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.