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Regulation of Focal Adhesion Formation and Filopodia Extension by the Cellular Prion Protein (Prpc)

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Regulation of Focal Adhesion Formation and Filopodia Extension by the Cellular Prion Protein (Prpc) Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC) Yvonne Schrock 1, Gonzalo P. Solis . ,1, Claudia A.O. Stuermer ' Department of Biology, Uni versity of Konstanz, Uni ve rsiraetsstmsse 10, 78464 KOllswnz, Germa ny ABSTRACT While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here. we demonstrate PrP functions in cell-substrate interaction in Drosop/lila 52. N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its down regulation leads to reduced FA numbers. increased FA length, along with Src and focal Edited by Jes us Avila adhesion kinase (FAI{) activation. Furthermore. its overexpression elicits the formation of novel FA-like structures, which require intact reggie/f1otillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAI( and Src. Keywords: PrPC Cell-substrate interaction Filopodia/lamellipodia Focal adhesion Signal transd uction Reggie/flotillin microdomain 1. Introduction uncover new roles of PrP in cell spreading and process extension, Remarkably, PrP modu'lates FA dynamics and the formation of The physiological role of the cellular prion protein (PrPC, hence­ FA-like structures, which seem to depend on reggie. forth called "PrP") remains poorly understood. However, it is thought to play roles in neuroprotection, cell adhesion and signal 2. Materials and methods transduction [1,2). Our previous results using T cells have revealed that PrP can induce signal transduction in association with reggie/ 2,1 . Reagents and antibodies f10tillin microdomains [3]. which are thought to serve as platforms for the assembly of multiprotein signalling complexes [4]. For in­ Cell culture reagents were purchased from Gibco BRL (Ger­ stance, reggies are known to interact with Src tyrosine kinases many). Antibody information is provided under Supplementary [5], the adaptor proteins CAP and vinexin [6]. and actin [7]. In addi­ material. tion, we recently showed that reggies are required for the recruit­ ment of CAP to focal adhesions (FAs) [8J and the regulation of cell 2.2, Plasm ids morphology [8,9J. Given the close association of PrP and reggies, we here investi­ The cloning procedures for the PrP, EGFP-PrP, EGFP-GPI, DsRed­ gated possible functions of PrP in cell-substrate interaction, FA and PrP, and ECFP-R 1 EA constructs are described in Supplementary process formation. To this aim, Droso phila 52, mouse N2a and material. The rat reggie-l -HA construct has been described human HeLa cells w ere used to study the effect of PrP overexpres­ previously [1 OJ. The GFP-paxillin and j33 -integrin-GFP vectors were sion and downregulation on cell-substrate interaction. Our results kindly provided by Yamada and Imhof, respectively. Abbreviations: PrP, prion prote in; FA. focal adhesion; FAI<' focal adhesion kinase; 2.3. Cell wlture, transfectiol1, and siRNA TIRFM. total internal refl ection fluorescence microscopy; Rl EA. reggie- l EA co nstruct; siRNA. small interfering RNA. N2a and HeLa cells were cultured in MEM supplemented • Co rres ponding authors, Fax: +49 753 1883894 (G, P, Solis), with 10% FCS, L-glutamine, pyruvate and penicillin/streptolllycin, E- mail addresses:Gonzalo.Soli s@uni-konstanz. de (G, P, Solis), Claudia, Stueroner@uni-konstan z, de (C.A.O, Stuermer), and transfected using Lipofectamine 2000 (Invitrogen). 52 cells I Authors made eq ual contribution, cultured in Schneider'S Mediulll supplemented with 10% FCS, 390 L-glutamine and penicillin/streptomycin, were transfected with 10% Glycerin ) supplemented with protease and phosphatase inhib­ Effectene (Qiagen). Duplexed small interfering RNAs (siRNAs) were itor cocktails (Calbiochem). Hundred microgram proteins were transfected at 100 pmol/ml. The target sequences for mouse and loaded per lane and immunoblotted as previously reported human PrPs were: 5-CfGATTGAAGGCAACAGGAAA-3 and 5-CAG­ (n = 4) [8]. CAAATAACCATTGGTTAA-3, respectively (Qiagen). siRNA against firefly luciferase (GL2, Dhannacon) served as control. 3. Results and discussion 2.4. Spreading assay 3. 1. PrP induces cell spreading and fil opodia forma tion 52 cells were transfected for 24 h, seeded on coverslips coated with alcian blue, poly-lysine, laminin or fibronectin (Sigma-Al­ To assess if PrP affects cell-substrate interaction, we used Dro­ drich) for 1 h, and stained with phalloidin. Cell area (excluding sophila 52 cell s, a PrP-negative non-adhesive cell line routinely filopodia) was scored from 20 randomly selected fields (>80 cell s). used to characterize adhesion molecules. Upon transfection, PrP­ expressing cells showed remarkable spreading on alcian blue, 2.5. Fluorescence, filopodia and FA quantification and produced abundant filopodia (Fig. 1A). These effects were also observed upon expression of an EGFP-PrP fusion protein but not of N2a cell s were transfected fo r 24-48 h, seeded on poly-lysine the EGFP-GPI control construct (Fig. 51). Since PrP interacts with coated coverslips for 24 h, and stained with anti-GFP or anti-PrP laminin but not fibronectin [11], we performed spreading assays antibodies (non-permeabilizing conditions), or with phalloidin or on these substrates. For all substrates tested, the area covered by anti-paxillin antibody (permeabilizing conditions). The membrane cells expressing non-tagged PrP or EGFP-PrP was 4-fold larger than expression levels of our constructs were controll ed by measuring that of control cells (transfected with EGFP-GPI or non-transfected; anti-GFP indirect fluorescence (50 cells/construct). Filopodial Fi g. 1 B). Thus, heterologous PrP expression in 52 cells triggers cell length was scored in two independent experiments (50 si ngle spreading and filopodia extension in a substrate-independent cell s). FA number and length were scored in four independent manner. experiments (>200 cell s). 3.2. PrP levels regulate process forl11ation 2.6. Microscopy The results obtained in 52 cells prompted us to a nalyze if PrP N2a, HeLa and 52 cell s were immunostained as previously de­ would also affect the morphology of mammalian cells. For this, scribed [7], and visualized using a Plan-Apochromat 63 x /1.4 objec­ we used mouse neuroblastoma N2a cell s, a cell line frequently em­ tive in a confocal microscope (LSM5l0 META) and/or Axioplan2 ployed for the characterization of PrP cellular properties. Upon equipped with an AxioCam HRm (widefield images). For total expression of EGFP-PrP, the spontaneous levels of spreading in internal reflection fluorescence microscopy (TIRFM ), the TIRF slider N2a cells were not altered (data not shown). However, we ob­ system and a (X-Plan Fluar 1 00x /l.45 objective were used with an served a signifi cant increase in the number of filopodia> 1 0 ~lm Axiovert 200M (all Zeiss). Images were processed using the Axiovi­ length (11.2 ± 3.3 filopodia/cell), as well as in the length of the lon­ sion 4.6 or LSM51 0 softwares (Zeiss ). gest filopodium (18.4 ± 2.8 !-.un ), compared to EGFP-GPI transfected cell s (2.0 ± 1.3 filopodia/cell and 11.4 ± 2.7 f.lm, respectively) 2.7. lml11ul1oblotting (Fig. 2A- D). Interestingly , PrP often accumulated in filopodial tips (Fig. 2A). Corresponding controls show ed that both EGFP-fusion N2a cell s were lysed using ice-cold lysis buffer (20 mM Tris-HCI proteins were effi ciently expressed (Fig. 52) and equally presented pH 7.5, 100 mM NaCl, 5 mM MgCI 2, 2 mM EDTA, 1 % Triton X-100, on the plasma membrane (Fig. S3A- E), and also that the endoge- B * * * * ...... 300 * * o Control N E lSI EGFP-GPI .2.: 200 • EGFP-PrP ro DPrP ~ ro 100 Q) U 0 alcian blue poly-lysine laminin fibronectin Fig. 1. PrP induces spreading and fi lopodia forma ti on in S2 cell s. (A) Transfected S2 cell s w ere transferred to al ci,111 blue coated coverslips, and stained w ith anti-!'rP antibody (green) and pha ll oidi n to vi suali ze F-actin (red). Confocal images of a ce ll expressing non-tagged PrP show increased cel l area and fo rmation of filopodia compared to non­ lransi'ectcd cell s (arrowheads). Scale bar: 5 ~tm . (B) Quantir.cation of the area shows that S2 cell s ex pressing non-tagged PrP or EGFP-l'rP spread equall y well on alci an blue, poly-lysine, laminin or fibronectin coated coversli ps. Non-transfe cted (Control) and transfected cell s expressing EGFP-GP I do not spread under these conditions. V" lu es shown are the mean ± S. D. '1' < 0.05, One-Way ANOVA. 391 nous PrP expression was not affected by the EGFP-G PI construct p5rc, and reggie-1 (Fig. 56A-F). PrP accumulation in FAs was not (Fig. 52F and G). Thus, PrP overexpression positively influences fi l­ observed in Hela cell s treated 'with siRNA, confirm ing the speci­ opodia formation and extension. To strengthen this observation, ficity of the immunostaining (Fig. 57). like in N2a cells, overex­ we used siRNAs to knockdown PrP in N2a cell s (>95% efficiency; pression of EGFP-PrP, but not of EGFP-GPI, resulted in the Fig. 54). Indeed, siRNA-treated cell s showed significantly few er fil­ appearance of FA-like structures in Hela cells ( Fig. 3E and F, opodia but extensive lamellipodial veils (Fig. 2E) compared to con­ and Fig. 55B), some of which partially co-locali zed w ith paxillin trol, wild type or PrP-overexpressing cells (Fig. 2A, B and E). Rescue (Fig. 3E). Moreover, in both cell types PrP often resided at the dis­ experiments performed by co-transfecting si RNA and EGFP-PrP tal end of FAs , much li ke pERI< in fibroblasts [1 2 ), suggesting a (lacking the siRNA binding site) considerably reverted this abnor­ signalling role of PrP in these structures.
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