A Network of Interactions Enables CCM3 and STK24 to Coordinate UNC13D-Driven Vesicle Exocytosis in Neutrophils

Developmental Cell Article

Yong Zhang,1,2,7 Wenwen Tang,1,2,7 Haifeng Zhang,2,3 Xiaofeng Niu,1,2 Yingke Xu,4 Jiasheng Zhang,5 Kun Gao,1,2 Weijun Pan,6 Titus J. Boggon,1 Derek Toomre,4 Wang Min,2,3,* and Dianqing Wu1,2,* 1Department of Pharmacology 2Department of Vascular Biology and Therapeutic Program 3Department of Pathology 4Department of Cell Biology 5Department of Internal Medicine Yale School of Medicine, New Haven, CT 06520, USA 6Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China 7These authors contributed equally to this work *Correspondence: [email protected] (W.M.), [email protected] (D.W.) http://dx.doi.org/10.1016/j.devcel.2013.09.021

SUMMARY et al., 2010; Zheng et al., 2010; Ceccarelli et al., 2011; Kean et al., 2011). Mutations in the CCM3 gene as well as in two other Neutrophil degranulation plays an important role in structurally unrelated genes, CCM1 and CCM2, were found in acute innate immune responses and is tightly regu- the familial forms of cerebral cavernous malformations (CCM) lated because the granule contents can cause tissue (Faurobert and Albiges-Rizo, 2010; Cavalcanti et al., 2012). damage. However, this regulation remains poorly un- CCM is a vascular pathological condition that affects the vascu- derstood. Here, we identify the complex of STK24 lature of the central nervous system and results in stroke, seizure and CCM3 as being an important regulator of neutro- and cerebral hemorrhage with a high prevalence. CCM3 has two domains—an N-terminal domain and a C-terminal focal adhe- phil degranulation. Lack of either STK24 or CCM3 in- sion targeting homology (FATH) domain. Global Ccm3 disruption creases the release of a specific granule pool without resulted in embryonic lethality probably due to defects in the car- affecting other neutrophil functions. STK24 appears diovascular system (He et al., 2010). STK24 and STK25 also to suppress exocytosis by interacting and competing appear to function in the same pathway as CCM3 in cardiovas- with UNC13D C2B domain for lipid binding, whereas cular development (Voss et al., 2009; Fidalgo et al., 2010; Zheng CCM3 has dual roles in exocytosis regulation. et al., 2010; Yoruk et al., 2012). However, the underlying Although CCM3 stabilizes STK24, it counteracts biochemical mechanisms for how GCKIII kinases or CCM3 regu- STK24-mediated inhibition of exocytosis by recruit- late these functions are still poorly understood. ing STK24 away from the C2B domain through its Exocytosis occurs in every cell and is a process by which a cell Ca2+-sensitive interaction with UNC13D C2A domain. directs the contents (secreted proteins, membrane proteins, and This STK24/CCM3-regulated exocytosis plays an lipids) of secretory vesicles toward extracellular space. Neutro- phils play important roles in innate immunity and utilize a regu- important role in the protection of kidneys from lated exocytic process of degranulation to execute some of their ischemia-reperfusion injury. Together, these findings functions. Degranulation results in the releases of various prote- reveal a function of the STK24 and CCM3 complex in ases and other cytotoxic agents, including matrix metalloprotei- the regulation of ligand-stimulated exocytosis. nases (MMPs) and myeloperoxidase (MPO) (Lacy and Eitzen, 2008). These granule contents are antimicrobial, but can also cause tissue damage and organ failure during ischemia-reperfu- INTRODUCTION sion that occurs in stroke or organ transplantation and during adapted immune responses in chronic inflammation and viral Serine/threonine protein kinase (STK) 24 (also known as infections (Lacy and Eitzen, 2008). mammalian sterile 20-like kinase 3 [MST3]), MST4, and STK25 Exocytosis is accomplished by the fusion of secretory vesicles belong to the germinal center kinase (GCK) III subfamily of sterile with the plasma membrane through the assembly of the SNARE 20 kinases (Pombo et al., 2007). These GCKIII subfamily kinases complex. Before membrane fusion, additional proteins mediate have been implicated in regulating a number of cellular functions and regulate the initial interaction between the vesicles and (Schinkmann and Blenis, 1997; Huang et al., 2002; Irwin et al., the acceptor membrane. They include the Rab family of 2006; Lu et al., 2006; Wu et al., 2008, 2011; Lorber et al., 2009; small GTPases, the exocyst, and numerous other regulatory pro- Fidalgo et al., 2010) and interact with CCM3 (also known as proteins (Sugita, 2008; He and Guo, 2009; Su¨ dhof and Rizo, 2011; grammed cell death 10 [PDCD10]) (Rual et al., 2005; Fidalgo Jahn and Fasshauer, 2012). Among these regulatory proteins is

Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. 215 Developmental Cell STK24 and CCM3 Regulate Exocytosis

Figure 1. STK24 Deficiency Increases Neutrophil Degranulation (A) Verification of STK24 deficiency in neutrophils. Neutrophils were isolated from WT and Stk24h/h mice and analyzed by western analysis using antibodies specific to STK24, MPO or Gb2. See also Figure S1A. (B) Lack of an effect of STK24 deficiency on the number of circulating leukocytes. PMN, neutrophils; MN, monocytes; LY, lymphocytes. The data are presented as means ± SD. (C–E) Lack of an effect of STK24 deficiency on chemotactic activities. Chemotactic responses of neutrophils were examined using a Dunn chamber containing an fMLP gradient. The data are presented as means ± SD. (F) Lack of an effect of STK24 deficiency on superoxide production upon stimulation by 1 mMfMLP. (G) Lack of an effect of STK24 deficiency on phagocytosis of opsonized E. coli. The data are presented as means ± SD. (H and I) Increased secretion of MMP and MPO from Stk24h/h neutrophils. Neutrophils were stimulated with 500 nM fMLP or 10 nM MIP2. The experiments were repeated three times. The data are presented as means ± SD (*p < 0.05, Student’s t test). (J) Suppression of MPO secretion by STK24 expression in Stk24h/h neutrophils. Stk24h/h neutrophils were transfected with GFP, GFP-STK24 (STK) or GFP-STK24-K53R (KR) plasmids, and GFP-positive cells were isolated by FACS. Cells were then stimulated with 500 nM fMLP and assayed for MPO secretion. The data are presented as means ± SD (*p < 0.05, Student’s t test; n = 3). See also Figure S1B. the UNC13 (Munc13) family of proteins, which consists of pools of exocytic granules that correspond to the two phases UNC13A-D (Koch et al., 2000; Feldmann et al., 2003). UNC13D of exocytosis observed upon ligand stimulation. One pool is (also known as Munc13-4), which is expressed at high levels in docked/primed and readily releasable. The other one is nonread- hematopoietic cells, contains two separate C2 domains (C2A ily releasable or reserved, which is not docked/primed before and C2B) and two Munc13-homology domains (MHD1 and stimulation and has to undergo docking/priming upon stimula- MHD2) (Fukuda, 2005). UNC13D binds to RAB27, through which tion (Sugita, 2008; Pores-Fernando and Zweifach, 2009; Seino it is tethered to vesicles, and also binds to syntaxins and DOC2a et al., 2011). In our investigation of the roles of STK24 and (Higashio et al., 2008; Boswell et al., 2012). Syntaxins are com- CCM3 in regulation of neutrophil functions, we unexpectedly ponents of SNARE complexes, which are involved in membrane discovered the STK24 and CCM3 complex as being a regulator fusion, whereas DOC2a is an exocytosis regulator. Both human of ligand/Ca2+-stimulated exocytosis that regulates the second and mouse genetic evidence has established an important role phase of degranulation in neutrophils. We elucidated the mech- of UNC13D in the regulation of exocytosis in cytotoxic T cells, anisms by which STK24 and CCM3 regulate the exocytic pro- mast cells, platelets, and neutrophils (Feldmann et al., 2003; Cro- cess through their interactions with UNC13D and demonstrated zat et al., 2007; Brzezinska et al., 2008; Pivot-Pajot et al., 2008; that CCM3 and STK24-regulated exocytosis play an important Ren et al., 2010; Elstak et al., 2011). UNC13D is involved in role in tissue injury protection. These results together have re- granule tethering to plasma membranes through the binding of vealed physiological and cellular roles of STK24 and CCM3 its C2B domain to membrane lipids during granule docking and provided mechanistic insights into ligand-regulated degran- and priming SNARE-mediated fusion (Me´ nager et al., 2007; de ulation/exocytosis. Saint Basile et al., 2010; Elstak et al., 2011; Boswell et al., 2012). Exocytic vesicles or granules exist in different forms, many of RESULTS which are released in a controlled manner, often by extracellular stimuli. Ligand-stimulated exocytosis, which neutrophil degran- STK24 Deficiency Enhances Degranulation ulation is a form of, plays important roles in various philological The Stk24 gene is expressed at a high level in human neutrophils and pathophysiological processes. A great deal of knowledge (Payton et al., 2009), and its protein was readily detected in has been gained on how ligands stimulate exocytosis particu- mouse neutrophils (Figure 1A). To investigate the importance larly through Ca2+ over the years (Pang and Su¨ dhof, 2010; Par- of Stk24 in neutrophils, a Stk24 mutant mouse line was acquired ekh, 2011; Yamashita, 2012). There appear to be at least two from the International Gene Trap Consortium. In this mouse line,

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Figure 2. STK24 Regulates the Release of the Reserved Pool of Granules (A) Colocalization of STK24 and MPO in mouse neutrophils. Mouse neutrophils were stained for STK24 and MPO and detected by a confocal microscope. Single optical sections are shown. (B) Lack of an effect of STK24 deficiency on the number of MPO-containing granules in resting neutrophils. Mouse neutrophils were stained for MPO and imaged using a TIRF microscope. The number of granules from 25 WT and mutant cells was counted and is presented as means ± SD (Student’s t test, n > 50). See also Figure S2A. (C–E) STK24 deficiency increases granule fusion events upon a follow-up stimulation. Individual WT and STK24-deficient neutrophils expressing VAMP8-pHluorin were stimulated with one puff of fMLP (10 mM) and CB (10 mM) from a micropipette (initial stimulation). Four minutes later, the neutrophils were stimulated with a follow-up puff (follow-up stimulation). Time-lapsed images were acquired using a TIRF microscope. Representative image series of single granule fusions are shown in (C). The image time interval is 200 ms. The durations of individual granule fusions for the initial and follow-up stimulations (D) are quantified, and the numbers of fusion events in per neutrophil after the initial stimulations and follow-up stimulations (E) are counted. Data are presented as means ± SD (*p < 0.05, Student’s t test; n = 9). See also Figure S2B. (F) Time-dependent effects of STK24 deficiency on MPO release. WT and STK-deficient neutrophils were treated with 500 nM fMLP and assayed for MPO secretion at different time points. Data are presented as means ± SD (*p < 0.05; ^p > 0.1, Student’s t test, n = 3). See also Figure S2C. the Stk24 gene was disrupted by an insertion, resulting in a hypo- different types of granules, a fraction of which contains MPO morphic allele (Figure S1A available online). The mice homozy- (Lacy and Eitzen, 2008), these results suggest that STK24 may gous for this hypomorphic allele, designated as Stk24h/h, were be involved in the regulation of different types of granules. This viable and showed no gross phenotypes. The STK24 protein concept is consistent with the finding that STK24 deficiency is content in the Stk24h/h neutrophils is greatly reduced (by more also associated with increased release of MMP (Figure 1I), which than 90%) (Figure 1A). STK24 deficiency did not appear to affect is in a different type of granules from those containing MPO (Lacy the numbers of neutrophils or other leukocytes including mono- and Eitzen, 2008). Using confocal microscopy, we compared the cytes or lymphocytes in the peripheral blood (Figure 1B). Addi- number of MPO-positive granules in WT and STK24-deficient tionally, STK24 deficiency did not cause significant alterations neutrophils and found no significant difference (data not shown). in the ability of the neutrophils to chemotax (Figures 1C–1E), We also compared the number of MPO-positive granules in produce superoxide (Figure 1F), or phagocytose (Figure 1G), close proximity (<200 nm) to plasma membranes as detected compared to those isolated from the wild-type (WT) littermates. by the total internal reflection fluorescence (TIRF) microscopy However, we found that Stk24h/h neutrophils released signifi- (Figure S2A) and again observed no significant difference cantly greater amounts of myeloperoxidase (MPO) (Figure 1H) between WT and Stk24h/h neutrophils (Figure 2B). and matrix metalloproteinases (MMPs) (Figure 1I) than the WT To further investigate the degranulation process, we visualized cells did in response to the stimulation of chemoattractant and compared the fusion processes of individual granules in the fMLP or MIP2. The MPO protein contents in WT and Stk24h/h WT and Stk24h/h neutrophils using live-cell TIRF microscopy. We neutrophils are similar (Figure 1A). Thus, these results suggest have previously used the fusion protein of the v-SNARE vesicle- that the lack of STK24 may result in enhanced neutrophil degran- associated membrane protein 2 (VAMP2) with a pH-sensitive ulation. In support of this conclusion, expression of STK24-GFP fluorescent protein, pHluorin, to monitor the exocytosis of fusion protein in the Stk24h/h neutrophils reduced MPO release GLUT4 storage vesicles (Xu et al., 2011). The fluorescence of (Figure 1J). Because expression of a kinase-dead mutant of pHluorin becomes much brighter when transferred from an STK24 (K53R) at a level similar to that of WT STK24-GFP (Fig- acidic (inside vesicles) to a neutral environment (after vesicle ure S1B) also led to a similar reduction in MPO release (Figure 1J), fusion with the plasma membrane) (Miesenbo¨ ck et al., 1998; STK24 probably regulates degranulation through a kinase activ- Jankowski et al., 2001; Sankaranarayanan and Ryan, 2001). ity-independent mechanism. Although VAMP2 is not associated with MPO-positive vesicles (data not shown), VAMP8 is (Figure S2B). We expressed STK24 Regulates the Release of a Specific Pool of VAMP8-pHluorin in neutrophils and locally stimulated each indi- Granules vidual neutrophil by fMLP using a micropipette. Shortly after the Immunostaining of mouse neutrophils with an anti-STK24 stimulation, there was an increase in the number of ‘‘flashes’’ as antibody revealed a punctate pattern of STK24 subcellular local- the result of a sudden increase in fluorescence upon granule ization, suggesting that STK24 may primarily be localized with fusion and dissipation of fluorescence due to lateral diffusion granules (Figure 2A). Costaining with an anti-MPO antibody of the VAMP molecules in the plasma membrane after granule showed that most of the MPO-positive granules were STK24- fusion (Figure 2C). Quantitative analysis of fusion events positive, whereas about a quarter of STK24-positive granules revealed no significant differences in either the total number were MPO positive. Because neutrophils are known to contain of fusion events or the kinetics of each fusion event between

Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. 217 Developmental Cell STK24 and CCM3 Regulate Exocytosis

Figure 3. CCM3 Deficiency Increases Neutrophil Degranulation (A) Colocalization of CCM3 and STK24 in mouse neutrophils. A representative neutrophil expressing GFP-STK24 and CCM3-mCherry was detected by a confocal microscope. Single optical sections are shown. (B) Validation of CCM3 deficiency in Ccm3m/m neutrophils. Ccm3m/m neutrophils were analyzed by western using an antibody to CCM3, STK24, or Gb2. (C and D) Increased secretion of MMP and MPO from Ccm3m/m neutrophils. Neutrophils were stimulated with 500 nM fMLP or 10 nM MIP2. The data are presented as means ± SD (*p < 0.05, n = 3, Student’s t test). See also Figure S3. (E–H) Effects of CCM3 deficiency on granule fusion. Cells were treated and analyzed as in Figure 1. The data are presented as means ± SD (*p < 0.05; Student’s t test, n = 9).

were given 20 min after the initial stimulations, the differences in fusion events became less apparent (Figure S2C). We interpret these results to suggest that STK24 primarily suppresses the release of the reserved pool of granules without an apparent role in the release of docked granules.

CCM3 Regulates Ligand- Stimulated Exocytosis STK24 forms a complex with CCM3 (Rual et al., 2005; Zheng et al., 2010; Ceccarelli et al., 2011; Kean et al., 2011). We confirmed the interaction in a coprecipi- WT or Stk24h/h neutrophils after the initial stimulation (Figures 2D tation assay (data not shown). In mouse neutrophils, GFP- and 2E). STK24 is also colocalized with CCM3-mCherry (Figure 3A). To Two types of secretory granules have been described. One is investigate whether CCM3 has a role in neutrophil degranulation, docked/primed and readily releasable. The other is nonreadily we generated myeloid-specific CCM3-deficient mice (Ccm3m/m) releasable or reserved, which is not docked/primed before stim- by crossing floxed CCM3 mice with the B6.129-Lyzstm1(cre)Ifo/J ulation and has to undergo docking/priming upon activation mice (Figure 3B). CCM3-deficient neutrophils also released (Sugita, 2008; Pores-Fernando and Zweifach, 2009; Seino greater amounts of MPO and MMP in response to chemoattrac- et al., 2011). We thus investigated whether STK24 deficiency tants than those of WT neutrophils (Figures 3C and 3D). Our data might affect the granule release from this reserved pool. We first also confirmed a previous observation that CCM3 stabilized examined the time course of MPO release upon uniform stimula- STK24 (Fidalgo et al., 2010), because the amount of STK24 pro- tion. Although there was no significant difference in the MPO tein was reduced by more than 60% in CCM3-deficient neutro- release between WT and Stk24h/h neutrophils after 1 min stimu- phils compared to that in WT neutrophils (Figure 3B). We also lation, significant differences were observed after longer stimula- examined VAMP8-positive granule fusion in CCM3-deficient tions (Figure 2F), suggesting that STK24 may be involved in neutrophils using live-cell TIRF microscopy (Figure 3E). Similar regulating the release of the reserved pool of granules. To further to what we observed with Stk24h/h neutrophils, CCM3 deficiency corroborate this conclusion, we performed sequential stimula- increased the number of fusion events in response to the follow- tions in the live TIRF experiment using micropipettes to tran- up stimulations compared to WT, without affecting the initial siently stimulate the cells. When the cells were stimulated at responses or the fusion kinetics (Figures 3F–3H). Thus, we 4 min after the initial stimulation, we observed a significant in- concluded that CCM3 and STK24 function similarly in neutrophil crease in the number of granule fusion events in STK24-deficient degranulation. Because neutrophils isolated from Stk24h/+ mice neutrophils compared to that in WT neutrophils in response to showed enhanced MPO secretion (Figure S3A), the enhanced this follow-up stimulation (Figure 2E), even though there was exocytosis phenotype of CCM3 deficiency, which resulted in no significant difference in the fusion kinetics between WT and more than one half reduction in STK24 protein content (Fig- mutant neutrophils (Figure 2D). When the follow-up stimulations ure 3B), may at least in part be due to reduced STK24 protein

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content. Consistent with this conclusion, overexpression of STK24 or CCM3, but not a CCM3 mutant (Figure S3B) that cannot bind to STK24 (Figure S3C), was able to revert CCM3 deﬁciency-induced increase in MPO release. This conclusion is further supported by the observation that overexpression of CCM3 in wild-type neutrophils, which led to an increase in STK24 protein, reduced MPO release (Figures S3D and S3E).

STK24 and CCM3 Interact with UNC13 To understand how STK24 and CCM3 regulate exocytosis, we used the tandem afﬁnity puriﬁcation (TAP) technique, followed by mass spectrometry, to identify proteins that may bind to STK24 in HEK293. CCM3 was the top hit. Among the other hits, there was UNC13B (Figure S4A). UNC13B (also known as Munc13-2) is a member of the UNC13 family of exocytic regula- tors (Koch et al., 2000; Feldmann et al., 2003; Li and Chin, 2003). Although Unc13b is expressed at a very low level in neutrophils, its family member Unc13d is expressed at a higher level in neutrophils (Figure S4B). In addition, UNC13D (also known as Munc13-4) has been shown to play a role in neutrophil degranulation (Pivot-Pajot et al., 2008; Johnson et al., 2011). Thus, we tested its interaction with STK24. UNC13D coimmunoprecipi- tated with WT or kinase-dead STK24, when expressed in HEK293 cells (Figure 4A). We also found that CCM3 and UNC13D interacted by coimmunoprecipitation (Figure 4A). To determine if these interactions are direct, recombinant proteins were used in a pull-down assay. UNC13D interacted with CCM3 as well as WT (Figure 4B) and kinase-dead (data not shown) STK24. The UNC13 family of proteins interacts with several exocytosis-related proteins, including RAB27, DOC2a, and syntaxins (STXs) (Neeft et al., 2005; Higashio et al., 2008; Boswell et al., 2012). Given that STK24 regulates degranulation independently of its kinase activity (Figure 1J), a possible mechanism for STK24 to inhibit exocytosis may be to disrupt one of these interactions with UNC13D. However, expression of STK24 and/or CCM3 did not attenuate the interaction of UNC13D with STX7, DOC2a,or RAB27 (Figures 4C–4E).

STK24 Inhibits UNC13D Binding to Liposomes UNC13D also binds with membrane lipids in a Ca2+-sensitive manner and has an important role in vesicle docking and SNARE-dependent vesicle fusion (Boswell et al., 2012). We Figure 4. STK24 and CCM3 Interact with UNC13D examined effects of STK24 and CCM3 on the binding of (A) Detection of the interactions by coimmunoprecipitation. HEK293 cells were cotransfected with plasmids as indicated in the figure, and immunoprecipi- UNC13D to lipids using buoyant density flotation of phosphati- tation was performed 24 hr later. Proteins were detected by western analysis. dylcholine (PC)/phosphatidylserine (PS) liposomes as previously See also Figure S4. described (Boswell et al., 2012). Although CCM3 had little effect (B). Detection of the direct interactions by GST pull-down using recombinant on the binding of UNC13D to liposomes regardless of the pres- proteins isolated from E. coli. Proteins were detected by western analysis. ence of Ca2+, STK24 strongly inhibited the binding both in the (C–E) STK24 and CCM3 do not interfere with the interaction of UNC13D with a presence and absence of Ca2+ (Figure 5A) and abrogated most RAB27, SYNTAXIN7, or DOC2 . HEK293 cells were cotransfected with plas- 2+ mids as indicated, followed by immunoprecipitation and western analysis. of the stimulatory effect of Ca on the binding (Figure 5B). STX7, SYNTAXIN7. Boswell et al. (2012) have shown that UNC13D bound to liposomes via its C2B domain in a Ca2+-sensitive manner. We found that mutating the two Ca2+-binding Asp residues (941 and 947) to both lipids and STK24. Consistent with this conclusion, into Asn in the C2B domain not only abrogated Ca2+-stimulated STK24, via its kinase domain, interacted with the C2B domain binding of UNC13D to liposomes as Boswell et al. (2012) showed in a coimmunoprecipitation assay (Figures S5B–S5D) and (Figure S5A), but also attenuated the interaction of UNC13D with pull-down assay using recombinant proteins (Figures 5D and STK24 (Figure 5C). This result suggests that these Asp residues S5E). In addition, STK24 without this kinase domain, unlike may be involved in Ca2+-mediated regulation of UNC13D binding WT STK24, was not able to revert STK24 deficiency-induced

Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. 219 Developmental Cell STK24 and CCM3 Regulate Exocytosis

derlie a mechanism by which degranulation of the reserved granule pool is regulated by chemoattractants. One possible way for CCM3 to counteract STK24-mediated inhibition is to disrupt the interaction between STK24 and UNC13D. Contrary to this possibility, the presence of CCM3 did not disrupt the STK24-UNC13D interaction (compare lanes 2 and 4 of Figure 6C), but rather markedly enhanced the interaction when Ca2+ was present (compare lanes 3 and 5 of Figure 6C). Because CCM3 interacts with UNC13D N-terminal (NT) region that contains the C2A domain (Figures S5B and S6A), we mutated two of the Ca2+-binding Asp residues (127 and 133) into Asn in this domain. Although these mutations did not affect Ca2+-dependent liposome binding (Figure S5A) as previously re- ported (Boswell et al., 2012), they, unlike those in the C2B domain that disrupt the interaction with STK24 (Figure 5C), unexpectedly augmented the interaction with CCM3 (Figure 6D). We do not understand the effect of these C2A mutations on the interaction. One simple explanation is that the mutated domain may mimic the Ca2+-bound state, given that Ca2+ also enhanced the interaction of UNC13D with CCM3 (Figure 6D). Nevertheless, this UNC13D mutant can help us to understand how CCM3 and its interaction with UNC13D are involved in counteracting STK24’s effect. Thus, we tested the effect of these C2A mutations on CCM3-mediated reversal of the inhibition by STK24. UNC13D carrying these C2A mutations appeared to bind to liposomes as strongly in the absence of Ca2+ as the WT UNC13D did Figure 5. STK24 Inhibits UNC13D Binding to Liposomes in the presence of Ca2+ in the liposome-binding assay in which (A and B) STK24, but not CCM3, inhibits UNC13D binding to liposomes. Re- both STK24 and CCM3 were present (compare lanes 1 and 4 combinant UNC13D (33 nM) and STK24 (66 nM) or CCM3 (66 nM) were of Figure 6E). In addition, the interaction of this mutant, unlike incubated with liposomes. After ultracentrifugation, liposome layers and inputs the WT UNC13D, with liposomes was largely insensitive to were analyzed by western blotting (A). The ratios of UNC13D contents in the 2+ liposome layers in the presence of Ca2+ to those in the presence of EGTA Ca (compare the difference between lanes 3 and 4 with that (Ca2+) were obtained from quantification of the blots from three independent between lanes 1 and 2 in Figure 6E). These results indicate a cor- experiments (B) and are shown as means ± SD (*p < 0.01; Student’s t test). relation between greater interaction of CCM3 with UNC13D, (C) Ca2+-binding site mutations in UNC13D C2B domain disrupt the interaction which can be the result of either increased Ca2+ concentration with STK24. GST pull-down was carried out using recombinant protein as or the mutations in the C2A domain, and stronger reversal of indicated in the presence of Ca2+ or EGTA (Ca2+). Proteins were detected by STK24-mediated inhibition of UNC13D binding to liposomes. western analysis. C2B*, UNC13D C2B mutant (D941N, D947N). See also Figure S5A. This correlation can be further interpreted to suggest that the (D) Direct interaction between SKT24 and UNC13D C2B. A pull-down assay enhanced interaction of CCM3 with UNC13D, which is mediated was carried out using recombinant proteins (40 nM C2B and 40 nM STK24), by the C2A domain, might be an important step in Ca2+-stimu- which was detected by Coomassie Blue staining. See also Figures S5–S5F. lated CCM3-mediated relief of STK24 inhibition of UNC13D-liposome binding. Together with the knowledge of the interactions increase in MPO release (Figure S5F), underscoring the impor- between CCM30s NT domain and STK24’s CT domain (Ceccar- tance of STK24-UNC13D C2B interaction in STK24-mediated elli et al., 2011) and between CCM3’s FATH domain and suppression of exocytosis. Therefore, putting these results UNC13D N terminus (Figure S6A), we propose a model to explain together, we conclude that STK24 may compete with lipids for how ligand/Ca2+ may regulate exocytosis through the CCM3 and binding to the C2B domain. STK24 complex (Figure 6F). In this model, STK24 binds to UNC13D C2B domain to inhibit UNC13D binding to liposomes CCM3 Counteracts STK24 in UNC13 Binding to at the resting state. When Ca2+ concentration arises upon ligand Liposomes stimulation, Ca2+ binds to UNC13D C2B domain and stimulates Although CCM3 alone had little effect on UNC13D-liposome the binding of the C2B domain to liposomes on one hand. On the binding (Figures 5A and 5B), it unexpectedly attenuated the inhi- other hand, Ca2+ binds to the C2A domain and enhances the bition of UNC13D-liposome binding by STK24 (Figure 6A). More- interaction of the C2A domain with CCM3. The binding of over, this attenuation was much greater in the presence of Ca2+ CCM3 to the C2A domain may recruit STK24 away from its bind- than in its absence, because there was significantly greater ing to the C2B domain, thus relieving STK24-mediated inhibition Ca2+-induced liposome binding of UNC13D in the presence of of liposome binding. This model is supported by the findings that CCM3 than that in the presence of STK24 alone (Figure 6B). In Ca2+ augmented the formation of a ternary complex of UNC13D other words, CCM3 appears to have a role in restoring Ca2+- C2A domain with CCM3 and STK24 (Figure 6G) and that the sensitivity suppressed by STK24. Given that chemoattractants CCM3 mutant that cannot interact with STK24 failed to reverse increase intracellular Ca2+ concentrations, this finding may un- STK24-mediated inhibition of lipid binding (Figure S6B).

220 Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. Developmental Cell STK24 and CCM3 Regulate Exocytosis

Figure 6. Ca2+ Stimulates CCM3-UNC13D Interaction to Relieve the Inhibition by STK24 (A and B) CCM3 counteracts STK24-mediated inhibition of UNC13D binding to liposomes. Recombinant UNC13D (33 nM), STK24 (66 nM), and CCM3 (66 nM) were incubated with liposomes. After ultracentrifugation, liposome layers and inputs were analyzed by western blotting (A). The ratios of UNC13D contents in the liposome layers in the presence of Ca2+ to those in the presence of EGTA (-Ca2+) were obtained from quantiﬁcation of the blots from three independent experiments (B) and are shown as means ± SD (*p < 0.01; Student’s t test). See also Figure S6A. (C) Ca2+ stimulates the formation of UNC13D, CCM3 and STK24 complex. GST pull-down was carried out using recombinant proteins as indicated in the presence of Ca2+ or EGTA (-Ca2+). Proteins were detected by western analysis. (D) Ca2+-binding site mutations in UNC13D C2A domain enhance the interaction with CCM3. GST pull-down was carried out using recombinant proteins as indicated in the presence of Ca2+ or EGTA (-Ca2+). C2A* stands for the UNC13D mutant carrying the D127N and D133N mutations. Proteins were detected by western analysis. (E) Ca2+-binding site mutations in UNC13D C2A domain enhance liposome binding. Recombinant proteins were incubated with liposomes in the presence of Ca2+ or EGTA (-Ca2+). After ultracentrifugation, liposome layers and inputs were analyzed by western blotting. Blots from three independent experiments were quantiﬁed, and the value for WT binding alone (Lane 6) was taken as 1. The data are shown as means ± SD (AU, arbitrary unit; *p < 0.05, Student’s t test). (F) A model depicting the regulation of degranulation of the reserved granule pool by STK24 and CCM3 in response to Ca2+. At the basal state, STK24 binds to the C2B domain of UNC13D and inhibits its binding to the plasma membrane lipid. When cells are stimulated, the intracellular Ca2+ concentrations increase. Ca2+ stimulates the interaction of UNC13D C2A domain with CCM3, which presumably leads to a reduction in STK24 binding to UNC13D at the C2B domain and hence STK24-mediated inhibition of UNC13D binding to lipid and granule docking/priming. F, FATH domain; NT, N-terminal domain; CT, C-terminal domain; K, kinase domain. See also Figure S6B. (G) Effect of Ca2+ on the formation of the ternary complex of UNC13D C2A domain (C2A), CCM3, and STK24. GST pull-down was carried out using recombinant proteins (20 nM C2A, 40 nM CCM3, and 40 nM STK24) in the presence or of Ca2+ or EGTA (Ca2+) and detected by Coo- massie staining.

STK24 or CCM3 Deficiency Exacerbates Tissue Injury in rial ischemia resulted in infiltration of neutrophils (Figures S7A an Ischemia-Reperfusion Model and S7B). Importantly, the reperfusion induced significantly We next examined the in vivo significance of enhanced degran- greater increases in serum creatinine contents (Figures 7A and ulation associated with the loss of STK24 or CCM3. A renal 7B) and the number of coagulative necrotic renal tubules (Fig- ischemia-reperfusion injury model was adopted, because neu- ures 7C–7F) in both Stk24h/h and Ccm3m/m mice than their trophils are important for the reperfusion-induced renal tissue wild-type controls. These results indicated that the lack of damage (Heinzelmann et al., 1999). Reperfusion after renal arte- STK24 or CCM3 was associated with increased renal tissue

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damages after ischemia-reperfusion. Given that STK24 or CCM3 deficiency did not significantly affect the numbers of neutrophils infiltrated into the kidneys (Figures S7C and S7D), augmented tissue damage occurred in these mutant mice should be at least in part attributed to the enhanced degranulation. In support of this idea, we found that there were significantly higher levels of plasma MPO in the STK24 or CCM3 mutant mice than their controls (Figures 7G and 7H).

DISCUSSION

In this study, we have uncovered an important cellular function for the protein complex of CCM3 and one of the GCK III subfamily members, STK24. We present solid evidence to demonstrate that the protein complex plays significant roles in ligand-stimulated exocytosis in neutrophils. Importantly, we have character- ized the biochemical mechanism by which CCM3 and STK24 regulate this cellular function. Most of the work on ligand-stimulated exocytosis has been done with neurons, endocrine cells, platelets, and cytotoxic T cells. Not much is known about the mechanisms for ligand- regulated exocytosis or degranulation in neutrophils, other than the involvement of UNC13D and RAB27 (Munafo´ et al., 2007; Brzezinska et al., 2008; Herrero-Turrio´ n et al., 2008; Pivot-Pajot et al., 2008; Elstak et al., 2011). In this study, we found that MPO secretion had at least two phases. The first phase probably corresponds to the release of the readily releasable pool, which is rapidly released upon ligand stimulation, whereas the second phase may be from the reserved pool, which may not be docked prior to the initial stimulation. The STK24-CCM3 complex seems to primarily regulate the release of the reserved pool. This conclusion is consistent with our mechanistic results, which show that STK24 appears to regulate steps upstream of vesicle fusion, because its deficiency doesn’t affect vesicle fusion kinetics. In further support of this conclusion, STK24 inhibits the binding of UNC13D to lipids, a step that is important for vesicle docking (Pivot-Pajot et al., 2008; Boswell et al., 2012). The involvement of CCM3 in exocytosis regulation seems more complicated than that of STK24. CCM3 appears to play dual roles. On one hand, it stabilizes the STK24 protein. This may explain the similar degranulation phenotypes of its deficiency to those of STK24 deficiency. On the other hand, CCM3 is required for restoration of Ca2+-stimulated binding of UNC13D to lipids that is strongly inhibited by STK24. This restoration provides a means for ligands that can increase intracellular Ca2+ concentrations to regulate the second phase of degranulation from the reserved pool, as de- picted in the model (Figure 6F). In this model, Ca2+ stimulates the interaction of CCM3 with UNC13D C2A domain, which recruits STK24 away from the C2B domain to allow the binding of the C2B domain to lipids. We, however, do not know exactly why the lack of either STK24 or CCM3 did not significantly impact Figure 7. Loss of STK24 or CCM3 Exacerbates Tissue Damages in a the first phase of exocytosis immediately after ligand stimulation. Renal Ischemia-Reperfusion Model (A and B) Blood creatinine contents from mice that were subjected to the renal ischemia-reperfusion procedure. Data are presented as means ± SEM (*p < were counted from six random PAS-stained sections per kidney and are 0.05, Student’s t test, n = 4). presented as means ± SEM (*p < 0.05, Student’s t test, n = 4). (C–F) Histological examination of renal tissue injury. Renal histological sections (G and H) Blood MPO contents from mice that were subjected to the renal from mice that were subjected to the renal ischemia-reperfusion procedure ischemia-reperfusion procedure. Data are presented as means ± SEM (*p < were stained with periodic acid-Schiff (PAS) dye, and the representative 0.05, Student’s t test, n = 4). sections are shown (C and D). The numbers of coagulative necrotic tubules See also Figure S7.

222 Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. Developmental Cell STK24 and CCM3 Regulate Exocytosis

A simple explanation is that certain equilibrium between STK24 pothesis that enhanced exocytosis may contribute to defective and membrane lipids for binding to UNC13D C2B domain may vessel tubular morphology observed in the CCM disease. have been reached at the resting state. Despite the shared heart development phenotypes in zebra- Our renal ischemia-reperfusion model study of STK24-defi- fish (Zheng et al., 2010) and exocytosis in cultured endothelial cient and particularly myeloid-specific CCM3-deficient mice cells by CCM3 and STK24 knockdown, we have not observed has demonstrated the in vivo significance of this degranulation overt cardiac or consistent vascular defects in the Stk24h/h regulation imposed by the STK24 and CCM3 complex. Given mice. The lack of the obvious cardiovascular phenotypes may the broad tissue expression patterns of CCM3 and GCKIII be attributed to greater molecular redundancy of the GCKIII sub- subfamily members, these proteins may also be involved in family members in mice, which is consistent with the weaker the regulation of exocytosis in other cell types. This may be effect of STK24 knockdown on P-selectin cell surface expres- particularly important for the understanding of the regulation sion than CCM3 knockdown (data not shown). Alternatively, of the second phase of exocytosis from the reserved pool, exocytosis may only be one of many cellular activities regulated because the mechanism for this regulation remains largely by CCM3, as CCM3 was found to regulate Golgi assembly with elusive (Sugita, 2008; Pores-Fernando and Zweifach, 2009; STK25 (Fidalgo et al., 2010). These questions need to be Seino et al., 2011). Another potential impact of this study may resolved in future investigation. be on the investigation of the pathogenic basis by which CCM3 mutations cause the CCM disease. Our preliminary evi- EXPERIMENTAL PROCEDURES dence suggests that CCM3 and STK24 are also involved in the regulation of endothelial cell exocytosis. Knockdown of Reagents and Constructs CCM3 or STK24 resulted in an increase in the level of cell sur- CCM3 antibody was generated as described before (He et al., 2010). Other antibodies were acquired commercially: STK24 antibodies (rabbit, 3723, face P-selectin upon TNFa stimulation without altering the Cell Signaling), GST antibody (2624, Cell Signaling), His antibody (2366, mRNA levels of P-selectin (data not shown). P-selectin is a Cell Signaling), MST4 antibody (3822, Cell Signaling), HA antibody (MMS- cell surface adhesion molecule and stored in endothelial gran- 101R, Covance), Myc antibody (MMS-150R, Covance), GFP antibody ules called Weibel-Palade bodies (WPBs). It is transported to (sc-9996, Santa Cruz Biotechnology), Gb2 antibody (sc-380, Santa Cruz cell surfaces through exocytosis in response to stimulation by Biotechnology), and MPO antibody (HM1051BT, HyCult Biotech). Protein proinflammatory ligands including TNFa (Valentijn et al., 2011). A/G-PLUS-agarose beads were acquired from Santa Cruz Biotechnology. Formyl-Met-Leu-Phe (fMLP) and Cytochalasin B (CB) were purchased from This preliminary observation not only indicates the importance Sigma. Human UNC13D cDNA, and mouse STK24 cDNA were acquired of this protein complex in exocytic regulation beyond neutro- from Open Biosystems. phils, but also provides a basis for a plausible hypothesis to explain the contribution of CCM3 inactivation to the CCM Mice pathogenesis. We hypothesize that enhanced exocytosis in STK24h/h mice were generated using a gene-trap ESC line AM0826 (Wellcome response to certain stimulations in the brain may contribute to Trust Sanger Institute) from International Gene Trap Consortium at the vascular defects observed in CCM disease. It is not difficult to Mutant Mouse Regional Resource Center (UC Davis) using the standard postulate how enhanced exocytosis, through which cell surface method. Mice heterozygous for the mutation were initially generated on a mixed 129/Ola and C57BL/6 background and were subsequently back- and secreted proteins and molecules are transported to their crossed to the C57BL/6 background for more than five generations. functional sites, causes the vasculature defects. For instance, Myeloid-specific CCM3 knockout mice were generated by crossing floxed enhanced release of angiopoietin-2, which is also stored from CCM3 mice (He et al., 2010) with the B6.129-Lyzstm1(cre)Ifo/J mice from Jack- WPBs, may cause capillary destabilization (Augustin et al., son Lab. All animal studies were approved by the Institutional Animal Care and 2009), whereas increased levels of cell adhesion or junction pro- Use Committees of Yale University. teins may result in defects in the formation and maintenance of vessel tubular morphology. This hypothesis is also compatible White Blood Cell Counts A Hemavet system (Drew Scientific), which is a multiparameter, automated for the anatomic restriction of the CCM lesions, which may be hematology analyzer designed for in vitro diagnostic use (Li et al., 2009), the result of the unique nature of the stimulations and/or effect was used to count the leukocyte numbers in circulating blood. To quantify of hyperexocytic activity on the blood vessels in the brain. neutrophil numbers in spleens, livers, and kidneys, cells were dissociated Therefore, more studies are warranted to further characterize from the tissues and total cell numbers were counted using a Guava the role of CCM3 and STK24 as well as other members of the EasyCyte System (Millipore). The cells were also stained with APC-CD11b GCKIII subfamily in endothelial cell exocytosis and determine antibody (17-0112, eBioscience) and Percp-Ly-6G (560602, BD) antibody if altered exocytosis contributes to the CCM phenotypes. Of and analyzed by a LSR II FACS analyzer. Neutrophils were defined as Ly- 6G+CD11b+ cells. note, our hypothesis is consistent with the findings from a recent fly genetic study. This fly work shows that the loss-of-function Neutrophil Preparation and Transfection mutants of fly CCM3 or GCKIII kinase ortholog have dilated Murine neutrophils were purified from bone marrows as previously described tracheal tubes, which resemble dilated vessels found in CCM (Zhang et al., 2010). Briefly, bone marrow cells collected from mice were patients. This tube dilation phenotype can be suppressed by treated with the ACK buffer (155 mM NH4Cl, 10 mM KHCO3 and 127 mM the reduction in the expression of N-ethylmaleimide sensitive EDTA) for red blood cell lysis, followed by a discontinuous Percoll density factor 2 (NSF2) (Song et al., 2013). Given the important role of gradient centrifugation. Neutrophils were collected from the band located between 81% and 62% of Percoll. For transient transfection of primary mouse NSF2 in SNARE recycling and the secondary phase of exocy- neutrophils, three million neutrophils were electroporated with 1.6 mg endo- tosis (Zhao et al., 2012), these fly results are in agreement toxin-free plasmids using the human monocyte nucleofection kit (Lonza) with our observation that the STK24-CCM3 complex regulates with an Amaxa electroporation system. The cells were then cultured for exocytosis of the reserved pool of granules and support our hy- overnight in the medium supplied with the kit containing 10% FBS and

Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. 223 Developmental Cell STK24 and CCM3 Regulate Exocytosis

25 ng/ml recombinant GM-CSF (PeproTech). GFP+ Cells were sorted by a TAP and Mass Spectrometry Analyses FACS Aria sorter (BD). TAP were performed with the InterPlay Mammalian TAP System (Agilent). The purified proteins were separated on SDS-PAGE, and the protein bands were Immunostaining of Neutrophils excised from the gel for MS analyses. An LTQ-Orbitrap was used for identifi- Neutrophils were placed onto the coverslips for 15 min before stimulation. The cation of CCM3 and STK24-associated proteins at W.M. Keck MS and Prote- cells were then fixed with 4% paraformaldehyde and permeabilized with omics Resource of Yale University. MS/MS spectra were searched against 0.01% saponin and blocked with 2% BSA in PBS (Brzezinska et al., 2008). Homo sapiens subset of the National Center for Biotechnology Information Neutrophils were stained with a STK24 antibody (3723, Cell Signaling, 1:100) (NCBI) protein sequence database (Release 20110610) using Mascot Daemon and/or biotinylated MPO antibody (HM1051BT, Hycult Biotech, 1:100), fol- software. lowed by Alexa-488-Goat-anti-Rabbit (Invitrogen, 1:300 for the STK antibody) and APC-streptavidin (BD, 1:300 for the MPO antibody) staining using a previ- Protein Preparation and Pull-Down ously described protocol (Xu et al., 2010). Mounted slides were observed using Recombinant proteins were expressed in E. coli BL21(DE3) and purified by a Leica SP5 confocal microscopy. affinity chromatography using glutathione agarose beads for GST-fusion proteins or Ni-NTA agarose beads (GE Healthcare) for His-tagged proteins. Pro- TIRF Microscopy teins were dialyzed to a buffer containing 20 mM HEPES, pH 7.5, 100 mM TIRF imaging was performed using an IX-70 inverted microscope (Olympus), KCl, 0.1% Triton X-100, 0.2 mM EGTA, 10% glycerol, and 1 mM DTT. For equipped with argon (488 nm) and argon/krypton (568 nm) laser lines (Melles the pull-down assays, His- and GST-tagged proteins were incubated with Griot), a 603 1.45 NA oil immersion objective lens (Plan-ApoN; Olympus), either glutathione-agarose or Ni-NTA agarose beads as indicated in the figures and a custom TIRFM condenser. Cells were imaged in one channel at at 4 C for 5 hr. After extensive washes, proteins on the beads were resolved by 5–10 Hz or two channels by sequential excitation at 3–5 Hz without binning SDS/PAGE and detected by western blot or Coomassie blue staining. and detected with a back-illuminated iXon887 EMCCD camera (512 3 512, 0.18 mm per pixel, 16 bits; Andor Technologies) with a 1.63 magnification Buoyant Density Flotation of Liposome Assay lens. The system was controlled by the Andor iQ software (Xu et al., 2011). The assay was performed as previously described (Boswell et al., 2012). Lipo- For live-cell imaging, neutrophils were resuspended in 2 ml Hank’s buffer somes were consisted of 85% PC and 15% PS which were purchased from with 0.25% BSA and seeded into 35 mm Petri dish with a No. 1.5 coverglass Avanti Polar Lipids and were incubated with UNC13D and indicated proteins at the bottom (MatTek). For cell stimulation, an Eppendorf Femotip was loaded (BSA or STK24/CCM3) for 30 min on ice in 75 ml of 25 mM HEPES, pH 7.4, with 50 mM fMLP and 50 mM CB, and ligands were loaded by applying air pres- 100 mM KCl, 10% glycerol, and 1 mM DTT (reconstitution buffer). Liposomes sure to the micropipette using a Picospritzer II microinjection system (Parker binding reactions were conducted in the absence of Ca2+ (0.2 mM EGTA) or Hannifin). Each puff lasts 1 s, and the volume for each puff is 5 nl. Time- presence of 100 mM of free Ca2+.75ml of 80% Accudenz was added to lapsed images were acquired at 200 ms intervals at 37C. For fixed cell imag- the binding reaction to yield a final concentration of 40% Accudenz. Thirty ing, neutrophils were stimulated with 1 mM fMLP and 10 mM CB, fixed, and percent Accudenz and reconstitution buffers were then layered on top and stained with anti-MPO antibody (HyCult Biotech) before imaging. centrifuged for 4 hr in a TLA100 rotor at 32,000 rpm. Either the floated fraction or inputs were collected and run on SDS-PAGE and analyzed by western MPO and MMP Detection blotting. One million neutrophils were incubated with 10 mM CB for 5 min at 37C prior to stimulation with fMLP (500 nM) or MIP2 (10 nM) for another 10 min. The reac- Murine Kidney Ischemia/Reperfusion Models tion was stopped by being placed on ice, and the suspension was centrifuged The left and right renal arteries of mutant and their littermate control mice at 500 3 g for 5 min at 4C. Supernatants were assayed for MPO and MMP (9–10 weeks old) were clamped for 30 min using vascular clamps (Fine Science contents using the EnzChek Myeloperoxidase Activity Assay Kit and EnzChek Tools) through a midline abdominal incision under general anesthesia. After Gelatinase/Collagenase Assay kit (Life Technologies), respectively (Lee et al., ischemia, the clamps were removed, the wounds were sutured, and the ani- 2007; Li et al., 2009). mals were allowed to recover for 24 hr before euthanization. Blood was collected by cardiac puncture in heparin-containing tube, and creatinine levels Immunoprecipitation were determined using the DetectX serum creatinine kit (Arbor Assays). Kid- HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium neys were dissected from mice, fixed in 4% paraformaldehyde, embedded (DMEM) with 4.5 g/liter glucose supplemented with 10% fetal bovine serum in paraffin, and stained for the periodic acid-Schiff (PAS) dye and a Ly6B (FBS). Transient transfection was carried out using Lipofectamine Plus (Life antibody. Plasma MPO levels were determined using an ELISA kit (HyCult Technologies), and samples were collected 24 hr after transfection. Cells Biotechnology). were lysed in a cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA, Roche’s protease inhibitor cocktail, Roche’s phospha- SUPPLEMENTAL INFORMATION tase inhibitor cocktail). After removing insoluble materials by centrifugation, immunoprecipitation was carried out by adding various antibodies and Supplemental Information includes seven figures and can be found with this Protein A/G-PLUS-agarose beads into supernatants. Immunocomplexes article online at http://dx.doi.org/10.1016/j.devcel.2013.09.021. were washed three times with lysis buffer before the SDS sample buffer was added for SDS-PAGE analysis. ACKNOWLEDGMENTS

Phagocytosis, Chemotaxis, and Superoxide Production Assays We thank Michelle Orsulak for technical assistance, Abdul Basit for discus- For testing phagocytosis, neutrophils (0.5 million) were incubated with 20 ml sion, and the Microsurgery Core of Yale Cardiovascular Research Center for pHrodo Escherichia coli BioParticles Conjugate (Invitrogen) at 37C. After fix- performing renal ischemic procedures. The work is supported by National ation, cells were analyzed by flow cytometry using a BD LSRII FACS analyzer Institutes of Health grants to D.W. (HL108430, HL070694, AI094525), W.M. (Hsieh et al., 2009). (HL115148, HL109420), T.J.B. (GM102262), D.T. (NIH DP2-OD002980), The chemotaxis assay was carried out using a Dunn chamber as previously H.Z. (AHA SDG 13SDG17110045), Yale/NIDA Neuroproteomics Center described (Zicha et al., 1997) with some modifications as detailed in (Xu et al., (DA018343), and W.P. (National Thousand Talents Program for Distinguished 2010; Zhang et al., 2010). Young Scholars). For superoxide production assay, neutrophils were suspended in Hank’s buffer at 1 3 106/ml on ice. Fifty microliters of cells were mixed with 20 ml Received: May 6, 2013 HRP (100 U/ml, Sigma), 1.5 ml luminol (10 mM, Sigma), and 28.5 ml Hank’s Revised: September 14, 2013 buffer in a well of a 96-well plate. Chemiluminescence was detected by a Accepted: September 22, 2013 Wallac plate reader (Dong et al., 2005). Published: October 28, 2013

224 Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. Developmental Cell STK24 and CCM3 Regulate Exocytosis

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