© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3018-3029 doi:10.1242/jcs.165365

RESEARCH ARTICLE The prion protein inhibits monocytic by stimulating β1 adhesion and uropod formation Dion D. Richardson1,*, Simon Tol1,*, Eider Valle-Encinas1, Cayetano Pleguezuelos1, Ruben Bierings2, Dirk Geerts3 and Mar Fernandez-Borja1,‡

ABSTRACT PrP is highly expressed in the hematopoietic system, particularly The broad tissue distribution and evolutionary conservation of the in hematopoietic stem cells and in mature mononuclear leukocytes glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP, also (Isaacs et al., 2006). Accordingly, PrP has been reported to have a in vivo known as PRNP) suggests that it plays a role in cellular homeostasis. role in several hematopoietic and immune cell functions , Given that integrin adhesion determines cell behavior, the proposed such as the self-renewal of long-term repopulating hematopoietic role of PrP in cell adhesion might underlie the various in vitro and stem cells (Kent et al., 2009; Zhang et al., 2006), macrophage in vivo effects associated with PrP loss-of-function, including the phagocytosis (de Almeida et al., 2005) and activation immune phenotypes described in PrP−/− mice. Here, we investigated (Ballerini et al., 2006). However, the role of PrP in the innate the role of PrP in the adhesion and (transendothelial) migration of immune response has hardly been explored with the exception of −/− human (pro). We found that PrP regulates β1-integrin- one study in which peritonitis was induced in PrP mice with −/− mediated adhesion of monocytes. Additionally, PrP controls the cell zymosan (de Almeida et al., 2005). PrP mice showed decreased morphology and migratory behavior of monocytes: PrP-silenced cells numbers of neutrophils and larger numbers of monocytes in show deficient uropod formation on immobilized VCAM and display peritoneal infiltrates. Although the basis for this phenotype was not bleb-like protrusions on the endothelium. Our data further show that investigated, one possibility is that PrP has an intrinsic role in PrP regulates ligand-induced integrin activation. Finally, we found leukocyte migration out of the circulation and/or into the in vivo in vitro that PrP controls the activation of several proteins involved in cell peritoneum. In addition, several and studies have – adhesion and migration, including RhoA and its effector cofilin, as well shown that PrP participates in the regulation of cell cell (Málaga- – as proteins of the ERM family. We propose that PrP modulates β1 Trillo et al., 2009; Mangé et al., 2002; Morel et al., 2008) and cell integrin adhesion and migration of monocytes through RhoA-induced matrix adhesion (Loubet et al., 2012; Schrock et al., 2009). actin remodeling mediated by cofilin, and through the regulation of Leukocyte extravasation takes place through the binding of ERM-mediated membrane– linkage. cytokine-activated leukocytes to the endothelium, followed by their migration across the endothelium and subsequently the vascular KEY WORDS: Prion protein, , Migration, Adhesion, RhoA, basement membrane (Nourshargh et al., 2010). Leukocyte integrin ERM binding to endothelial ICAM-1 and VCAM-1 receptors is crucial for leukocyte arrest and firm adhesion to the endothelium, allowing INTRODUCTION leukocytes to resist flow-induced detachment. This requires integrin The cellular prion protein (PrP, also known as PRNP) is a clustering upon ligand binding in order to increase avidity and glycosylphosphatidylinositol (GPI)-anchored glycoprotein that strengthen adhesiveness, which is mediated by integrin association resides in detergent-resistant membrane domains. The conversion with the actin cytoskeleton through adaptor proteins (Alon et al., of PrP to a misfolded insoluble isoform (PrP Scrapie) occurs at 2005). Migrating leukocytes polarize, with one side of the cell the onset of neurodegenerative disorders known as transmissible forming a leading edge and the other forming a uropod. The uropod spongiform encephalopathies (Colby and Prusiner, 2011). However, is an adhesive structure containing cholesterol-rich membrane its physiological function is largely unclear (Biasini et al., 2012), domains, β1 and other adhesion receptors (i.e. ICAM-1 although it has been suggested that it is involved in multiple cellular and ICAM-3) as well as actin-membrane linker proteins of the ezrin, processes such as survival, , differentiation and growth radixin and moesin (ERM) family (Friedl and Weigelin, 2008). To (Linden et al., 2008). This variety of functions together with the high migrate, leukocytes need to detach and retract their uropod, a evolutionary conservation and wide tissue expression of PrP suggest process regulated by the small GTPase RhoA (Liu et al., 2002; that this protein has a role in cellular homeostasis. Worthylake et al., 2001). Here, we addressed the role of PrP in monocyte adhesion and migration and show that PrP silencing impairs monocyte adhesion to β1 integrin ligands and to endothelial cells while increasing their 1Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam motility. We show that PrP regulates ligand-induced integrin 1066CX, The Netherlands. 2Department of Plasma Proteins, Sanquin Research and activation and provide evidence that ERM proteins and the Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, RhoA–cofilin pathway are involved. Amsterdam 1066CX, The Netherlands. 3Department of Pediatric Oncology/ Hematology, Erasmus University Medical Center, Rotterdam 3015 CN, The Netherlands. RESULTS *These authors contributed equally to this work PrP colocalizes with integrins at cholesterol-rich domains in ‡ Author for correspondence ([email protected]) monocytic cells In vivo experiments in mice have shown that PrP regulates the

Received 4 November 2014; Accepted 3 July 2015 recruitment of monocytes to inflamed peritoneum and the Journal of Cell Science

3018 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3018-3029 doi:10.1242/jcs.165365

Fig. 1. PrP resides in cholesterol-rich membrane domains on the surface of human pro-monocytic U937 cells. (A) Detection of PrP in live cells by flow cytometry using the SAF32 antibody. Mouse IgG2b was used as an isotype control. (B) Triton X-100 cell lysates were fractionated in Optiprep density gradients. Fractions were collected from the top, and proteins were precipitated with trichloroacetic acid and analyzed by immunoblotting for the presence of PrP, the lipid raft-marker flotillin-1 and the non- lipid raft marker transferrin receptor (TfR). PrP and flotillin-1 were enriched in the top fractions, consistent with their localization in lipid rafts. (C) U937 cells were stimulated or not with 100 ng/ml CXCL12 for 15 min at 37°C and then cooled on ice for PrP and integrin detection with SAF32 (PrP) and P5D4 (β1 integrin subunit) antibodies. Pearson’s colocalization coefficients (mean±s.d., n=3) between PrP and β1 integrin were determined in the selected ROIs (A and B) depicted in the scheme, both in polarized and non-polarized cells (≥25 cells analyzed per experiment). In polarized cells, A included the integrin cluster and B, the remaining membrane. In non-polarized cells, each A and B value correspond to one half of the membrane. The A:B ratio (A/B) indicates the relative colocalization measured in A with respect to B. Scale bars: 10 µm. (D) Stable U937 cell lines were generated by puromycin-selection of cells transduced with lentiviruses expressing shRNAs against human PrP (shPrP-1, shPrP-2 and shPrP-3) or with non- targeting shRNA (shCtrl). PrP expression was examined by flow cytometry using the SAF32 anti-PrP antibody. Graphs represent the mean±s.d. of three independent transduction or selection experiments. MFI, mean fluorescence intensity. ***P<0.001 (one-way ANOVA).

phagocytosis of dead cells by macrophages, suggesting a role for Both the localization of PrP in lipid rafts and its colocalization PrP in innate immunity (de Almeida et al., 2005). However, the with integrins suggest a role for PrP in the regulation of monocyte mechanism underlying these effects has not been explored. Here, adhesion and migration. To investigate this, we generated stable we first investigated the role of PrP in the adhesion and migration of U937 cell lines with reduced PrP expression by transduction with the human pro-monocytic cell line U937. This model has been used lentiviruses expressing three different short hairpin RNAs extensively to study monocyte adhesion and CXCL12-induced (shRNAs) against human PrP. In parallel, stable cell lines were migration (Chan et al., 2001; Hyduk and Cybulsky, 2009) and generated by transduction with lentivirus expressing a non-targeting allows for gene expression manipulation by transfection and shRNA (shCtrl-U937) as a negative control. Flow cytometry transduction techniques. detection of surface PrP showed that shPrP-1 (complementary to the U937 cells express PrP on their cell surface as assayed by flow 3′UTR of PrP mRNA) was able to reduce PrP expression to ∼35%, cytometry (Fig. 1A). To determine whether PrP accumulates in whereas shPrP-2 and shPrP-3 (complementary to the PrP coding cholesterol-rich membrane domains (‘lipid rafts’), we performed sequence) reduced PrP expression to ∼70% (Fig. 1D). Considering a membrane flotation assay. PrP was primarily found in the low- the moderate efficiency of shPrP-2 and shPrP-3 in silencing PrP density fractions together with the lipid raft marker flotillin-1 expression, we continued our work using stable shPrP-1-transduced (Fig. 1B), indicating that PrP resides in lipid rafts in U937 cells. cell lines (shPrP-U937 cells). Further analysis of PrP distribution by confocal microscopy PrP silencing did not affect the proliferation rate of U937 cells as showed that a fraction of U937 cells displayed polarized PrP evidenced by routine cell counting of cells transduced with control localization on the membrane (Fig. 1C). This fraction increased shRNA (shCtrl-U937) and shPrP-U937 cell cultures (data not from ∼20% to ∼50% upon CXCL12 stimulation. In polarized shown). Accordingly, staining of apoptotic cells with annexinV– cells, both PrP and β1 integrins clustered together in membrane FITC showed no significant differences between shCtrl- and caps where they colocalized (Pearson’s coefficient larger than shPrP-U937 cells (0.3% and 0.2% positive cells, respectively). In 0.5) (Fig. 1C, polarized cell, region of interest A). In contrast, PrP addition, there was no difference in CXCL12-induced activation of and β1 integrins failed to colocalize in the membrane area devoid the survival kinase Akt between control and PrP-silenced cells of clustered integrins (Fig. 1C, polarized cell, region of interest B) (data not shown). and in non-polarized cells (Fig. 1C, non-polarized cell, regions of interest A and B) (Pearson’s coefficient smaller than 0.5). PrP PrP regulates β1 integrin adhesion of monocytes also colocalized with the lipid-raft-resident protein flotillin-1 in We used stable shCtrl- and shPrP-U937 cell lines to explore the polarized cells (supplementary material Fig. S1). These data effect of PrP depletion on CXCL12-induced cell in a indicate that integrins are clustered in a fraction of U937 cells in Transwell assay using fibronectin-coated filters. shPrP-U937 steady-state conditions and that, upon cell polarization, PrP and cells showed a significant increase in chemotaxis when

β1 integrins are recruited to flotillin-1 lipid raft domains. compared with shCtrl-U937 cells (Fig. 2A), suggesting that Journal of Cell Science

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Fig. 2. PrP regulates U937 cell and primary monocyte adhesion to β1 integrin ligands. (A) CXCL12-induced migration of shCtrl- and shPrP-U937 cells was determined using a Transwell assay. Owing to the inter-experimental variation of absolute migration values, the migration values of shCtrl-U937 were set to 100% to determine statistical significance of the differences. Data are mean±s.d. from three independent experiments performed in triplicate. **P<0.01 (two-tailed unpaired Student’s t-test). (B) CXCL12- induced migration of U937 cells incubated with either anti- PrP SAF61 antibody or IgG2a was determined using a Transwell assay as in A. ***P<0.001 (two-tailed unpaired Student’s t-test). (C) Representative ECIS experiment showing real-time changes in electrical resistance upon adhesion of shCtrl- and shPrP-U937 cells to fibronectin or BSA. The black arrow indicates addition of cells to the electrodes and the green arrow indicates PMA addition to stimulate cell adhesion. FN, fibronectin. (D) PMA-stimulated adhesion of shCtrl- and shPrP-U937 to BSA and fibronectin was determined by dividing the resistance at t=1 h by the resistance at t=0 after PMA addition (n=3). **P<0.01 (two- way ANOVA). (E) PMA-stimulated shCtrl- and shPrP-U937 cell adhesion to VCAM-1 and ICAM-1 was calculated as in D (n=6 for VCAM; n=4 for ICAM). *P<0.05 (two-way ANOVA). (F) ECIS adhesion assay of shCtrl- and shPrP-primary human monocytes as in C. (G) Quantification of PMA- stimulated adhesion as in D (n=3). *P<0.05, **P<0.01 (unpaired two-tailed Student’s t-test). Results in D, E and G show the mean and interquartile range.

PrP is a negative regulator of chemotaxis. To confirm this, we fibronectin-coated electrodes showed a larger increase in PMA- induced PrP-dependent signaling by cross-linking of surface PrP stimulated adhesion than shPrP-U937 cells (Fig. 2C). U937 cells with the SAF61 antibody, previouslyshowntotriggersignal express β1[α4β1 (VLA-4) and α5β1 (VLA-5)] and β2 integrins transduction in T lymphocytes and in a neuronal cell line [αLβ2 (LFA-1) and αMβ2 (Mac-1)] (Prieto et al., 1994), and under (Stuermer et al., 2004; Mouillet-Richard et al., 2000). U937 our experimental conditions, preferentially adhered to β1 integrin cells incubated with SAF61 displayed impaired migration ligands (fibronectin and VCAM-1) (Fig. 2D,E). Consistent with a (Fig. 2B), confirming the negative regulatory effect of PrP on role for PrP in the regulation of β1-integrin-mediated adhesion, monocytic cell migration and supporting the specificity of the shPrP-U937 cells showed reduced adhesion to fibronectin (Fig. 2D) shRNA-mediated PrP silencing. These effects of PrP on cell and Fc-VCAM-1 (Fig. 2E). Thus, PrP silencing impairs β1-integrin- chemotaxis were not due to changes in CXCR4 surface mediated adhesion of monocytic cells. This reduced integrin- expression or CXCL12-induced receptor internalization as mediated adhesion might facilitate leukocyte motility and tested by flow cytometry (data not shown). migration, which could explain our findings. We then investigated whether changes in U937 cell migration To confirm that our findings also apply to primary monocytes, we were paralleled by changes in cell adhesion by recording the transduced freshly isolated human monocytes with shPrP-1. ECIS electrical resistance at 32 kHz over time of cells adhering to electric adhesion assays showed similar differences in adhesion to Fc- cell–substrate impedance sensing (ECIS) electrodes coated with VCAM between shCtrl- and shPrP-transduced monocytes to that in integrin ligands (fibronectin, Fc-ICAM-1 or Fc-VCAM-1) or with U937 cells (Fig. 2F,G). Similar results were obtained using the bovine serum albumin (BSA) (Fig. 2C–E). This assay is based on human T lymphocyte cell line Jurkat lymphocytic cells (data not the increase in electrical resistance when cells spread on the shown). To establish that the differences using the ECIS adhesion electrodes following cell adhesion (Wegener et al., 2000). Addition assay were due to differences in adhesion strength and not in cell of cells to the electrode arrays resulted in a slight increase in shape (i.e. spreading), we performed conventional adhesion assays electrical resistance, which was consistently higher for shCtrl-U937 by seeding cells on Fc-VCAM-coated plates and counting adherent cells compared to shPrP-U937 cells (Fig. 2C), although differences cells after thorough washing (supplementary material Fig. S2). were not significant (data not shown). Comparatively, a large Taken together, these data indicate that PrP regulates β1-integrin- increase in resistance was observed upon the stimulation of cell mediated adhesion of monocytes and suggest that it has a similar adhesion with PMA (Fig. 2C). shCtrl-U937 cells seeded on role in other leukocyte types. Journal of Cell Science

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numbers when compared with shCtrl-U937 cells, both after cell stimulation with CXCL12 (Fig. 3B) and with PMA (Fig. 3C). Our data show a significant reduction in firm adhesion of shPrP-U937 cells to the endothelium under flow in both conditions (Fig. 3D, CXCL12; Fig. 3E, PMA). Next, we transduced freshly isolated human monocytes with shPrP-1 or with shCtrl and quantified the percentage of monocytes that adhered to CXCL12-coated endothelium in the same assay as described for U937 cells. Our data show that PrP silencing also impaired firm adhesion of primary monocytes to the endothelium (Fig. 3F). We performed similar experiments with PMA-activated monocytes; however, we obtained no significant differences between the adhesion of shCtrl- and shPrP-transduced monocytes. We think this can be explained by the low numbers of adherent cells owing to monocyte activation and adhesion to the tubing of the flow system. Finally, similar results were obtained using PrP-silenced Jurkat cells (supplementary material Fig. S3A). These findings suggest that PrP positively regulates the firm adhesion of monocytes, and possibly other leukocyte types, to inflamed endothelium under physiological conditions of flow-induced shear stress.

PrP regulates cell shape and migratory behavior of U937 cells on immobilized VCAM Our data above show that PrP regulates monocyte chemotaxis (Fig. 2). To analyze further the effects of PrP silencing on cell migration, we examined the migratory behavior of shCtrl- and shPrP- U937 cells on immobilized Fc-VCAM and CXCL12 by time-lapse video recording. Migrating shCtrl-U937 cells formed long and persistent uropods and moved in a polarized fashion, extending lamellipodia and filopodia at the cell front (Fig. 4A; supplementary material Movie 1). In contrast, shPrP-U937 cells failed to form long uropods (Fig. 4A; supplementary material Movie 1). Manual tracking of migrating cells and subsequent calculation of cell velocity and the accumulated distance of cell trajectories showed that shPrP-U937 Fig. 3. PrP regulates U937 cell and primary monocyte adhesion to endothelial cells under shear stress. (A) Mixed populations of red-labeled cells were faster and covered longer distances than shCtrl-U937 cells shCtrl- and green-labeled were incubated with PMA or CXCL12 for 10 min (Fig. 4A). Lack of significant differences in directionality suggests before being perfused over TNFα-stimulated endothelial monolayers. Upper that PrP silencing does not affect cell polarization (Fig. 4A). These panels show DIC and fluorescence snapshots of 30-min video recordings of data corroborate our findings using Transwell chemotaxis assays and U937 adhesion to the endothelium (arrow indicates the direction of flow). Lower suggest that PrP silencing enhances leukocyte motility through the panels show the result of image processing with ImageJ software to downregulation of integrin-mediated adhesion. automatically count adherent U937 cells. Scale bars: 50 µm. (B) Graph β showing the total number of CXCL12-stimulated cells adhering to the Both 1 integrins and phosphorylated ERM (phospho-ERM) endothelium in three microscopic fields of a representative flow experiment. proteins localize to the uropod of migrating leukocytes (Lee (C) Same as in B for PMA-stimulated cells. (D) Percentage of CXCL12- et al., 2004; Martinelli et al., 2013). Similarly, phospho-ERM stimulated shCtrl- and shPrP-U937 cells adhering to the endothelium per proteins accumulated in the uropod and partially colocalized with microscopic field after 10 min of flow (n=3). (E) Same as in D for PMA- β1 integrins in shCtrl-U937 cells migrating on immobilized stimulated cells. (F) Same as in D for CXCL12-stimulated primary monocytes. VCAM-1 (Fig. 4B). In contrast, shPrP-U937 cells mostly lacked Results are mean±s.d. **P<0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed Student’s t-test). uropods and contained reduced levels of phospho-ERM proteins, which localized in discrete spots and in thin membrane protrusions (Fig. 4B). Measurement of uropod length in cells PrP regulates monocyte adhesion to the endothelium under migrating on Fc-VCAM-1 showed that the average length of the flow uropod was reduced from 26.5 µm to 14.5 µm upon PrP silencing To assess the effects of PrP silencing in monocyte–endothelium (Fig. 4B). To test whether uropod loss could be rescued by interactions under near-physiological flow conditions we used overexpression of PrP in shPrP-U937 cells, we took advantage of parallel flow chamber assays. Mixed suspensions of shCtrl- and the fact that shPrP-1 targets the 3′-UTR of PrP mRNA allowing shPrP-U937 cells (1:1), differently labeled with either Calcein for rescue with human PrP cDNA. We transfected cells with Red-Orange AM or Calcein Green were stimulated with either GFP–PrP or GFP cDNAs by electroporation and subsequently CXCL12 or PMA. After stimulation, labeled cells were perfused selected GFP-positive cells by flow cytometry. Given that GFP– over TNFα-stimulated human umbilical vein endothelial cells PrP mainly localizes to the cell middle plane (in the Golgi, not (HUVECs), while recording fluorescent and bright-field time- shown), and the signal was therefore low at the bottom plane, we lapse movies (a movie snapshot is shown in Fig. 3A). Adherent co-detected PrP with SAF32 in non-permeabilized cells to cells were quantified in each video frame as described in the clearly show distribution of PrP on the membrane. Uropod

Materials and Methods. shPrP-U937 cells adhered in lower formation in PrP-silenced cells was rescued by overexpression of Journal of Cell Science

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Fig. 4. Defective uropod formation in migrating PrP-silenced cells. (A) shCtrl and shPrP-U937 cells were seeded on Fc-VCAM-1 or CXCL12-coated plates and recorded using a wide-field microscope. The figure shows snapshots of these video recordings. Migrating shCtrl-U937 cells develop long uropods (arrowheads). In contrast, uropod formation is impaired in shPrP-U937 cells. Bar graphs represent the mean±s.d. [n=3; ≥60 cells (each for shCtrl- and shPrP-U937 cells) per experiment] quantification of accumulated distance, cell velocity and the degree of directional migration (directness) by manual cell tracking using Gradientech Tracking Tool software. **P<0.01 (two-tailed unpaired Student’s t-test). (B) shCtrl- and shPrP-U937 cells were seeded on Fc-VCAM-1- or CXCL12-coated IBIDI µ-slides, allowed to migrate for 20 min and processed for immunofluorescence detection of phospho- ERM proteins and β1 integrins. The bar graph shows the mean±s.d. length of the uropod in cells migrating on VCAM as described above [n=3, ≥40 cells (each for shCtrl- and shPrP-U937 cells) per experiment]. ***P<0.001 (two-tailed unpaired Student’s t-test). (C) shPrP-U937 cells were transfected with GFP–PrP or GFP (negative control) cDNA by electroporation and sorted by flow cytometry to enrich for transfected cells. Sorted cells were seeded on immobilized Fc-VCAM-1 or CXCL12 and fixed after for 20 min for the immunodetection of β1 integrins and PrP. The bar graph shows mean±s.d. uropod length in GFP–PrP- and GFP-expressing cells [n=3; ≥30 cells (each for GFP-PrP- and GFP-expressing cells) per experiment]. **P<0.01 (unpaired two-tailed Student’s t-test). Scale bars: 50 µm (A); 20 µm (B); 10 µm (C).

GFP–PrP, but not GFP (Fig. 4C). In addition, PrP on the cell lower in PrP-silenced cells when compared to control. These data surface (detected with anti-PrP antibodies in non-permeabilized suggest that PrP regulates uropod formation in monocytic cells cells) partially co-localized with endogenous β1 integrin through the regulation of ERM protein phosphorylation. (Pearson’s correlation coefficient=0.65±0.01, mean±s.e.m.), ERM proteins are known to link the uropod marker CD44 to the which corroborates our findings for cells in suspension. actin cytoskeleton (Friedl and Weigelin, 2008; Fehon et al., 2010). ERM proteins link membrane proteins to the actin cytoskeletal To determine whether the reduction in ERM protein activation cortex (Fehon et al., 2010) and active phospho-ERM proteins observed in shPrP-U937 cells had any effects on CD44 distribution, localize to and regulate formation of the uropod in migrating we immunodetected CD44 and flotillin-1 in cells migrating on lymphoblasts (Lee et al., 2004; Martinelli et al., 2013). We observed immobilized Fc-VCAM. In shCtrl-U937 cells, CD44 was enriched that the intensity of phospho-ERM labeling appeared to be reduced in uropods together with flotillin-1 (Fig. 5B). In contrast, CD44 was in shPrP-U937 cells (Fig. 4B). To substantiate this observation, we more uniformly distributed on the membrane of shPrP-U937 cells, assessed ERM protein phosphorylation by immunoblotting in cells although flotillin-1 still showed a polarized distribution (Fig. 5B). stimulated with CXCL12 or PMA in a timecourse experiment. As These data indicate that PrP silencing alters CD44 membrane moesin is the predominant ERM isoform expressed in leukocytes distribution. (Ivetic and Ridley, 2004), we detected this protein as a control for Next, we examined in detail the migratory behavior of shCtrl- and equal protein loading. CXCL12 induced ERM phosphorylation shPrP-U937 cells arrested on the endothelium under near- whereas PMA reduced phospho-ERM levels in a time-dependent physiological flow conditions as described above. Firm adhesion of manner (Fig. 5A). Consistent with the role of ERM proteins in leukocytes to the endothelium is enhanced by PMA treatment, which uropod formation, levels of phospho-ERM proteins were stimulates integrin adhesion and consequently inhibits leukocyte significantly reduced in shPrP-U937 cells stimulated with motility (Cinamon et al., 2001). As expected, PMA-stimulated shCtrl- CXCL12 and slightly decreased after stimulation with PMA U937 cells remained mostly round and immotile after arrest and

(Fig. 5A). In addition, basal phospho-ERM levels were 1.5-fold adhesion to the endothelium. In contrast, PMA-stimulated shPrP-U937 Journal of Cell Science

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U937 cells undergoing diapedesis across a TNFα-activated CXCL12-coated endothelium under flow conditions (Grabovsky et al., 2000). Predictably, a significantly higher percentage of shPrP- U937 cells transmigrated across the endothelium when compared to control cells (Fig. 6C). Thus, following arrest on the endothelium, PrP-silenced cells appear to have a diminished capacity to establish firm adhesion, displaying increased motility together with high membrane protrusive activity, as well as increased diapedesis.

PrP regulates integrin activity Integrin-mediated adhesion is regulated by conformational changes (affinity) as well as by the number of integrin molecules engaged in ligand binding (avidity) (Carman and Springer, 2003). Our data show that PrP silencing leads to reduced monocyte adhesion to β1 integrin ligands. To gain insight into the molecular mechanism underlying these effects, we first investigated whether PrP silencing impaired integrin affinity upregulation in in cells stimulated or not with CXCL12, PMA or Mn2+ for 10 min. PMA and CXCL12 induce intracellular signaling that leads to integrin activation, whereas Mn2+ binds to surface integrins triggering a signaling- independent conformational change (Carman and Springer, 2003). Total and activated β1 integrin on the surface of shCtrl- and shPrP- U937 cells were assessed by flow cytometry. No differences in surface expression of total or activated integrins were detected between shCtrl- and shPrP-U937 cells (Fig. 7A).To further examine the effect of PrP silencing on stimulus-induced upregulation of high-affinity β1 integrins, we stimulated cells with CXCL12, PMA or Mn2+ in the presence of soluble Fc-VCAM. Cell-bound Fc- VCAM was subsequently detected using APC-labeled F(ab′)2 goat anti-human-IgG antibody and flow cytometry analysis (Chan et al., 2003). shPrP-U937 cells consistently bound less Fc-VCAM (Fig. 7B). Direct integrin activation with Mn2+ induced higher Fc- VCAM binding and reduced the differences between shCtrl- and Fig. 5. PrP silencing impairs ERM protein activation. (A) Phosphorylated shPrP-U937 cells (Fig. 7B). These data suggest that PrP silencing ERM proteins were detected with an antibody that recognizes a does not affect the number of integrins on the cell surface but phosphorylated threonine residue conserved in all three proteins (P-ERM). decreases ligand-induced integrin activation. We assessed this Blots were reprobed with antibodies against total moesin. Quantification (mean±s.e.m.) of band density was performed as described in the Materials further by using multimeric Fc-VCAM complexes. For this, and Methods section (n=5). *P<0.05, **P<0.01, ****P<0.0001 (two-way preformed soluble multimeric ligand complexes were generated ANOVA with Bonferroni’s multiple comparisons test). Given that this test takes by incubation of Fc-VCAM with IgG F(ab′)2 goat anti-human-IgG into account all the time points, differences at t=0 of CXCL12 stimulation, antibody (Konstandin et al., 2006). This assay was previously although having similar values as t=0 for PMA stimulation, are not significant shown to detect both integrin affinity and avidity in leukocytes when compared with later time points. (B) CD44 and flotillin-1 were based on the fact that polyclonal F(ab′)2 fragments recognize immunodetected in shCtrl- and shPrP-U937 cells migrating on immobilized different epitopes of the human Fc moiety of Fc-VCAM VCAM. Scale bars: 10 µm. (Konstandin et al., 2006). The differences in ligand binding between control and PrP-deficient cells were more pronounced cells crawled over the endothelium while forming dynamic bleb-like when multimeric Fc-VCAM complexes were used (Fig. 7C). The membrane protrusions (Fig. 6A; supplementary material Movies 2–4). exacerbation of the differences between control and PrP-silenced On average, 30% of shPrP-U937 cells arrested on the endothelium cells when using a multivalent ligand suggests that PrP silencing displayed dynamic protrusions in contrast to only 10% of shCtrl-U937 primarily impairs ligand-induced integrin avidity. cells (Fig. 6B). PMA-activated shPrP-monocytes also showed increased membrane activity compared to shCtrl-transduced PrP regulates RhoA activation and actin polymerization monocytes (supplementary material Movie 5), although differences Integrin avidity is enhanced upon increased lateral mobility of were smaller (the percentages of adherent cells with membrane integrins on the plasma membrane, which allows them to form protrusions were 38.5% for shCtrl- transduced monocytes and 53.3% microclusters (Bakker et al., 2012; Buensuceso et al., 2003). for shPrP-transduced monocytes). However, these data should be Integrin binding to the actin cytoskeleton might impose constraints taken with caution owing to the low number of PMA-activated to the lateral mobility of integrins and reduce avidity. In addition, monocytes that were flown over the endothelium. Finally, this PrP-silencing has previously been shown to stabilize actin filaments phenotype appears not to be unique to monocytic cells because PrP- in neuronal cells through a RhoA-dependent pathway (Loubet et al., silenced Jurkat cells displayed similar bleb-like motility on activated 2012). Therefore, we explored RhoA activation in shCtrl- and endothelium to that of U937 cells (supplementary material Fig. S3B). shPrP-U937 cells stimulated with CXCL12 or PMA. shPrP-U937 Finally, we assessed the role of PrP in leukocyte transendothelial cells displayed significantly elevated GTP-RhoA levels after 2 and migration by quantification of the number of shCtrl-and shPrP- 5 min of CXCL12 stimulation (Fig. 8A). In contrast to CXCL12, Journal of Cell Science

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Fig. 6. Increased motility and membrane protrusive activity of PrP-deficient U937 cells on the endothelium under shear stress. (A) DIC and fluorescence snapshots of a video recording of PMA-stimulated shCtrl-U937 cells labeled with Calcein Red-Orange and shPrP-U937 cells labeled with Calcein Green on TNFα- activated endothelial cells under shear stress. shPrP-U937 cells undergo shape changes while crawling over the endothelium (lower green cell) whereas the shCtrl-U937 cells are motionless and maintain a spherical shape. Time is indicated on the figures in min:sec. (B) Percentage of shCtrl- and shPrP- U937 cells that displayed dynamic membrane protrusions after adhesion to endothelial cells under flow (mean±s.d., n=3). (C) Percentage of adherent shCtrl- and shPrP-U937 cells that migrated across the endothelium (mean±s.d., n=3). *P<0.05, ****P<0.0001 (unpaired two-tailed Student’s t-test).

PMA stimulation decreased RhoA activation. However, the levels stability by PrP in neurons (Loubet et al., 2012). To of GTP-RhoA were also slightly elevated in PMA-stimulated PrP- determine whether this pathway is operational in monocytes, we silenced cells when compared to control cells (Fig. 8A). determined the levels of phosphorylated (inactive) cofilin 1 in cells RhoA regulates the actin cytoskeleton by inducing both actin adhering to immobilized Fc-VCAM. We found that PrP-silencing polymerization and actin microfilament crosslinking and increases phosphorylated cofilin 1 levels (Fig. 8D) and thus inactivates remodeling. Therefore, we investigated actin polymerization cofilin1. Taken together, these findings provide evidence suggesting dynamics in CXCL12-stimulated suspension cells using a more that PrP regulates monocyte adhesion and migration through the sensitive flow-cytometry-based assay (Voermans et al., 2001). In regulation of actin remodeling by the RhoA–cofilin pathway. agreement with previous published data (Voermans et al., 2001), we observed a burst of F-actin after 0.5 min stimulation, followed by a DISCUSSION decrease to unstimulated levels at 10 min (Fig. 8B). PrP-silenced Leukocyte migration across the vascular wall is of crucial importance cells showed higher F-actin content at every time point (Fig. 8B). for the immune response. It has been previously shown that PrP is Low concentrations of cytochalasin B are reported to enhance actin involved in the influx of immune cells into the peritoneal cavity remodeling, whereas at high concentrations, cytochalasin B causes during zymosan-induced peritonitis, where monocytes are recruited actin cytoskeleton depolymerization. To test whether altered actin in larger numbers in PrP−/− mice compared to wild-type controls dynamics underlies the adhesion defects of PrP-silenced cells, we (de Almeida et al., 2005). Our study now provides evidence for performed an ECIS adhesion assay in the presence of low the molecular mechanisms underlying the effects of PrP silencing concentrations of cytochalasin B. At 1 µM, cytochalasin B on monocyte adhesion and migration, and corroborates the increased adhesion of shPrP-U937 cells to control levels immunomodulatory role of PrP. (Fig. 8C). At 5 µM, cytochalasin B, reduced PMA-induced Our findings show that PrP silencing increases the chemotaxis adhesion of both shCtrl and shPrP-U937 cells (Fig. 8C). These and motility of monocytic U937 cells on β1 integrin ligands in data suggest that reduced actin dynamics mediates the effects of PrP static conditions as well as on endothelium in near-physiological silencing in cell adhesion. flow conditions. In addition, we show that PrP-deficient U937 It has been shown previously that the RhoA-regulated actin- cells are able to transmigrate more efficiently than control cells. severing protein cofilin 1 is involved in the regulation of actin In contrast, activation of endogenous PrP signaling by antibody- Journal of Cell Science

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Fig. 7. PrP regulates integrin avidity. (A) Flow cytometry analysis of total and activated β1 integrins on shCtrl- (black line) and shPrP- (red line) U937 cells treated or not with CXCL12, PMA or Mn2+ for 10 min at 37°C. Cells were incubated with mouse IgG1 as isotype control (gray shaded area). No differences for total or activated integrins were detected between shCtrl- and shPrP- U937 cells. (B) Cells were stimulated for different times with CXCL12, PMA or Mn2+ in the presence of Fc-VCAM. Cell-bound ′ Fc-VCAM was detected with APC-labeled F(ab )2 goat anti- human-IgG antibody and analyzed by flow cytometry (mean±s.d., n=3). (C) Similar experiment as in B except that cells were mixed ′ with pre-formed multimeric Fc-VCAM/ F(ab )2-APC complexes (mean±s.d., n=3). *P<0.05, ***P<0.001, ****P<0.0001 (using two-way ANOVA).

mediated crosslinking of surface PrP impairs U937 cell remodeling might be required to allow disengagement of integrins chemotaxis. Collectively, these data suggest that PrP is a from cortical actin and allow them to cluster, therefore increasing the negative regulator of monocyte (transendothelial) migration. strength of ligand binding (Bakker et al., 2012; Worthylake and Leukocyte migration is dictated by the strength of integrin-mediated Burridge, 2003). We show here that PrP silencing increases CXCL12- cell adhesion. Although leukocyte attachment to the endothelial induced RhoA activation as well as F-actin content. Interestingly, surface is required to resist flow-induced shear stress forces, excessive although CXCL12-induced actin polymerization follows similar adhesion abrogates leukocyte motility on the endothelium as well as kinetics in shCtrl- and shPrP-U937 cells, actin depolymerization is diapedesis across the endothelial monolayer (Cinamon et al., 2001). slower in PrP-silenced cells (Fig. 8). Notably, increasing actin We show that PrP silencing impairs pro-monocyte adhesion to β1 depolymerization rates with low concentrations of cytochalasin B integrin ligands (fibronectin and VCAM-1) in static conditions, as abrogatesthe effects of PrP silencing on cell adhesion. Consistent with well as to TNFα-activated endothelial cells under flow. In addition, we this, we found that PrP silencing induces the inactivation of the actin- show that primary monocytes and Jurkat cells also display defective severing protein cofilin 1. Inactivation of cofilin by phosphorylation adhesion and enhanced motility upon PrP silencing, suggesting that on Ser3 is mediated by LIM kinase, which in turn is activated by RhoA PrP regulates β1-integrin-mediated interactions of monocytes (and (Bravo-Cordero et al., 2013). In view of the above findings, we probably T cells) with the endothelium in vivo. propose that the stabilization of actin filaments by RhoA-induced The regulation of integrin-mediated adhesion takes place through cofilin inactivation prevents the release of activated integrins from the changes in affinity, avidity and/or trafficking (Margadant et al., 2011). cytoskeleton in PrP-deficient cells, therefore impairing integrin We observed no changes in integrin affinity in PrP-silenced cells by microclustering and adhesiveness. Our findings are in agreement using either an antibody specific for the high-affinity conformation of with previously reported data showing increased cytoskeletal stability, β1 integrin or a soluble β1 integrin ligand (Fc-VCAM1). Likewise, we RhoA activation and cofilin inactivation in PrP-deficient neuronal detected no differences between control and PrP-silenced cells in β1 cells (Loubet et al., 2012). Interestingly, overexpression of PrP in integrin internalization by using an antibody-feeding assay (data not primary rat hippocampal neurons induces cofilin activation and the shown). However, PrP silencing impaired the binding of multimeric formation of actin–cofilin rods (Walsh et al., 2014). These structures Fc-VCAM complexes to cells. These data suggest that PrP controls β1 are found in Alzheimer disease brain and have been shown to impair integrin adhesiveness through the regulation of ligand-induced synaptic function (Minamide et al., 2000). It is tempting to speculate changes in integrin activation, likely through the regulation of that cofilin regulation is crucial both for the physiological function of integrin avidity, which is determined by the number of integrins PrP and for prion-induced neurodegeneration. engaged in ligand binding (Carman and Springer, 2003). VLA-4 We found that PrP colocalizes with integrins in pre-formed flotillin- (α4β1) integrin microclustering can induce adhesiveness by 1 domains (Langhorst et al., 2006). In contrast to in T cells, where PrP increasing valency independently of changes in integrin affinity recruitment to flotilin-1 membrane caps can be induced by antibody- (Grabovsky et al., 2000). The attachment of the integrin cytosolic tail mediated PrP crosslinking (Stuermer et al., 2004), we observed that to the actin cytoskeleton restrains the lateral mobility of integrins on PrP co-clustered with integrins in a fraction of U937 cells in the the membrane and consequently impairs microclustering (Bakker absence of stimulation or antibody-mediated crosslinking. This et al., 2012; Buensuceso et al., 2003). Thus, local microfilament suggests that a percentage of U937 cells are ‘primed’ in resting Journal of Cell Science

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Fig. 8. PrP silencing enhances RhoA activation. (A) A pulldown assay was used to assess the levels of GTP-bound active RhoA in shCtrl- and shPrP-U937 stimulated with CXCL12 or PMA for the indicated times (in min). Total RhoA was detected in the corresponding whole-cell lysates for input. Graphs represent mean±s.e.m. band density of the bands as described in the Materials and Methods section (n=6). (B) Cell F-actin content was determined at different time points of CXCL12 stimulation by flow cytometry using Alexa-Fluor-488- labeled phalloidin (mean±s.e.m., n=3). MFI, mean fluorescence intensity. (C) Cells were incubated for 30 min at 37°C with 1 or 5 µm cytochalasin B and subsequently seeded on electrode arrays for the ECIS adhesion assay. The figure shows a representative graph corresponding to one of three independent experiments showing changes in adhesion from the moment of cell addition. Bar graph represents the mean±s.e.m. increase in resistance from the moment of cell addition until 1 h after PMA addition (n=3). (D) Phospho-cofilin (P-cofilin) and total cofilin were detected by immunoblotting of cell lysates of cells after 30 min adhesion to immobilized Fc-VCAM. Bar graphs show the quantification (mean±s.d.) of band densities of three independent experiments. *P<0.05, **P<0.01 [two- way ANOVA (A,B,C); two-tailed Student’s t-test (D)]. conditions, which could explain the differences observed in F-actin crosslink membrane receptors with the underlying actin content and ligand binding in unstimulated cells. Despite the cytoskeleton (Fehon et al., 2010). ERM protein activation is colocalization of PrP and β1 integrin in flotillin-1 domains, we were crucial for uropod formation (Ivetic and Ridley, 2004; Lee et al., unable to show association of PrP and β1integrininco- 2004; Martinelli et al., 2013) and for β1-integrin-dependent T cell immunoprecipitation assays (data not shown), although β1 integrin adhesion and polarization (Chen et al., 2013; Liu et al., 2002). was previously identified as a PrP-interacting partner in a proteomic Therefore, the inhibition of ERM phosphorylation in the absence of study (Watts et al., 2009). We found that PrP silencing dramatically PrP likely contributes to both the lack of uropods and the reduced β1 altered the morphology and migratory behavior of cells moving on 2D integrin adhesion observed for PrP-deficient monocytes. surfaces. shCtrl-U937 cells migrating on immobilized VCAM RhoA is crucial for uropod de-adhesion and retraction during accumulated β1 integrins, phospho-ERM, flotillin-1 and CD44 in migration (Liu et al., 2002; Smith et al., 2003; Worthylake et al., the uropod, all bona fide uropod markers (Shulman et al., 2009; 2001). In neutrophils, activation of by RhoA has been Gómez-Moutón and Mañes, 2007). In contrast, PrP-depleted cells implicated in actomyosin-driven uropod retraction (Eddy et al., generally failed to form uropods and displayed a round shape, 2000). However, we found that phospho-myosin levels were equal although β1 integrin and flotillin-1 still showed a polarized in shCtrl- and shPrP-U937 cells, and that they did not increase upon distribution. Flotillins are essential for uropod formation in stimulating migration (data not shown). This suggests that neutrophils (Ludwig et al., 2010). Although PrP colocalizes with actomyosin contraction does not play a role in uropod retraction flotillin in U937 cells, flotillin distribution is not affected upon PrP in monocytic cells. In line with this, myosin inhibition did not affect silencing, which suggests that PrP does not regulate uropod formation uropod retraction in migrating monocytes (Worthylake et al., 2001). through the disruption of flotillin domains. PMA-dependent integrin activation induces firm leukocyte The uropod is an adhesive structure that needs to be retracted adhesion to the endothelium and blocks motility and migration during migration (Liu et al., 2002; Smith et al., 2003; Worthylake (Cinamon et al., 2001). Thus ‘excessive’ integrin activation et al., 2001). In agreement with this, PrP-deficient cells migrated prevents migration. In line with this, PMA-stimulated control cells faster than control cells. We found that PrP silencing alters the firmly adhered to the endothelium, whereas PMA-stimulated activation of ERM proteins and RhoA, which are regulators of PrP-deficient cells crawled over the endothelium while protruding uropod dynamics. First, PrP silencing reduces ERM protein dynamic bleb-like extensions. This motility could be prompted by phosphorylation. ERM proteins are activated by phosphorylation the weaker adherence of PMA-simulated PrP-silenced cells. In of a conserved C-terminal threonine residue, which enables them to addition, decreased membrane tension due to lower levels of active Journal of Cell Science

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ERM proteins might also contribute to this phenotype. In support of trichloroacetic acid and pelleted at 10,000 g for 15 min at 4°C. Pellets were this, constitutively active ezrin has been shown to increase washed with cold acetone and solubilized in SDS sample buffer. lymphocyte membrane tension, reduce chemokine-induced shape changes and block transendothelial migration (Liu et al., 2002). Chemotaxis In summary, we propose that PrP has a role in integrin-mediated Transwell chemotaxis assays were performed and analyzed as previously described (Lorenowicz et al., 2006). Crosslinking of surface PrP was adhesion and motility through the regulation of ERM-membrane– – performed by incubating cells for 30 min at 37°C with 0.5 µg/ml of the cortex adaptors and the RhoA cofilin pathway, which controls SAF61 anti-PrP antibody. cytoskeleton remodeling. This might be a general mechanism for the reported regulation of cell–cell and cell–matrix adhesion by PrP ECIS adhesion assays in different cell types (Petit et al., 2013). In addition, our data Cell adhesion to fibronectin, BSA, Fc-VCAM or Fc-ICAM was measured contributes to the understanding of additional roles of PrP in by using the electrical cell–substrate impedance sensing system (ECIS, neurodegeneration. Applied Biophysics). ECIS 8W10E electrode arrays were coated with 20 µg/ ml fibronectin or BSA for 16 h at 4°C. For Fc-ICAM and VCAM coating, MATERIALS AND METHODS electrode arrays were first coated with 4 µg/ml of AffiniPure F(ab′)2 goat Cell culture and lentiviral transduction anti-human-IgG antibody (Jackson ImmunoResearch) for 1 h at 37°C in Human pro-monocytic U937 cells were transduced with lentivirus 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM CaCl2 and 2 mM MgCl2. containing the validated Sigma MISSION shRNA plasmids targeting BSA (1%) was added for 30 min at 37°C to block non-specific binding. human PrP (shPrP-1, TRCN0000083488; shPrP-2, TRCN0000083490; or After blocking, human recombinant Fc-ICAM-1 or Fc-VCAM-1 were shPrP-3, TRCN0000083491) or non-targeting shRNA (shCtrl, SHC002) as added at a concentration of 1 µg/ml and incubated for 16 h at 4°C. The a negative control. Subsequently shRNA-expressing cells were selected by electrical resistance of cells (500,000 cells per well) seeded on protein- culturing in RPMI medium containing 10% fetal calf serum and 1.5 µg/ml coated arrays was measured in real-time at a frequency of 32 kHz. puromycin. Before each experiment, cells were cultured in puromycin-free medium for at least 24 h. PrP expression in control and knockdown cell lines Plate adhesion assays was tested before each experiment by flow cytometry with the SAF32 anti- Cells (100,000 cells per well) were added to F(ab′)2 and Fc-VCAM-coated PrP antibody. HUVEC (Primary Human Umbilical Vein Endothelial Cells) wells of flat-bottomed 96-well Maxisorp plates (coating was performed as were cultured in endothelial growth medium-2, both from Lonza. described for the ECIS arrays). Immediately after cell seeding, stimuli were Human monocytes were isolated from PBMCs by positive selection with added to the wells and cells were allowed to adhere for 30 min at 37°C. magnetic CD14 beads (Milteny Biotech). For transduction, shRNA Subsequently, non-adherent cells were removed by washing three times with lentiviruses were incubated with 4 µg/ml polybrene for 10 min at room PBS containing 0.5% BSA. Adherent cells were fixed with 3.7% temperature prior to their addition to monocyte suspensions (2×106 cells/ml) formaldehyde, incubated with Hoechst 33342 to stain nuclei and counted in complete RPMI. M-CSF was added at a concentration of 10 ng/ml to keep on an Axiovert wide-field microscope (Zeiss). monocytes steady without inducing differentiation (Troegeler et al., 2014). After 48 h, PrP expression was analyzed by flow cytometry. The maximum Adhesion and transendothelial migration under flow efficiency of PrP knockdown was ∼30–60%. Only those monocytes with at Confluent HUVEC monolayers on fibronectin-coated parallel-plate flow least 50% knockdown were used for experimental assays. chamber slides (µ-slide VI0.4, Ibidi) were treated with 10 ng/ml TNFα for 16 h. Flow chambers were mounted on an inverted wide-field fluorescence Antibodies and reagents microscope (Axiovert 200, Zeiss) fitted in a 37°C and 5% CO2 chamber. Mouse monoclonal antibodies against PrP (SAF32 and SAF61) were from Spi- shCtrl- and shPrP-U937 cells were differently labeled with either Calcein Bio. Anti-β1 integrin antibody (clone P5D4) was from Abcam. Activated β1 Green AM or Calcein Red-Orange AM (2.5 µM, Life Technologies). After integrins were detected with HUTS-4 (Millipore). Flotillin-1 rabbit polyclonal washing, cells were resuspended in assay buffer [20 mM Hepes pH 7.4, antibody was from Abcam and transferrin receptor mouse monoclonal 132 mM NaCl, 6 mM KCl, 1.2 mM K2HPO4, 1 mM MgSO4, 1 mM CaCl2, antibody from Life Technologies. RhoA monoclonal mouse antibody was 0.1% D-(+)-glucose and 2.5% human albumin]. Differently calcein-labeled from Santa Cruz Biotechnology. Rabbit antibodies against phospho-ERM shCtrl- and shPrP-U937 cells were mixed at 1:1 ratio to a final total cell proteins [ezrin (Thr567), radixin (Thr564), moesin (Thr558)], phospho-cofilin concentration of 1×106 cells/ml and subsequently stimulated with 100 ng/ml (Ser3), cofilin and myosin light chain (MLC) as well as mouse monoclonal PMA or with 100 ng/ml CXCL12 for 10 min. Stimulated cell mixes were antibody against phosphorylated myosin light chain (Ser19) were from Cell flown over TNFα-activated HUVEC monolayers at a flow rate of 0.5 ml/min, Signaling. Mouse monoclonal anti-moesin was from BD Biosciences. FITC- corresponding to a shear stress of 0.9 dynes/cm2.Time-lapsebright-fieldand labeled anti-CXCR4 mouse monoclonal was from R&D Systems. Mouse fluorescence images were acquired from four random microscope fields for monoclonal anti-actin was from Sigma. Horseradish peroxidase (HRP)- and 30–60 min using an Orca R2 digital CCD camera (Hamamatsu). Alexa-Fluor-dye-conjugated secondary antibodies were from Dako and Life Quantification of the number of adherent cells per frame was performed Technologies, respectively. Human fibronectin was from Biopur. Recombinant using the ‘analyze particles’ application of ImageJ after thresholding of the human Fc-ICAM and Fc-VCAM were from R&D Systems. The APC-labeled fluorescence video images. Transendothelial migration of U937 cells was polyclonal goat antibody human IgG F(ab′)2 was from Jackson assessed in similar experiments as above except for the immobilization of ImmunoResearch Laboratories. AnnexinV–FITC was from Pharmingen. CXCL12 on the endothelial surface (100 ng/ml, 15 min at 37°C) prior to U937 Human recombinant TNFα and CXCL12 were from Peprotech. Phorbol-12- cell perfusion. Time-lapse microscopy was performed with a LSM510 META myristate-13-acetate (PMA) and cytochalasin B were from Sigma. Calcein confocal microscope (Zeiss). Scoring of transmigrating cells was based Green AM and Calcein Red-Orange AM were from Life Technologies. on shape changes (from round to spread) and loss of brightness in the DIC channel. Membrane flotation assay 20×106 cells were lysed on ice for 15 min in TXNE buffer containing Cell motility on immobilized Fc-VCAM-1 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% IBIDI µ-slides were coated with F(ab′)2 goat anti-human-IgG and Fc- Triton X-100 and cOmplete Protease Inhibitor Cocktail Tablets (Roche). VCAM-1 as described for the ECIS arrays. Subsequently, CXCL12 was Lysates were mixed with 1.8 ml of 60% OptiPrep (Sigma) in TXNE buffer. absorbed on the dishes by incubation for 1 h at 37°C with 100 ng/ml Subsequently, 7.5 ml of 28% OptiPrep and 1.8 ml of TXNE buffer were CXCL12 in assay buffer. Motility of cells on Fc-VCAM-1 was recorded layered on top. Gradients were centrifuged at 200,000 g for 3 h at 4°C. 1-ml from four microscopic fields with a wide-field microscope as described fractions were taken from the top of the gradient and proteins were above. Cell tracking on videos and calculation of accumulated distance and precipitated for 30 min on ice by addition of an equal volume of 20% velocity was performed using Gradientech Tracking Tool software. Journal of Cell Science

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Flow cytometry Tablets, Roche. RhoA-GTP was isolated from cell lysates using a pulldown Cells were stimulated with CXCL-12(100 ng/ml) for 15 min at 37°C,cooled on assay as previously described (Alblas et al., 2001). ice and labeled with SAF32 anti-PrP antibody and anti-β1- or anti-β2-integrin antibodies for 1 h at 4°C. Cells were then fixed in 4% paraformaldehyde, Immunoblotting blocked in 50 mM NH4Cl and incubated with Alexa-Fluor-dye-labeled Cells were stimulated in suspension and lysed in cold NP-40 buffer (25 mM secondary antibodies. Labeled cells were analyzed by flow cytometry using a Tris-HCl, pH 7.2, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2 1% NP-40 FACSCanto flow cytometer (BD Biosciences). Integrin activation was induced and 5% glycerol) containing Halt™ protease and phosphatase inhibitor with 100 ng/ml PMA or with 1 mM MnCl2 for 10 min at 37°C in Hank’s cocktail (Thermo Scientific). Phospho-ERM and total moesin levels were balanced salt solution containing 1 mM Ca2+ and 1 mM Mg2+. determined by western blotting. Pixel density was determined using ImageJ. Timecourse experiments for Fc-VCAM binding to cells were performed Data represent phospho-ERM-to-moesin ratios and were normalized to the as described previously (Chan et al., 2003). For quantification of the binding value at t=0 in shCtrl cells. to cells of Fc-VCAM and APC-labeled (Fab')2 complexes, we followed the To determine phospho-cofilin and total cofilin levels, U937 cells were protocol described by Konstandin et al. (Konstandin et al., 2006). lysed in sample buffer after 30 min of adhesion to VCAM-coated plates. Fluorescence was measured by flow cytometry using a FACSCanto flow Equal loading was controlled by detection of total cofilin. Pixel density cytometer (BD Biosciences). values was determined using ImageJ. Data represent phospho-cofilin-to- cofilin ratios. Values were normalized to shCtrl. Immunofluorescence For confocal microscopy analysis, antibody-labeled cells as above were Actin polymerization assay resuspended in mounting medium (Vectashield) containing DAPI, and CXCL12-induced changes in intracellular content of F-actin were measured – mounted on a glass slide. Alternatively, cells were seeded on Fc-VCAM and by Alexa-Fluor-488 phalloidin staining as described previously (Voermans CXCL12-coated IBIDI µ-slides, allowed to migrate and/or polarize for et al., 2001). Labeled cells were analyzed by flow cytometry using a 20 min before being fixed with 4% paraformaldehyde in assay buffer, FACSCanto flow cytometer (BD Biosciences) blocked using 50 mM NH4Cl in PBS and subsequently permeabilized with PBS-0.1% Triton X-100 for 2 min. After blocking in PBS containing 1% Statistical analysis BSA, cells were subsequently incubated with primary antibodies for 30 min Data are represented as mean±s.d. or mean±s.e.m. Statistical comparisons of ’ t at room temperature and Alexa-Fluor-488-labeled goat anti-mouse-IgG means were performed using a two-tailed Student s -test, one-way ANOVA ’ antibodies. To stain nuclei, Hoechst 33342 was added in the washing buffer. with a Bonferroni post hoc test or two-way ANOVA with a Bonferroni s Fluorescence images were obtained with the LSM510 META (Zeiss) or multiple comparisons post hoc test. Statistical significance is represented by P P P P with a SP8 (Leica) confocal microscopes. asterisks (* <0.05, ** <0.01, *** <0.001, **** <0.0001). For colocalization analysis between PrP and β1 integrin, regions of ’ Acknowledgements interest (ROIs) were selected on digital pictures and Pearson s coefficients We are grateful to Dr Peter Hordijk for critically reading our manuscript. We also were determined using the Colocalisation Test plugin from ImageJ (Costes thank Erik Mul for his assistance with flow cytometry assays and Nicoletta Sorvillo for randomization method). In polarized cells, ROIs were selected in the helping with monocyte isolation. We are grateful to Maaike Scheenstra for sharing capped and non-capped membrane domains. In non-polarized cells, ROIs with us her protocol for monocyte transduction and to Dr Keith Burridge and were made around the membrane of each cell halves. We considered that Dr Metello Innocenti for their helpful comments. colocalization existed at Pearson’s coefficient values from 0.5 to 1.0. Competing interests The authors declare no competing or financial interests. DNA constructs Monomeric EGFP (mEGFP) was inserted between the signal peptide Author contributions – – (amino acids 1 22) and mature PrP (amino acids 23 253) by ligation D.D.R., S.T., E.V.-E., C.P. and R.B. performed the research and analyzed the data; independent cloning. A 121-bp fragment containing the PrP signal peptide M.F.-B. designed the research, analyzed the data and wrote the paper. D.G. was PCR amplified from human PrP cDNA (TrueClone from Origene) contributed reagents. D.D.R. and S.T. contributed equally to this study. using primers MFB001 (5′-GCAGGGGCGCAACAGACCCCGGTG- CCACCATGGCGAACCTTGGCTGCTGGATGC-3′) and MFB002 Funding (5′-CCACCAGGCCGGCCAGCACCCGGTCCGCAGAGGCCCAGG- This work was financed with a grant from Sanquin Blood Supply [grant number TCACTCCATGTG-3′) and inserted in frame in front of mEGFP in XmaI- PPOC 11-006]. R.B. was supported by a European Hematology Association Research Fellowship. digested mEGFP-LIC as described previously (Bierings et al., 2012), resulting in sp-mEGFP-LIC. A 754-bp fragment containing mature PrP was Supplementary material ′ PCR amplified using primers MFB003 (5 -GGGCGCGCCTGGTGGGG- Supplementary material available online at CCAAGAAGCGCCCGAAGCCTGGAGGATG-3′) and MFB004 (5′-GC- http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.165365/-/DC1 GGCCGCCTGCTCGTCCATCATCCCACTATCAGGAAGATGAGG-3′) and inserted in frame behind mEGFP in SacII-digested sp-mEGFP-LIC References using ligation independent cloning. All plasmids were verified by sequence Alblas, J., Ulfman, L., Hordijk, P. and Koenderman, L. (2001). Activation of Rhoa analysis. and ROCK are essential for detachment of migrating leukocytes. Mol. Biol. Cell 12, 2137-2145. Alon, R., Feigelson, S. W., Manevich, E., Rose, D. M., Schmitz, J., Overby, D. R., Rescue of PrP expression Winter, E., Grabovsky, V., Shinder, V., Matthews, B. D. et al. (2005). U937 cells stably expressing shRNA targeting human PrP were cultured for Alpha4beta1-dependent adhesion strengthening under mechanical strain is 48 h in puromycin-free medium. After 48 h cells were transfected by regulated by paxillin association with the alpha4-cytoplasmic domain. J. Cell electroporation (Neon Transfection System, Life Technologies) with a GFP– Biol. 171, 1073-1084. ’ Bakker, G. J., Eich, C., Torreno-Pina, J. A., Diez-Ahedo, R., Perez-Samper, G., PrP or GFP construct according to the manufacturer s recommendations. van Zanten, T. S., Figdor, C. G., Cambi, A. and Garcia-Parajo, M. F. (2012). GFP-positive cells were sorted using a FACSAria II cell sorter (BD Lateral mobility of individual integrin nanoclusters orchestrates the onset for Biosciences). Cells were allowed to recover for 2 h in complete medium leukocyte adhesion. Proc. Natl. Acad. Sci. USA 109, 4869-4874. before seeding on Fc-VCAM-coated IBIDI µ-slides. Ballerini, C., Gourdain, P., Bachy, V., Blanchard, N., Levavasseur, E., Gregoire, S., Fontes, P., Aucouturier, P., Hivroz, C. and Carnaud, C. (2006). Functional implication of cellular prion protein in antigen-driven interactions between T cells and GTPase activation assay dendritic cells. J. Immunol. 176, 7254-7262. Cells were lysed in 25 mM Tris-HCl pH 7.2, 150 mM NaCl, 10 mM MgCl2, Biasini, E., Turnbaugh, J. A., Unterberger, U. and Harris, D. A. (2012). Prion

1% NP-40 and 5% glycerol and cOmplete Protease Inhibitor Cocktail protein at the crossroads of physiology and disease. Trends Neurosci. 35, 92-103. Journal of Cell Science

3028 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3018-3029 doi:10.1242/jcs.165365

Bierings, R., Hellen, N., Kiskin, N., Knipe, L., Fonseca, A.-V., Patel, B., Meli, A., (2012). Neuritogenesis: the prion protein controls beta1 integrin signaling activity. Rose, M., Hannah, M. J. and Carter, T. (2012). The interplay between the FASEB J. 26, 678-690. Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade Ludwig, A., Otto, G. P., Riento, K., Hams, E., Fallon, P. G. and Nichols, B. J. body exocytosis. Blood 120, 2757-2767. (2010). Flotillin microdomains interact with the cortical cytoskeleton to control Bravo-Cordero, J. J., Magalhaes, M. A. O., Eddy, R. J., Hodgson, L. and uropod formation and neutrophil recruitment. J. Cell Biol. 191, 771-781. Condeelis, J. (2013). Functions of cofilin in cell locomotion and invasion. Nat. Málaga-Trillo, E., Solis, G. P., Schrock, Y., Geiss, C., Luncz, L., Thomanetz, V. Rev. Mol. Cell Biol. 14, 405-417. and Stuermer, C. A. O. (2009). Regulation of embryonic cell adhesion by the Buensuceso, C., de Virgilio, M. and Shattil, S. J. (2003). Detection of integrin prion protein. PLoS. Biol. 7, e55. alpha IIbbeta 3 clustering in living cells. J. Biol. Chem. 278, 15217-15224. Mangé, A., Milhavet, O., Umlauf, D., Harris, D. and Lehmann, S. (2002). PrP- Carman, C. V. and Springer, T. A. (2003). Integrin avidity regulation: are changes in dependent cell adhesion in N2a neuroblastoma cells. FEBS Lett. 514, 159-162. affinity and conformation underemphasized? Curr. Opin. Cell Biol. 15, 547-556. Margadant, C., Monsuur, H. N., Norman, J. C. and Sonnenberg, A. (2011). Chan, J. R., Hyduk, S. J. and Cybulsky, M. I. (2001). Chemoattractants induce a Mechanisms of integrin activation and trafficking. Curr. Opin. Cell Biol. 23, 607-614. rapid and transient upregulation of monocyte alpha4 integrin affinity for vascular Martinelli, S., Chen, E. J. H., Clarke, F., Lyck, R., Affentranger, S., Burkhardt, cell adhesion molecule 1 which mediates arrest: an early step in the process of J. K. and Niggli, V. (2013). Ezrin/Radixin/Moesin proteins and flotillins cooperate emigration. J. Exp. Med. 193, 1149-1158. to promote uropod formation in T cells. Front. Immunol. 4, 84. Chan, J. R., Hyduk, S. J. and Cybulsky, M. I. (2003). Detecting rapid and transient Minamide, L. S., Striegl, A. M., Boyle, J. A., Meberg, P. J. and Bamburg, J. R. upregulation of leukocyte integrin affinity induced by chemokines and (2000). Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that chemoattractants. J. Immunol. Methods 273, 43-52. disrupt distal neurite function. Nat. Cell Biol. 2, 628-636. Chen, E. J. H., Shaffer, M. H., Williamson, E. K., Huang, Y. and Burkhardt, J. K. Morel, E., Fouquet, S., Strup-Perrot, C., Pichol Thievend, C., Petit, C., Loew, D., (2013). Ezrin and moesin are required for efficient T cell adhesion and homing to Faussat, A.-M., Yvernault, L., Pinçon-Raymond, M., Chambaz, J. et al. (2008). lymphoid organs. PLoS. ONE 8, e52368. The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells Cinamon, G., Shinder, V. and Alon, R. (2001). Shear forces promote lymphocyte and in the distribution of junction-associated proteins. PLoS ONE 3, e3000. migration across vascular endothelium bearing apical chemokines. Nat. Immunol. 2, Mouillet-Richard, S., Ermonval, M., Chebassier, C., Laplanche, J. L., Lehmann, 515-522. S., Launay, J. M. and Kellermann, O. (2000). Signal transduction through prion Colby, D. W. and Prusiner, S. B. (2011). Prions. Cold Spring Harb. Perspect. Biol. protein. Science 289, 1925-1928. 3, a006833. Nourshargh, S., Hordijk, P. L. and Sixt, M. (2010). Breaching multiple barriers: de Almeida, C. J. G., Chiarini, L. B., da Silva, J. P., e Silva, P. M. R., Martins, M. A. leukocyte motility through venular walls and the interstitium. Nat. Rev. Mol. Cell and Linden, R. (2005). The cellular prion protein modulates phagocytosis and Biol. 11, 366-378. Petit, C. S. V., Besnier, L., Morel, E., Rousset, M. and Thenet, S. (2013). Roles of inflammatory response. J. Leukoc. Biol. 77, 238-246. the cellular prion protein in the regulation of cell-cell junctions and barrier function. Eddy, R. J., Pierini, L. M., Matsumura, F. and Maxfield, F. R. (2000). Ca2 Tissue Barriers 1, e24377. +-dependent myosin II activation is required for uropod retraction during neutrophil Prieto, J., Eklund, A. and Patarroyo, M. (1994). Regulated expression of integrins migration. J. Cell Sci. 113, 1287-1298. and other adhesion molecules during differentiation of monocytes into Fehon, R. G., McClatchey, A. I. and Bretscher, A. (2010). Organizing the cell macrophages. Cell. Immunol. 156, 191-211. cortex: the role of ERM proteins. Nat. Rev. Mol. Cell Biol. 11, 276-287. Schrock, Y., Solis, G. P. and Stuermer, C. A. O. (2009). Regulation of focal Friedl, P. and Weigelin, B. (2008). Interstitial leukocyte migration and immune adhesion formation and filopodia extension by the cellular prion protein (PrPC). function. Nat. Immunol. 9, 960-969. FEBS Lett. 583, 389-393. Gómez-Moutón, C. and Mañes, S. (2007). Establishment and maintenance of cell Shulman, Z., Shinder, V., Klein, E., Grabovsky, V., Yeger, O., Geron, E., polarity during leukocyte chemotaxis. Cell Adh. Migr. 1, 69-76. Montresor, A., Bolomini-Vittori, M., Feigelson, S. W., Kirchhausen, T. et al. Grabovsky, V., Feigelson, S., Chen, C., Bleijs, D. A., Peled, A., Cinamon, G., (2009). Lymphocyte crawling and transendothelial migration require chemokine Baleux, F., Arenzana-Seisdedos, F., Lapidot, T., van Kooyk, Y. et al. (2000). triggering of high-affinity LFA-1 integrin. Immunity 30, 384-396. Subsecond induction of alpha4 integrin clustering by immobilized chemokines Smith, A., Bracke, M., Leitinger, B., Porter, J. C. and Hogg, N. (2003). LFA-1- stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion induced T cell migration on ICAM-1 involves regulation of MLCK-mediated molecule 1 under flow conditions. J. Exp. Med. 192, 495-506. attachment and ROCK-dependent detachment. J. Cell Sci. 116, 3123-3133. Hyduk, S. J. and Cybulsky, M. I. (2009). Role of alpha4beta1 integrins in Stuermer, C. A. O., Langhorst, M. F., Wiechers, M. F., Legler, D. F., Von chemokine-induced monocyte arrest under conditions of shear stress. Hanwehr, S. H., Guse, A. H. and Plattner, H. (2004). PrPc capping in T cells Microcirculation 16, 17-30. promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads Isaacs, J. D., Jackson, G. S. and Altmann, D. M. (2006). The role of the cellular to signal transduction. FASEB J. 18, 1731-1733. prion protein in the immune system. Clin. Exp. Immunol. 146, 1-8. Troegeler, A., Lastrucci, C., Duval, C., Tanne, A., Cougoule, C., Maridonneau- Ivetic, A. and Ridley, A. J. (2004). Ezrin/radixin/moesin proteins and Rho GTPase Parini, I., Neyrolles, O. and Lugo-Villarino, G. (2014). An efficient siRNA- signalling in leucocytes. 112, 165-176. mediated gene silencing in primary human monocytes, dendritic cells and Kent, D. G., Copley, M. R., Benz, C., Wohrer, S., Dykstra, B. J., Ma, E., Cheyne, macrophages. Immunol. Cell Biol. 92, 699-708. J., Zhao, Y., Bowie, M. B., Zhao, Y. et al. (2009). Prospective isolation and Voermans, C., Anthony, E. C., Mul, E., van der Schoot, E. and Hordijk, P. (2001). molecular characterization of hematopoietic stem cells with durable self-renewal SDF-1-induced actin polymerization and migration in human hematopoietic potential. Blood 113, 6342-6350. progenitor cells. Exp. Hematol. 29, 1456-1464. Konstandin, M. H., Sester, U., Klemke, M., Weschenfelder, T., Wabnitz, G. H. Walsh, K. P., Kuhn, T. B. and Bamburg, J. R. (2014). Cellular prion protein: a co- and Samstag, Y. (2006). A novel flow-cytometry-based assay for quantification of receptor mediating neuronal cofilin-actin rod formation induced by beta-amyloid affinity and avidity changes of integrins. J. Immunol. Methods 310, 67-77. and proinflammatory cytokines. Prion 8, 375-380. Langhorst, M. F., Reuter, A., Luxenhofer, G., Boneberg, E.-M., Legler, D. F., Watts, J. C., Huo, H., Bai, Y., Ehsani, S., Jeon, A. H., Shi, T., Daude, N., Lau, A., Plattner, H. and Stuermer, C. A. O. (2006). Preformed reggie/flotillin caps: stable Young, R., Xu, L. et al. (2009). Interactome analyses identify ties of PrPC and its priming platforms for macrodomain assembly in T cells. FASEB J. 20, 711-713. mammalian paralogs to oligomannosidic N-glycans and - Lee, J.-H., Katakai, T., Hara, T., Gonda, H., Sugai, M. and Shimizu, A. (2004). derived chaperones. PLoS. Pathog. 5, e1000608. Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod Wegener, J., Keese, C. R. and Giaever, I. (2000). Electric cell-substrate impedance formation. J. Cell Biol. 167, 327-337. sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to Linden, R., Martins, V. R., Prado, M. A. M., Cammarota, M., Izquierdo, I. and artificial surfaces. Exp. Cell Res. 259, 158-166. Brentani, R. R. (2008). Physiology of the prion protein. Physiol. Rev. 88, 673-728. Worthylake, R. A. and Burridge, K. (2003). RhoA and ROCK promote migration by Liu, L., Schwartz, B. R., Lin, N., Winn, R. K. and Harlan, J. M. (2002). Requirement limiting membrane protrusions. J. Biol. Chem. 278, 13578-13584. for RhoA kinase activation in leukocyte de-adhesion. J. Immunol. 169, 2330-2336. Worthylake, R. A., Lemoine, S., Watson, J. M. and Burridge, K. (2001). RhoA is Lorenowicz, M. J., van Gils, J., de Boer, M., Hordijk, P. L. and Fernandez-Borja, required for monocyte tail retraction during transendothelial migration. J. Cell Biol. M. (2006). Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis. 154, 147-160. J. Leukoc. Biol. 80, 1542-1552. Zhang, C. C., Steele, A. D., Lindquist, S. and Lodish, H. F. (2006). Prion protein is Loubet, D., Dakowski, C., Pietri, M., Pradines, E., Bernard, S., Callebert, J., expressed on long-term repopulating hematopoietic stem cells and is important for Ardila-Osorio, H., Mouillet-Richard, S., Launay, J.-M., Kellermann, O. et al. their self-renewal. Proc. Natl. Acad. Sci. USA 103, 2184-2189. Journal of Cell Science

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