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PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury Graphical Abstract

Authors Qianying Yuan, Chunguang Ren, Wenwen Xu, ..., Paul Kubes, Dianqing Wu, Wenwen Tang

Correspondence [email protected] (D.W.), [email protected] (W.T.)

In Brief Yuan et al. elucidate a mechanism by which PKN1 regulates directional trafficking of RAB21 vesicles via phosphorylation RPH3A, a RAB21 effector. This mechanism underlies the polarized distribution of PIP5K1C90 in neutrophils and is important for neutrophil recruitment during inflammation.

Highlights d

RPH3A is an effector of RAB21

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PKN1 phosphorylates RPH3A to enhance its binding to GTPbound Rab21

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PIP5K1C90 polarization is regulated by PKN1 polarized RAB21 vesicle trafficking

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This pathway is critical for neutrophil adhesion and recruitment during inflammation

Yuan et al., 2017, Cell Reports 19, 2586–2597 June 20, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.05.080

Cell Reports

Article PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury Qianying Yuan,1,5 Chunguang Ren,1,5 Wenwen Xu,1 Bjo¨rn Petri,2,3 Jiasheng Zhang,4 Yong Zhang,1 Paul Kubes,2 Dianqing Wu,1,* and Wenwen Tang1,6,* 1Department

of Pharmacology, Vascular Biology and Therapeutic Program, Yale School of Medicine, New Haven, CT 06520, USA Institute for Chronic Diseases Mouse Phenomics Resource Laboratory 3Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine University of Calgary, Calgary, AB T2N 4N1, Canada 4Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520, USA 5These authors contributed equally 6Lead Contact *Correspondence: [email protected] (D.W.), [email protected] (W.T.) http://dx.doi.org/10.1016/j.celrep.2017.05.080 2Snyder

SUMMARY

Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to guanosine triphosphate (GTP)-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusionrelated tissue injury. INTRODUCTION The neutrophil is a key player in inflammatory responses. It not only plays an important role in host defense but also contributes to inflammation-related tissue damages in various chronic and acute inflammatory diseases, including ischemia reperfusion (Kolaczkowska and Kubes, 2013; Nauseef and Borregaard, 2014). During the inflammation, neutrophils are recruited to the sites of injury and infection. They extravasate across the

endothelium that lines the blood vessel wall through a multistep process, which includes rolling, firm adhesion, intravascular crawling, diapedesis, and extravascular chemotaxis (Abram and Lowell, 2007; Gerhardt and Ley, 2015; Imhof and Aurrand-Lions, 2004; Luo et al., 2007; Nourshargh and Alon, 2014; Rose et al., 2007; Williams et al., 2011). This infiltration process, which is also shared by other types of leukocytes, requires neutrophil polarization, namely the transformation of the apolar, circulating cells to being polarized through a spatial reorganization of signaling and cytoskeletal molecules. Chemoattractant stimulation can polarize the neutrophils anteroposteriorly to form the lamellar F-actin-rich leading edge and actomyosin-containing uropod, and this polarization is crucial for neutrophil migration (Afonso and Parent, 2011; Bear and Haugh, 2014; Graziano and Weiner, 2014; Ko¨lsch et al., 2008; Ridley, 2011; Shelef et al., 2013; Swaney et al., 2010; Weninger et al., 2014). However, in vivo before neutrophils undergo chemoattractant-stimulated migration, they have to first adhere to the endothelium via integrin engagement, which can induce a different form of neutrophil polarization from that induced by chemoattractants. Integrins induce the polarization of PIP5K1C90 independently of chemoattractant signaling initially at the site of attachment, which becomes the uropod when the cell polarizes anteroposteriorly upon chemoattractant stimulation (Xu et al., 2010). Polarization of PIP5K1C90, which is a lipid kinase involved in PtdIns4,5P2 synthesis, leads to polarized RHOA activation and increases in integrin affinity and neutrophil adhesion to the endothelium (Nourshargh and Alon, 2014; Williams et al., 2011; Xu et al., 2010). One mechanism for establishment and maintenance of cell polarity is polarized vesicle trafficking. Secretory vesicles generated from the trans-Golgi network carrying newly synthesized cargos or from endosomal compartments often carrying recycled cargos are transported to specific areas of the cell, leading to cell polarization. The evolutionarily conserved exocyst, an octameric protein complex, plays an important role in polarized exocytosis during many cellular activities, including cell migration and epithelial polarization (He and Guo, 2009;

2586 Cell Reports 19, 2586–2597, June 20, 2017 ª 2017 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

filament proteins, neurofilaments, vimentin, and the microfilament protein a-actinin (Mukai, 2003). The role of PKN1 in myeloid functions has not been investigated. PIP5K1C90 is a plasma-membrane-associated protein and interacts with talin and endocytic structural protein adaptin-2 (AP2) (Di Paolo et al., 2002; Lee et al., 2005; Ling et al., 2002; Thieman et al., 2009; Xu et al., 2010). Upon b2 integrin engagement, PIP5K1C90 is endocytosed with integrins and shows polarized localization in mouse neutrophils (Xu et al., 2010). Polarization of PIP5K1C90 leads to polarized RHOA activation and is important for neutrophil firm adhesion to the endothelium (Nourshargh and Alon, 2014; Williams et al., 2011; Xu et al., 2010). Although vesicle trafficking is implicated for its involvement in PIP5K1C90 polarization (Xu et al., 2010), the mechanism for this polarization is unknown. In this study, we elucidate the mechanism by which PKN1 regulates directional trafficking of RAB21 vesicles via its phosphorylation of a newly characterized RAB21 effector RPH3A, which underlies PIP5K1C90 polarization. RESULTS Figure 1. PKN1 Deficiency Attenuates Neutrophil Adhesion to Endothelial Cells (A) Validation of the lack of PKN1 proteins in neutrophils isolated from Pkn1m/m mice by western blot analysis. PKN2 and tubulin were detected as internal controls. (B–D) PKN1 deficiency impairs neutrophil infiltration into inflamed mouse peritonea (B), adhesion to endothelial cells under shear flow (C), and binding to ICAM1 (D). Data are presented as means ± SEM (*p < 0.05; Student’s t test; n > 5). (E–G) Effect of PKN1 deficiency on neutrophil-endothelial cell interaction in inflamed cremaster muscle venues. Adhesion (E), transmigration of neutrophils (F), and rolling flux (G) were determined after stimulation of the cremaster muscle with TNF-a (0.5 mg) for 4 hr. All values are means of n = 5 animals (with five to seven vessels per animal) ± SEM (*p < 0.05; **p < 0.01; Student’s t test).

Mukherjee et al., 2014; Orlando and Guo, 2009), including the control of directional vesicle trafficking to leading edges in migrating cells (Fletcher and Rappoport, 2010; He and Guo, 2009; Ulrich and Heisenberg, 2009). One of the components of the exocyst is the RAB small guanosine triphosphatases (GTPases). In addition to their involvement in the exocyst, the superfamily of the RAB small GTPases is the master regulator of vesicle trafficking in almost every step of the process. However, the role of RAB GTPases or directional vesicle trafficking in neutrophil protein polarization and infiltration has not yet been well investigated. Protein kinase N (PKN)1 together with its close homologs PKN2 and 3 are also known as protein-kinase-c-related kinases (PRKs) (Mukai, 2003). They belong to the AGC superfamily of protein kinases. PKN1 was reported to be involved in several functions, including smooth muscle migration and division (Singh et al., 2012), cardiac myocyte survival (Kajimoto et al., 2011), neurofilament organization and axonal transport in neurons, and enhancement of germinal center formation (Yasui et al., 2012). Known substrates for PKN1 are the intermediate

PKN1 Deficiency Impedes Neutrophil Adhesion to Endothelial Cells The Pkn1 gene is expressed at high levels in mouse leukocytes and the highest in neutrophils among the leukocytes based on gene expression data of the Immunological Genome Project (http://www.immgen.org). The PKN1 protein was also readily detected in mouse neutrophils by western blot analysis (Figure 1A). To investigate the importance of Pkn1 in mouse myeloid cells, we generated the myeloid-specific knockout mouse line (Pkn1m/m) by crossing the Pkn1-floxed mouse line (Figure S1A) with the Lyzstm1(cre)Ifo/J mice. The PKN1 protein was not detectable in the Pkn1m/m neutrophils by western blot analysis (Figure 1A). Myeloid-specific PKN1 deficiency did not affect the numbers of neutrophils or monocytes in circulation (Table S1). Next, we investigated the effect of PKN1 deficiency on neutrophil recruitment in vivo by using a peritonitis model. When isolated wild-type (WT) and PKN1-null neutrophils were labeled with different fluorescent dyes, mixed, and injected intravenously to WT mice in which peritonitis was elicited by thioglycollate, significantly fewer PKN1-null neutrophils were detected than the WT cells in the peritonea (Figure 1B). To understand how PKN1 deficiency affects neutrophil infiltration in vivo, we first examined whether PKN1 deficiency affects neutrophil chemotaxis in vitro using a Dunn chamber. PKN1 deficiency did not significantly affect the motility, even though it increased the directional errors (Figure S1B). The defect in chemotactic directionality reflected by the increase in the directional errors does not appear to be substantial enough to explain the strong in vivo infiltration defects. We thus examined whether PKN1 deficiency affected the neutrophil adhesion to endothelial cells, another key step in neutrophil infiltration. We first assessed the adhesion in vitro using a flow chamber coated with mouse endothelial cells, a condition resembling what occurs in vivo. We found that PKN1 deficiency significantly decreased the number of neutrophils adherent to endothelial cells under shear flow Cell Reports 19, 2586–2597, June 20, 2017 2587

(Figure 1C). Consistent with this observation, we also found that PKN1-null neutrophils showed significantly reduced binding to the integrin ligand ICAM-1 (Figure 1D), suggesting that PKN1 may regulate the adhesion through the regulation of integrin activity. To assess the role of PKN1 in neutrophil adhesion to endothelial cells in vivo, we performed intravital microscopic examination of the cremaster muscle venules. PKN1 deficiency markedly reduced the number of cells adherent to the endothelium of the venules treated with tumor necrosis factor alpha (TNF-a) (Figure 1E) and the number of transmigrated cells (Figure 1F) while increasing the number of rolling cells on the endothelium (Figure 1G). The decrease in the adhesion would result in a decrease in transmigration and an increase in rolling. Therefore, these intravital observations are consistent with those made in the flow chambers, and they together demonstrate that PKN1 is important for firm neutrophil adhesion to endothelial cells. PKN1 Is Required for PIP5K1C90 Polarization The adhesion-related phenotypes described in Figures 1B–1G for PKN1 deficiency are very similar to what we have previously reported for PIP5K1C deficiency (Xu et al., 2010). We hence wondered whether PKN1 was involved in PIP5K1C90 polarization. PIP5K1C90 polarization is induced by b2 integrin engagement and has an important role in firm adhesion of neutrophils to endothelial cells (Xu et al., 2010). Indeed, PKN1 deficiency significantly inhibited PIP5K1C90 polarization in response to fibrinogen (Fb), an integrin ligand (Figures 2A– 2C). Instead of being localized at one side of the WT cells, PIP5K1C90 was primarily localized at the vicinity of EEA1-postive early endosomes with a partial colocalization with EEA1 staining in the majority of PKN1-null neutrophils (Figures 2A and 2B). This result, together with the knowledge of the association of PKN1 with endosomes (Mellor et al., 1998) and involvement of vesicle transport in PIP5K1C90 polarization (Xu et al., 2010), prompted us to postulate that PKN1 may regulate PIP5K1C90 polarization via its regulation of vesicle trafficking.

Figure 2. PKN1 Deficiency Impairs PIP5K1C90 Polarization in Neutrophils (A and B) WT (A) or PKN1-defient (B) mouse neutrophils transfected with HAPIP5K1C90 were placed on Fb-coated coverslips for 30 min. They were then fixed and stained by anti-HA and anti-EEA1 antibodies followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Cell contours are outlined, and scale bars are 2.5 mm for all of the figures. (C) Quantification of the effect of PKN1 deficiency on PIP5K1C90 polarization. The quantification was done from three independent observations, and more than 20 cells were examined in each observation. Data are presented as means ± SD (**p < 0.01; Student’s t test).

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RAB21 Is Polarized and Regulates PIP5K1C90 Polarization Given the central role of the RAB GTPases in directing vesicle trafficking, we reasoned a likely involvement of at least one RAB GTPase in PIP5K1C90 polarization. Identification of the RAB GTPase may help us to understand how PKN1 regulates PIP5K1C90 polarization. We tested eight RAB GTPases, including RAB3D, RAB21, RAB11A, RAB9, RAB14, RAB32, RAB35, and RAB22A that are highly expressed in neutrophils (Carroll et al., 2001; Fletcher and Rappoport, 2010; He and Guo, 2009; Kauppi et al., 2002; Klinkert and Echard, 2016; Lu and Wilson, 2016; Millar et al., 2002; Ortiz Sandoval and Simmen, 2012; Simpson et al., 2004; Stenmark, 2009; Ulrich and Heisenberg, 2009). Each of the dominant negative (DN) mutants of these small GTPases was expressed in neutrophils, and PIP5K1C90 polarization on Fb was quantified (Figure S1C). We found that the DN mutant of RAB21 (T31N) inhibited the PIP5K1C90 polarization induced by Fb (Figures 3A–3C, S1C,

Figure 3. RAB21 Is Polarized and Involved in PIP5K1C90 Polarization in Neutrophils (A–C) RAB21DN mutant inhibits PIP5K1C90 polarization in mouse neutrophils. Mouse neutrophils were cotransfected with HA-PIP5K1C90 and GFP (A) or RAB21DN-GFP (B) and sorted by FACS. GFP-positive cells were stimulated by Fb for 30 min and stained by anti-HA and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Two representative cells are shown in (A) and (B). (C) shows the quantification of the percentage of neutrophils showing PIP5K1C90 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (*p < 0.01; Student’s t test). Figure S2A shows localization of PIP5K1C90 and EEA1 staining in representative cells placed on PK-coated surfaces as a control. (D–F) Polarization of RAB21 in mouse neutrophils upon Fb stimulation. Mouse neutrophils were placed on coverslips coated with PK (D) or Fb (E) for 30 min and stained by anti-RAB21 and Alexa488-conjugated secondary antibody (green) in presence of Alexa Fluor 647-phalloidin (red). (F) shows the quantification of the percentage of neutrophils showing RAB21 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (*p < 0.01; Student’s t test). (G) Polarization of RAB21 and PIP5K1C90 in neutrophils. Mouse neutrophils transfected with HAPIP5K1C90 were placed on Fb-coated coverslips for 30 min and stained by anti-HA and anti-RAB21 antibody followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Figure S2C shows localization of PIP5K1C90 and RAB21 staining in cells placed on PK-coated surfaces as a control. (H) PKN1 deficiency impairs RAB21 polarization. Mouse neutrophils were placed on Fb-coated coverslips for 30 min and fixed and stained by anti-RAB21 and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). The quantification for percentage of neutrophils showing RAB21 polarization was done from three independent observations, and more than 20 cells were examined in each observation. Data are presented as means ± SD (*p < 0.01; Student’s t test).

and S2). RAB21 belongs to the RAB5 subfamily of small GTPases that includes RAB5, RAB21, and RAB22. These RABs are localized to early endosomes and plasma membrane and play an important role in vesicle trafficking (Caswell et al., 2009; Pellinen et al., 2006; Stenmark, 2009). However, RAB5 (Xu et al., 2010) and RAB22 (Figure S1C) are not involved in PIP5K1C90 polarization in mouse neutrophils. Of note, neither RAB21DN expression nor PKN1 deficiency affected static adhesion of neutrophils to Fb or poly-lysine (PK)-coated surfaces; hence, their effects on PIP5K1C90 polarization cannot be due to a secondary effect of reduced neutrophil adhesion (Figure S2B). Importantly, the localization of RAB21 was also polarized in the majority of cells placed on Fb-coated surfaces compared to cells placed on PK-coated surfaces that do not

elicit strong integrin signaling (Figures 3D–3F, S2F, and S2G; Movie S1). In addition, RAB21 and PIP5K1C90 were polarized toward the same locations in Fb-stimulated neutrophils (Figure 3G). Concordantly, there was increased colocalization of RAB21 and PIP5K1C90 in Fb-stimulated cells compared to cells on a PK-coated surface (Figures 3G and S2C). Unlike Fb, which does not stimulate overtly polarized F-actin formation (Xu et al., 2010; Figure 3E), chemoattractants, such as fMLP, stimulate strong F-actin formation at the leading edge (Figure S2E). Chemoattractant-stimulated leading edge F-actin serves as a useful reference marker for neutrophil polarity. In fMLP-treated neutrophils, PIP5K1C90 polarized to the uropods opposite to the leading edge F-actin (Xu et al., 2010) and so did RAB21 (Figure S2E). ICAM-1 is a more specific b2-integrin ligand expressed on the Cell Reports 19, 2586–2597, June 20, 2017 2589

Figure 4. RPH3A Binds to RAB21 RAB21 binds to RPH3A via RPH3A’s C2 domain in a Ca2+ and GTP-dependent manner. Purified recombinant proteins were used in a GST-pull-down assay under the conditions described in the figure. Proteins were then detected by western blot analysis. C2, the C2 domain; Zn, the Zinc binding domain.

endothelial cell surfaces. We previously showed that it also could promote PIP5K1C90 polarization as Fb does (Xu et al., 2010). It stimulated polarization of RAB21 as well (data not shown). Moreover, the addition of chemoattractant CXCL2 to neutrophils seeded on an ICAM-1-coated surface, which is more closely relevant to the in vivo settings, resulted in RAB21 polarization to the uropods (Figure S2D) as occurred under the Fb and fMLP stimulation. These results together demonstrate that PIP5K1C90 and RAB21 share the same polarity in polarized neutrophils. In contrast to RAB21, RAB5 showed a different localization pattern (Figure S2E). Thus, we conclude that RAB21 is involved in PIP5K1C90 polarization in mouse neutrophils. Like PIP5K1C90 localization in PKN1-null neutrophils, PIP5K1C90 was also found in the vicinity of EEA1-positive endosomes with partial colocalization in neutrophils expressing RAB21DN (Figure 3B). This result suggests that functional RAB21, like PKN1, is required for trafficking of PIP5K1C90associated vesicles to their polarized destination from the endosomes. However, when a constitutively active form of RAB21 (Q76L) was expressed in neutrophils, PIP5K1C90 polarization was impaired as well, in contrast to normal PIP5K1C90 polarization in neutrophils expressing WT RAB21 (Figure 3C). This result suggests that the GTP/guanosine diphosphate (GDP) cycling may be important for RAB21 to regulate PIP5K1C90-associated vesicle trafficking. We next examined whether PKN1 regulated RAB21 polarization and found that PKN1 deficiency inhibited RAB21 polarization (Figure 3H). The localization of RAB21 in the vicinity of EEA1 endosomes in PKN1-null neutrophils (Figure 3H) implies that PKN1 may regulate RAB21-associated vesicle trafficking away from the early endosomes for PIP5K1C90 polarization. RPH3A Interacts with RAB21 and Regulates the Polarization To understand how RAB21 regulates PIP5K1C90 polarization, a search for RAB21 effectors was conducted. The recombinant protein of a constitutively active form (Q76L) of RAB21 fused to glutathione S-transferase (GST) was used in a pull-down experiment with the cell extract of HEK293 cells, followed by mass 2590 Cell Reports 19, 2586–2597, June 20, 2017

spectrometry (MS) analysis. Among the proteins identified by MS (Table S2), rabphilin 3A (RPH3A) (Figure S3A) caught our attention for its role in vesicle trafficking in neurons (Gonzalez and Scheller, 1999; Sasaki et al., 1997) and importantly for being one of numerous potential substrates of PKN1 identified in a previous proteomic study (Amano et al., 2010). Given that PKN1 deficiency and RAB21DN acted similarly in impairment of PIP5K1C90 polarization, we suspected that RPH3A might be the link between RAB21 and PKN1. We went on to validate the direct interaction between RAB21 or RAB21QL and RPH3A using purified recombinant proteins (Figures 4A and S3B). RPH3A showed a preference for GTP-bound RAB21 or RAB21-QL in a Ca2+-dependent manner (Figures 4A and S3B). Deletional mutagenesis revealed that RAB21 directly binds to the region that contains the C2 domains (Figures 4B and S3C). The finding of the interaction of the C-terminal portion of RPH3A with RAB21 is rather unexpected, as the Zn-binding domain at RPH3A N terminus was shown to interact with RAB3A and RAB27 previously (Fukuda et al., 2004; Gonzalez and Scheller, 1999). The role of RPH3A polarization in neutrophils was then €ter et al., examined using a RPH3A-null mouse line (Schlu 1999). RPH3A deficiency significantly impeded polarization of PIP5K1C90 and RAB21 in mouse neutrophils (Figures 5A and 5B). The lack of RPH3A also caused the localization of PIP5K1C90 and RAB21 in the vicinity of the EEA1-positive endosomes (Figures 5A and 5B). These results indicate that RPH3A regulates the polarization of both PIP5K1C90 and RAB21 in mouse neutrophils. Supporting this conclusion, RPH3A deficiency neutrophils showed phenotypes similar to those of PIP5K1C-null and PKN1-null neutrophils: RPH3A-null neutrophils showed significant reductions in binding to ICAM and adhesion to endothelial cells under flow in vitro and infiltration to inflamed peritonea in mice (Figures 5C–5E). Moreover, RPH3A-deficient neutrophils showed significantly reduced adhesion to the WT endothelium of inflamed cremaster muscle venules in WT mice receiving transfer from Rph3a / bone marrow compared to WT neutrophils in mice receiving transfer from WT bone marrow (Figure 5F). In addition, RPH3A-deficient neutrophils showed reduced transmigration and increased rolling in the same intravital microscopic observations (Figures 5G and 5H). These phenotypes are similar to those of PKN1 deficiency described in Figures 1B–1G and PIP5K1C deficiency described previously (Xu et al., 2010), further confirming that these proteins function in the same pathway.

Figure 5. RPH3A Is Involved in PIP5K1C90 and RAB21 Polarization (A and B) RPH3A deficiency impairs polarization of PIP5K1C90 and RAB21 in mouse neutrophils. Mouse neutrophils transfected with HAPIP5K1C90 were placed on Fb-coated coverslips for 30 min and stained by anti-EEA1 and anti-HA (A) or anti-RAB21 (B) antibodies, followed by secondary antibodies conjugated with Alexa 488 (colored in green) and Alexa Fluor 633 (colored in red). The percentage of neutrophils showing polarization was quantified from three independent observations, and more than 15 cells were examined in each observation. Data are presented as means ± SD (**p < 0.01; Student’s t test). (C–E) RPH3A deficiency impairs neutrophil binding to ICAM1 (C), adhesion to endothelial cells under shear flow (D), and infiltration into inflamed mouse peritonea (E). Data are presented as means ± SEM (*p < 0.05; **p < 0.01; Student’s t test; n > 4). (F–H) Effect of RPH3A deficiency on neutrophilendothelial cell interaction in inflamed cremaster muscle venues. Adhesion (F), transmigration of neutrophils (G), and rolling flux (H) were determined after stimulation of the cremaster muscle with TNF-a (0.5 mg) for 4 hr. All values are means of n = 3 animals (with five to seven vessels per animal) ± SEM (**p < 0.01; Student’s t test).

In addition to regulating integrin-mediated neutrophil firm adhesion during infiltration, PIP5K1C90 was reported to regulate myosin-II-mediated uropod retraction during chemotaxis in differentiated HL-60 cells (Lokuta et al., 2007). This might be another mechanism accounting for our neutrophil recruitment defect in PKN1-deficient or RPH3A-deficient mice. However, we did not observe obvious neutrophil retraction defect in our intravital microscopy examination. We also performed in vitro transendothelial cell migration assay using transwells covered with mouse endothelial cells. No significant differences were observed between WT and PKN1-null or RPH3A-null neutrophils (Figure S3D). Thus, it is very unlikely that PKN1 or RPH3A deficiency causes strong uropod retraction defects that would affect neutrophil transendothelial migration. PKN1-Mediated Phosphorylation of RPH3A Regulates the Polarization We next investigated whether RPH3A is an actual substrate for PKN1. We found that PKN1 phosphorylated RPH3A in an in vitro kinase assay, and the phosphorylation could be detected

by an anti-phospho-AKT substrate motif antibody (Figure 6A). Both PKN1 and AKT belong to the AGC protein kinase superfamily, and some of these kinases share similar substrate specificity. We mapped RPH3A residues phosphorylated by PKN1 by mutating the Ser or Thr residues within each of the putative AKT substrate motifs (RXXS/T) found in RPH3A. When RPH3A residues Ser-234 and 271 were both mutated, the protein could no longer be phosphorylated by PKN1 in an in vitro kinase assay (Figure 6B). Of note, residue Ser-234 was actually recognized by the anti-AKT substrate motif antibody because when it was mutated the protein could not be detected by the antibody in western blot analysis (data not shown). Furthermore, an antibody specific to phosphorylated Ser-234 of RPH3A failed to detect RPH3A in PKN1-deficient neutrophils (Figure 6C), suggesting that PKN1 is the key kinase that phosphorylates RPH3A in neutrophils. We next tested the importance of RPH3A phosphorylation in neutrophil polarization. We found that expression of WT RPH3A in RPH3A-null neutrophils could rescue RAB21 polarization in response to Fb stimulation (Figure 6D). In addition, RPH3A was also polarized (Figures S4A and S4B; Movie S2) together with RAB21 (Figure 6D) and PIP5K1C90 (Figure S4C), and overexpressing RAB21DN can impair the polarization of RPH3A (Figures S4D and S4E). However, the RPH3A mutant with the phosphorylation sites mutated failed to polarize or rescue RAB21 polarization in RPH3A-null neutrophils upon Fb stimulation (Figure 6E). Instead, the mutant protein is localized in the vicinity of RAB21 Cell Reports 19, 2586–2597, June 20, 2017 2591

Figure 6. PKN1-Mediated Phosphorylation of RPH3A Is Required for Polarization (A) PKN1 phosphorylates RPH3A. Phosphorylation of recombinant RPH3A protein by recombinant PKN1 protein was performed in an in vitro kinase assay, and RPH3A phosphorylation was detected by western blot analysis using an anti-phosphoAKT substrate motif antibody. (B) RPH3A residues S234 and S271 are phosphorylated by PKN1. WT RPH3A and its mutants SA1 (S234A) and SA (S234 and 271A) were subjected to an in vitro kinase assay using [32P]ATP. Phosphorylated RPH3A ([32P]RPH3A) was detected by a phosphoimager, whereas the inputs were detected by western blot analysis. (C) RPH3A phosphorylation at Ser 234 depends on PKN1. WT and PKN1-null neutrophils were analyzed by western blot analysis using an antiphospho-Ser234 RPH3A, anti-RPH3A, PKN1, or actin antibody. (D and E) WT RPH3A, but not its phosphorylationdefective mutant, can rescue RAB21 polarization in RPH3A-null cells. RPH3A-null neutrophils were transfected with RPH3A-HA (D) or RPH3A-SA-HA (E). The cells were then stained with an anti-HA antibody and anti-RAB21 antibody. Only anti-HAstaining-positive cells were examined. (F) Phosphorylated RPH3A shows enhanced binding to RAB21. Purified RPH3A protein was first incubated with PKN1 in the presence or absence of ATP in a kinase assay buffer for 30 min at 37 C. Phosphorylated RPH3A (pRPH3A) is evidenced by its upshift in the blot compared to unphosphorylated RPH3A (RPH3A). These RPH3A proteins were then subjected to a pull-down assay with GTPgS-bound RAB21. The proteins were detected by western blot analysis.

(Figure 6E) in these RPH3A-null neutrophils, suggesting that PKN1-mediated phosphorylation may be required for polarized trafficking of RAB21 and its associated cargo PIP5K1C90. To understand why the phosphorylation is important, we examined the effect of the phosphorylation on RAB21 and RPH3A interaction. RPH3A that was pre-phosphorylated by PKN1 showed enhanced interaction with RAB21 compared to non-phosphorylated RPH3A in the absence of calcium (Figure 6F). We have tested and found that phosphorylated RPH3A can bypass the need for calcium to interact with RAB21 (data not shown). We also examined localization of PKN1 in neutrophils and found that it showed polarized localization together with RPH3A in stimulated WT neutrophils (Figure S4F). Similar to RAB21, RPH3A, and PIP5K1C90, PKN1 shows no polarization in RPH3A-null neutrophils (Figure S4G) and is localized in the proximity to the phosphorylation-defective RPH3A mutant (Figure S4G) and probably RAB21 by inference (Figure 6E). We have previously shown that b2-integrin exhibited polarized colocalization with PIP5K1C90 in Fb-stimulated neutrophils (Xu et al., 2010), but in PKN1 or RPH3A-deficient neutrophils b2-integrin polarization was also impaired (Figure S4H). These results together suggest a model as depicted in Figure 7A; PKN1-mediated phosphorylation of 2592 Cell Reports 19, 2586–2597, June 20, 2017

RPH3A facilitates the recruitment of RAPH3A by RAB21, which is required for polarized transport of the vesicles and their associated cargos from endosomes to their polarized destinations in neutrophils. Disruption of any step of this mechanism, by expression of a DN RAB21, genetic inactivation of PKN1 or RPH3A, or substitution of a phosphorylation-defective mutant for WT RPH3A, would halt the polarized trafficking process and cause localization of these proteins at or near the endosomes. In addition, this pathway is independent of chemoattractant-induced neutrophil polarization epitomized by leading edge F-actin polarization as neither PKN1 nor RPH3A deficiency affects fMLPinduced F-actin polarization (Figure S4I). PKN1 Deficiency Ameliorates Renal Ischemia-Reperfusion Injury Neutrophils play important roles in reperfusion-induced renal tissue damage (Heinzelmann et al., 1999). Thus, we examined the effects of myeloid-specific PKN1 deficiency on neutrophil recruitment and tissue damage using the renal ischemia-reperfusion injury model in an attempt to provide additional in vivo validation of the PIP5K1C90 polarization pathway we characterized in this study. Reperfusion after renal arterial ischemia resulted in

Figure 7. PKN1 Deficiency Attenuates Renal Ischemia-Reperfusion Injury (A) Schematic presentation of a model for RPH3A and its phosphorylation to regulate directional vesicle trafficking and PIP5K1C90 polarization. Integrin engagement triggers endocytosis of PIP5K1C90, which traffics through EEA1-positive endosomes and emerges in RAB21-positive recycling vesicles. GTP-bound RAB21 exits from the endosomes and recruits PKN1-phosphorylated RHP3A. This process is required for polarized RAB21 vesicle trafficking and PIP5K1C90 polarization. (B and C) Loss of PKN1 reduces renal neutrophil infiltration. Neutrophil infiltration into reperfused kidneys was evaluated by flow cytometry after dissociated kidney cells were stained with CD11b and Ly6G (B) and by immunostaining of renal tissue sections with anti-Ly6B (C). Representative flow cytometric charts are shown in Figure S5. Data in (B) are presented as means ± SEM (**p < 0.01; Student’s t test; n = 5). (D) Blood creatinine contents from mice that were subjected to the renal ischemia-reperfusion procedure. Data are presented as means ± SEM (**p < 0.01; Student’s t test; n = 5). (E) Histological examination of renal tissue injury. Renal histological sections from mice that were subjected to the renal ischemia-reperfusion procedure were stained with periodic acid-Schiff (PAS) dye, and the representative sections are shown.

DISCUSSION

marked infiltration of neutrophils detected by flow cytometry (Figures 7B and S5) and immunohistostaining (Figure 7C) of kidney samples. Accompanied by neutrophil infiltration, there was renal damage evidenced by the increases in serum creatinine content (Figure 7D) and the number of coagulative necrotic renal tubules (Figure 7E). Both neutrophil infiltration and renal damage were significantly reduced in Pkn1m/m mice subjected to the same ischemia-reperfusion procedure (Figures 7B–7E and S5). Thus, PKN1 in myeloid cells plays an important role in renal ischemia-reperfusion injury.

In this study, we characterized a phosphorylation-regulated polarization mechanism in mouse neutrophils. This mechanism involves polarization of RAB21 most likely through RAB21-associated vesicles in a manner depending on PKN1-mediated phosphorylation of a newly identified RAB21 effector RPH3A. This mechanism is important for integrin activation, endothelial cell adhesion, and neutrophil infiltration in inflammation models. We previously showed that b2 integrin activation triggers polarization of PIP5K1C90 in neutrophils via vesicle transport (Xu et al., 2010). In this study, we show that PKN1-phosphorylation-regulated RAB21 polarization underlies PIP5K1C90 polarization in neutrophils. These data together allow us to propose a model as depicted in Figure 7A, in which integrin engagement triggers endocytosis of PIP5K1C90, a plasma-membrane-associated protein, which interacts with talin and endocytic structural protein AP2 (Di Paolo et al., 2002; Lee et al., 2005; Ling et al., 2002; Thieman et al., 2009; Xu et al., 2010). PIP5K1C90 likely traffics through EEA1-positive endosomes and emerges largely in RAB21-positive recycling Cell Reports 19, 2586–2597, June 20, 2017 2593

vesicles. GTP-bound RAB21 exits from the endosomes (Jean et al., 2012) and preferentially recruits phosphorylated RHP3A as PKN1-mediated RPH3A phosphorylation enhances RPH3A interaction with RAB21. We believe that the interaction has an important role in transport of RAB21 vesicles carrying PIP5K1C90 from endosomes to its polarized destination on the plasma membrane. Whereas RAB21- and PKN1-mediated phosphorylation of RPH3A are involved in trafficking of PIP5K1C90 away from the endosomes, additional mechanisms are likely to be needed for PIP5K1C90 to reach its final destination on plasma membranes. One reason for this assertion is that expression of the constitutively active mutant of RAB21, like the RAB21DN mutant, impaired PIP5k1C90 polarization in contrast to the expression of the WT RAB21 (Figure 3C), suggesting that GTP/GDP cycling is required for the polarization. In addition, the phosphor-mimetic mutant of RPH3A, unlike WT RPH3A, failed to rescue the polarization defect of RPH3A-deficient neutrophils and it cannot rescue the polarization defect of PKN1-deficient neutrophils either (data not shown). These suggest a possibility that subsequent dephosphorylation of RPH3A may be needed for the vesicles targeted to their final destination. Moreover, in order for PIP5K1C90 to be targeted to its polarized final destination on the plasma membranes, something on the plasma membrane has to mark the destination. Future studies will address these questions. PIP5K1C90 promotes RHOA activation and increases integrin affinity and neutrophil adherence to endothelial cells (Xu et al., 2010). Polarized PIP5K1C90 leads to polarized activation of RHOA, thus increasing the local active RHOA concentration that would result in greater integrin activation and stronger adhesion, which is needed by the inflamed endothelium to capture and hold on neutrophils under the shear flow of blood stream. Given that PKN1 and RPH3A deficiency shows neutrophil phenotypes, including reduced integrin binding, endothelium adhesion, and neutrophil infiltration, consistent with PIP5K1C deficiency or loss of PIP5K1C90 polarization (Xu et al., 2010), PKN1- and RPH3A-mediated PIP5K1C90 polarization may largely be accounted for these neutrophil phenotypes. RAB21 is also known to be involved in integrin recycling (Alanko et al., 2015; Mai et al., 2011; Pellinen et al., 2006, 2008; Stenmark, 2009). We previously showed that b2-integrin also showed posterior polarization in activated neutrophils (Xu et al., 2010). The localization of b2-integrin is more diffused than PIP5K1C90, and there is partial colocalization with PIP5K1C90 (Xu et al., 2010). Thus, there is a possibility that the directional vesicle-trafficking mechanism characterized in this study may also have a role in integrin recycling. This will be further investigated in the future. Ischemia-reperfusion injury occurs following myocardial infarction, transplantation, stroke, and trauma. It can lead to organ failure and remains the major cause of death in critically ill patients. There is a lack of effective therapeutic strategies for combatting ischemia-reperfusion injury. Neutrophils are the primary responders following ischemia and reperfusion, and their infiltration is positively correlated to the inflammatory reaction and injury severity associated with ischemia-reperfusion. In this study, we show that PKN1 -deficiency significantly amelio2594 Cell Reports 19, 2586–2597, June 20, 2017

rates tissue injury in a renal ischemia-reperfusion model. PKN1 is a serine/threonine protein kinase, which is a druggable target. In addition, global knockout of PKN1 shows no developmental or gross phenotypes (Que´tier et al., 2016; Yasui et al., 2012; data not shown), suggesting low likelihood of severe on-target side effects from its inhibition. Thus, PKN1 may be a potential therapeutic target for combating ischemia-reperfusion injury. However, because mice lacking its close homolog PKN2 are embryonically lethal (Que´tier et al., 2016), specific inhibitors of PKN1 may be needed. EXPERIMENTAL PROCEDURES Reagents and Constructs Antibodies to the following antigens were acquired commercially: GST (2624; Cell Signaling Technology); His (2366; Cell Signal); hemagglutinin (HA) (MMS101R; Covance); RAB21 (R4405; Sigma); RAB5 (D-11; Santa Cruz Biotechnology); EEA1 (612006; BD Biosciences); p-S234-RPH3A (Thermo Fisher Scientific; PA1-4693); RPH3A (Aviva System Biology; ARP59498_P050); and PIP5K1C90 (Di Paolo et al., 2002; kindly provided by Dr. Pietro De Camilli). Protein A/G-PLUS-agarose beads were acquired from Santa Cruz. formyl-MetLeu-Phe (fMLP) was purchased from Sigma. The cDNAs for RAB21 and RPH3A were acquired from Open Biosystems. Mice The floxed PKN1 mice were generated using a gene-targeting construct as depicted in Figure S1A. The construct was transfected in the embryonic stem cells (ESCs), and cells in which desirable homologous recombination has occurred were selected and used for blastocyst injection to produce chimeric mice, which were backcrossed into C57BL background for at least five generations. The floxed mice were then crossed with the B6.129-Lyzstm1(cre)Ifo/J mice from The Jackson Laboratory. The floxed PKN1 mice are available at The Jackson Laboratory now. The RPH3A-null mice were obtained from The Jackson Laboratory. WT C57BL mice were purchased from The Jackson Laboratory and used for neutrophil isolation. All animal studies were approved by the institutional animal care and use committees of Yale University. Neutrophil Preparation and Transfection Murine neutrophils were purified from bone marrows as previously described (Zhang et al., 2010). Briefly, bone marrow cells collected from mice (8–10 weeks old) were treated with the ACK buffer (155 mM NH4Cl, 10 mM KHCO3, and 127 mM EDTA) for red blood cell lysis, followed by a discontinuous Percoll density gradient centrifugation. Neutrophils were collected from the band located between 81% and 62% of Percoll. For transient transfection of primary mouse neutrophils, three million neutrophils were electroporated with 1.6 mg endotoxin-free plasmids or 300 nM of small interfering RNA (siRNA) using the human monocyte nucleofection kit (Lonza) with an Amaxa electroporation system. The cells were then cultured for overnight in the medium supplied with the kit containing 10% fetal bovine serum (FBS) and 25 ng/mL recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech). Cell sorting was done by a FACS Aria sorter (BD Biosciences). We have used this method to overexpress protein in primary neutrophils for many years, and electroporated neutrophils have good response to chemoattractant stimulation (Gao et al., 2015; Tang et al., 2011; Xu et al., 2010; Zhang et al., 2013). And this method is also widely used by other groups (Loison et al., ~es et al., 2007; Sun et al., 2007). 2014; Magalha Immunostaining and Observation of Neutrophils Neutrophils were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.01% saponin for 5 min and blocked with 2% BSA in PBS for 1 hr. Cells were then incubated with 1:100 diluted primary antibodies in blocking buffer at 4 C for overnight. Next day, secondary antibodies with conjugated fluorescent probes (Alexa 488 colored in green and Alexa Fluor 633 colored in red in the figures) were 1:200 in blocking buffer and incubated with cells for 1 hr at room temperature. Mounted slides were observed using

a Leica SP5 confocal microscopy. Because the fluorescence of the GFP expressed in neutrophils quenches after the fixation, permeabilization, overnight primary antibody staining, and repeated washes, the presence of GFP does not interfere with Alexa 488 signals. For quantification of colocalization of Rab21 with PIP5K1C-90, z stack images of consecutive optical planes spaced by 0.2 mm were acquired to cover the whole cell volume using confocal microscopy. Pearson’s coefficient was determined using Imaris 7.2.3. For quantification of polarized distribution, target proteins were stained with appropriate antibodies and imaged under Leica SP5 confocal microscopy. Cell peripheries were traced using the ImageJ Segment line tool with a width of 5 (examples are shown in Figure S6). Florescence intensities were plotted along the lines (Figure S6). The ratios of fluorescence intensities of 25% cell periphery surrounding the highest fluorescence intensity to total fluorescence intensities were calculated (see examples in Figure S6). We arbitrarily take cells with the ratios over 0.5 as being polarized and below 0.5 as being non-polarized. In other words, if the fluorescence intensity in 25% of the cell periphery accounts for more than 50% of the total florescence intensity of the cell periphery, we call the cell polarized. Preparation and Purification of Recombinant Proteins and Pull-Down Assay Full-length RPH3A and its mutants and segments as well as RAB21 were expressed in E. coli and purified by affinity purification. For the pull-down assays, recombinant GST-GTPase RAB21 protein (0.2 mg/mL in 10 mL dissolved in buffer A containing 10 mM phosphate buffer [pH 7.4], 10% glycerol, and 0.2 mM EDTA) was added with 1 mL of 10 mM GDP or GTPgS stock (dissolved in 50 mM Tris-HCl [pH 7.8]) for a final concentration of 1 mM guanine nucleotides and incubated on ice for 15 min. Then, RPH3A (0.2 mg/mL in 10 mL dissolved in buffer A) and 180 mL buffer B (10 mM phosphate buffer [pH 7.4], 10% glycerol, 1% Triton X-100, 0.1% SDS, 3 mM DTT, protease inhibitor cocktail, 10 mM MgCl2, and 0.1 mM GTPgS or GDP, with or without 0.1 mM CaCl2) were added to RAB21 protein and incubated at 4 C on a rotating platform for overnight. The protein mixture was then added to glutathione beads pre-blocked with 1% BSA. After the beads were washed six times with buffer B, proteins bound to the beads were analyzed by western blotting. Neutrophil Infiltration into Inflamed Peritonea and Flow Chamber Adhesion Assay For the peritonitis infiltration model, purified WT and mutant neutrophils were labeled with 2.5 mM CFSE (5-[and 6]-carboxyfluorescein diacetate succinimidyl esters) and 2.5 mM Far-Red DDAO SE, respectively, and vice versa. The WT and mutant cells with different fluorescence labels were mixed at a 1:1 ratio and injected into retro-orbital venous sinus of WT littermates (8–10 weeks old; female), which were injected with 2 mL of 3% thioglycolate (TG) 2 hr earlier. The mice were euthanized 1.5 hr later. Cells in their peritonea were collected and analyzed by cell counting and flow cytometry. The data presented are the combination of the experiments with reciprocal fluorescence labeling. To examine neutrophil adherence to endothelial cells under shear stress, mouse endothelial cells (Wang et al., 2008) were cultured to confluency on 10 mg/mL fibronectin-coated coverslips and treated with 50 ng/mL TNF-a for 4 hr. The coverslips containing the endothelial cell layer were washed with PBS and placed in a flow chamber apparatus (GlycoTech). The WT and mutant-cells-labeled different fluorescence labels as described above were mixed at a 1:1 ratio and flowed into the chamber at a shear flow rate of 1 dyn/cm2. The adherent cells were then examined and counted under a fluorescence microscope. ICAM-Binding Assay The assay was carried out as previously described (Konstandin et al., 2006). The ICAM-1-Fc-F(ab’)2 complexes were generated by incubating Cy5conjugated AffiniPure goat anti-human Fcg fragment-specific immunoglobulin G (IgG) F(ab’)2 fragments (Jackson Immunobiology) and ICAM-1-Fc (100 mg/mL; R&D Systems) at 4 C for 30 min in PBS. Neutrophils, which were resuspended at 0.5 3 106 cells/mL in PBS containing 0.5% BSA, 0.5 mM Mg2+, and 0.9 mM Ca2+ were mixed with the ICAM-1-Fc-F(ab’)2 complexes in the presence or absence of fMLP for durations specified in the figure

legends. The reactions were terminated by adding 4% paraformaldehyde. After 5 min, fixation was stopped by adding 3 mL ice-cold fluorescence-activated cell sorting (FACS) buffer. Cells were pelleted, resuspended in 300 mL of FACS buffer, and analyzed on a flow cytometer. Bone Marrow Transplantation and Intravital Microscopy Bone marrows from RPH3A-deficient mice and their littermates were transplanted into WT recipient mice (C57/BL; 8 weeks old; male) that had been subjected to 1,000 Gy X-ray irradiation. Eight weeks later, the transplanted mice were used for intravital observation. For intravital observations, mice were injected intrascrotally with recombinant mouse TNF-a (0.5 mg; R&D Systems) in 200 mL of saline for 4 hr prior to recording. To visualize neutrophils, rat monoclonal antibody RB6-8C5 against mouse Ly-6G (Gr-1) conjugated to Alexa Fluor 647 (2 mg in 200 mL saline; eBioscience) was injected intravenously into the tail vein after TNF-a stimulation. A mixture of 10 mg/kg xylazine (Bayer; Animal Health) and 200 mg/kg ketamine hydrochloride (Rogar/STB) was injected intraperitoneally to anesthetize mice. In all protocols, the left jugular vein was cannulated to administer additional anesthetics or antibodies. To visualize the vasculature of the cremaster muscle, mice were injected with 50 mg rat monoclonal antibody (mAb) against mouse PECAM-1 (eBioscience; clone 390, which has been previously reported not to interfere with leukocyte recruitment; ChristofidouSolomidou et al., 1997; Tasaka et al., 2003) conjugated to Alexa Fluor 594 (Invitrogen). The mouse cremaster muscle was used to study neutrophil recruitment as previously described (Liu et al., 2005) with the exception that neutrophil extravasation was visualized with spinning-disk confocal microscopy using a 203 water/0.95 immersion objective to focus the excitation onto the sample (Olympus). A 512 3 512 pixel back-thinned electron-multiplying charge-coupled device camera (C9100-13; Hamamatsu) was used for fluorescence detection. Volocity software (Improvision) was used to acquire and analyze images. A neutrophil was considered to be adherent if it remained stationary for more than 30 s, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-mm length of venule during 5 min. Leukocyte emigration was defined as the number of cells in the extravascular space within a 200 3 300 mm area (0.06 mm2) adjacent to the observed venule and was normalized against the number of adherent cells. In Vitro Chemotaxis Assay in a Dunn Chamber The chemotaxis assay using a Dunn chamber was carried out as previously described (Zicha et al., 1997) with some modifications as detailed in Zhang et al. (2010). We analyzed WT and mutant neutrophils simultaneously by labeling the cells with different tracing dyes. We alternated the labeled group in the study to completely eliminate the possibility of any influence from the dye. Time-lapse image series were acquired at 30-s intervals for 30 min and were analyzed using the MetaMorph image analysis software as described in Zhang et al. (2010). We normally obtain two parameters to quantify neutrophil chemotaxis: average directional errors and motility. The average directional error measures the angle between the cell migration direction, and the gradient direction and reflects how well a cell follows the gradient. Motility is cell migration speed. Transendothelial Migration Assay Mouse endothelial cells (Wang et al., 2008) were seeded into the upper well of a 24-well Boyden chamber until confluent, followed by incubation with 10 ng/mL TNF-a for 4 hr. The WT or mutant neutrophils were plated in the upper well, and medium with or without 5 mM fMLP was added to the lower chamber. Neutrophils that migrate across the endothelial cells were recovered from the lower chamber and quantified using ATPlite system (PerkinElmer). Murine Kidney Ischemia-Reperfusion Models The left and right renal arteries of mutants and their littermate control mice (9 or 10 weeks old; male) were clamped for 30 min using vascular clamps (Fine Science Tools) through a midline abdominal incision under general anesthesia. After ischemia, the clamps were removed, the wounds were sutured, and the animals were allowed to recover for 24 hr before euthanization. Blood was collected by cardiac puncture in heparin-containing tube, and creatinine levels were determined using the DetectX serum creatinine kit (Arbor Assays).

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Kidneys were dissected from mice. One kidney of a mouse was fixed in 4% paraformaldehyde, embedded in paraffin, and stained for the periodic acidSchiff (PAS) dye and a Ly6B antibody. The other kidney from the same mouse was digested with collagenase I (300 U) and analyzed by flow cytometry. Statistical Analysis Statistical analyses were performed using a two-tailed Student’s t test. The results are indicated as mean ± SD or mean ± SEM. p < 0.05 was considered statistically significant. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, two tables, and two movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2017. 05.080. AUTHOR CONTRIBUTIONS D.W. and W.T. supervised the project and developed the concepts. D.W., W.T., Q.Y., C.R., B.P., and P.K. designed the experiments. W.T., Q.Y., C.R., W.X., B.P., J.Z., and Y.Z. performed the experiments. D.W., W.T., Q.Y., C.R., and B.P. analyzed the data. D.W. and W.T. wrote the manuscript. All the authors contributed to the manuscript and approved the final version. ACKNOWLEDGMENTS We thank Michelle Orsulak for technical assistance and the Microsurgery Core of Yale Cardiovascular Research Center for performing renal ischemic procedures. The work is supported by NIH grants to D.W. (HL108430, HL120465, and HL135805), an AHA grant to W.T. (14SDG20490020), the Yale/NIDA Neuroproteomics Center (DA018343), and the Snyder Mouse Phenomics Resources Laboratory and Live Cell Imaging Facility, both of which were funded by the Snyder Institute for Chronic Diseases at the University of Calgary. Received: February 24, 2017 Revised: April 18, 2017 Accepted: May 24, 2017 Published: June 20, 2017

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