neely JD, et al., 2001, Procl Natl Acad Sci USA 98: 14108
    a hypothetical working model for AQP9 induced lopodia based on the in-                                                                                                                                                                                                                                                                                     Guadango E and Moukhles H, 2004, Glia 47: 138                               Pietrement C, et al., 2008, J Biol Chem 283: 2986
    terplay between increased water ux and other proteins interacting with                                                                                                                                                                                                                                                                                     Hiroaki Y, et al., 2006, J Mol Biol 355: 628
    AQP9 to enable polarized distribution of this protein together with other
    players involved in cell adhesion and motility.                                                                                                                                                           AQP9 augments osmotic susceptibility in HL60 cells

                                                                                                                                                                                                  313mOsm              AQP9 -GFP                                      325mOsm                                             350mOsm
        AQP9 induces highly dynamic lopodia in di erent cell types                                                                                                                                                     GFP

                                                                                                                                                                                                                                                                                                                                                                                               Working model for AQP9 induced lopodia
                                                                                                                                               Volume (%)




   A)   HEK-293 AQP9-GFP                                                B) HEK-293 AQP9-GFP



                                                                                                                                                                                                                                                                                                                                                                                                                                                                   Filopodium
                                                                                                                                                                                                                                                                                                                                                                              (1)                                                                       (4)
                                                                                                                                                                                                            Time (s)                                                            Time (s)                                            Time (s)
                                                                                                                                                            HL60 cells overexpressing AQP9-GFP or GFP only were exposed to solutions with increasing osmolarity and
                                                                                                                                                            relative volume was measured with a coulter counter. Both AQP9-GFP and GFP expressing cells responded
                                                                                                                                                            with a decreased cell volume when exposed to a hyperosmotic condition. However, AQP9-GFP expressing                                                                                                                   dystrophin     α-dys                    α-dys
                                                                                                                                                            cells reacted faster with cell volume decrease than GFP transfected cells when placed in a 313mOsm solu-                                                                                                                α-s              α-s                      α-s
                                                                                                                                                                                                                                                                                                                                                                                        yn                yn                      yn
                                                                                                                                                            tion.                                                                                                                                                                                                                                   PDZ                     PDZ
                                                                                                                                                                                                                                                                                                                                                                                  PDZ




                                                                                                                                                                                                                                                                                                                                                                                                                    (3)
                                                                                                                                                   Increased adhesiveness, integrin expression and migration in HL60 cells
                                                                EpiFL
                                                                                                                                                   overexpressing AQP9                                                                                                                                                                                                               (2)
   C)   MDCK-1 AQP9-GFP                                                  D) HL60 AQP9-GFP                                                                                                        Adhesion                                                        CD11b presentation                                                  Migration
                                                                                                                                                                                                                                                                                                                                                                 Extracellular
                                                                                                          percentage of AQP9 cells attached




                                                                                                                                                                                                                                                                                **                                                                   **
                                                                                                                                                             Percentage of AQP9 cells attached




                                                                                                                                                                                                                                   Fluorescent intensity (a.u)




                                                                                                                                                                                                                                                                                                         migrated cells/image




                                                                                                                                                                                                                                                                                                                                                                 Intracellular
                                                                TIRF



                                                                                                         EpiFL
                                                                                                                                                                                                            ***




                                                                         E) HEK-293 GFP
                                                                                                                                                                                                                                                                                                                                                                                Lipid raft                          Water
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Pressure




                                                                                                                                                                                                                                                                                                                                                                                                          a
                                                                                                                                                                                                                                                                                                                                                                                                              b
                                                                                                                                                                                                                                                                                                                                                                                CAM                                 F-actin
                                                                                                                                                                                                      P                                                                       P   P                                             ion       ion   LP        LP
                                                                                                                                                                                                 9- GF GFP                                                                  GF GF                                  t    at    M fM
                                                                                                                                                                                                                                                                                                                gra migr P + f FP +
                                                                                                                                                                          P                                                                                             P9-                                   mi m       F    G
                                                                                                                                                                       AQ                                                                                            AQ                                    om ndo P9-G
                                                                                                                                                                                                                                                                                                         d                                                                       Aquaporin-9                      Lipid membrane
                                                                                                                                                                                                                                                                                                    P ran FP ra AQ
                                                                                                                                                                                                                                                                                                   F    G
                                                                                                                                                                                                                                                                                              P9-G
                                                                                                                                                                                                                                                                                           AQ
                                                                EpiFL                                   EpiFL                                               Di erentiated HL60 cells were seeded onto glass coverslips and unbound cells were washed away. Cells over-
  When GFP-fused AQP9 is overexpressed it induces numerous of lopodia. This is seen in many di erent                                                        expressing AQP9 showed increased adhesive abilities compared to Mock transfected cells. This might depend
  cell-types such as the epithelilal cell lines HEK-293 (A, B), MDCK-1 (C) and in the neutrophil-like cell line                                             on a more rm attachment by AQP9 overexpressing cells through increased presentation of adhesion recep-                                                                                               (1) AQP9 is anchored to specialized membrane micro-domains through PDZ domain interaction with alpha-syntrophin that in
  HL60 (D). Long protrusions extending from the apical side of the cell appears to bend down towards the                                                    tors such as CD11b (β -integrins) but might also be a ected by a more morphologically polarized phenotype.                                                                                           turn binds α-dystrobrevin. (2) In ux of water across the membrane increases the local hydrostatic preassure at the membrane,
  glass (C upper panel). The AQP9 induced lopodia appears to be highly dynamic and sequentially pro-                                                        Furthermore, in transwell experiments, cells overexpressing AQP9 show increased migratory ability towards a                                                                                          which tears the membrane apart from the cytoskeleton creating the intial stage of a membrane protrusion. (3) The membrane
  truding and retracting in AQP9 transfected HEK-293 cells (B, Time (s)). When cells were transfected with                                                  chemotactic gradient that could rely on the highly dynamic membrane enabling rapid cellular shape changes                                                                                            protrusion makes space for actin polymerisation at the site of water in ux and (4) a growing lopodia is formed that further in-
  only GFP, lopodia are abscent (E). Unlabeled scale bar 10um                                                                                               when squeezing through the narrow pores of the transwell membrane.                                                                                                                                   creases the surface area of the cell and thereby also the area of integrin insertion.

				
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posted:3/6/2010
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