PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization

This information is current as Hai-Min Sun, Xin-Lei Chen, Xin-Jie Chen, Jin Liu, Lie Ma, of September 29, 2021. Hai-Yan Wu, Qiu-Hua Huang, Xiao-Dong Xi, Tong Yin, Jiang Zhu, Zhu Chen and Sai-Juan Chen J Immunol 2017; 199:1817-1826; Prepublished online 24 July 2017;

doi: 10.4049/jimmunol.1602018 Downloaded from http://www.jimmunol.org/content/199/5/1817

Supplementary http://www.jimmunol.org/content/suppl/2017/07/23/jimmunol.160201 Material 8.DCSupplemental http://www.jimmunol.org/ References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/199/5/1817.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization

Hai-Min Sun,1 Xin-Lei Chen,1 Xin-Jie Chen,1 Jin Liu, Lie Ma, Hai-Yan Wu, Qiu-Hua Huang, Xiao-Dong Xi, Tong Yin, Jiang Zhu, Zhu Chen, and Sai-Juan Chen

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid–induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator Downloaded from of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization. The Journal of Immu- nology, 2017, 199: 1817–1826.

ALLD is an actin-associated protein and crucial to Mature myeloid cells maintain tissue homeostasis, repair, and establishing cellular morphology and maintaining cyto- remodeling, as well as eliminating foreign materials or pathogens P skeletal organization in various cell types (1, 2). A single through the specialized function of phagocytosis (18, 19). Phago- http://www.jimmunol.org/ PALLD gene gives rise to several isoforms, some of which are cytosis is the receptor-mediated engulfment of large particles expressed in tissue-specific patterns. The most common PALLD (diameter $0.5 mm) by plasma membrane–derived vacuoles isoform is 90–92 kDa and contains a proline-rich domain in the called phagosomes (20, 21). Phagocytosis begins as phagocytic N-terminal and three C-terminal IgC2 domains (third, fourth, and receptors are bound with certain signal moieties, such as certain fifth IgC2 domain) (3). PALLD interacts with proteins involved in intrinsic components, IgG, or complement opsonins, on the sur- actin polymerization and cross-linking, acting as a molecular scaf- face of particles. The occupied phagocytic receptors then trigger a folding protein that links proteins with different functional modali- series of complex intracellular signaling events to orchestrate ef- ties into large complexes (3–15). ficient particle internalization. The details of this process vary by guest on September 29, 2021 PALLD is absent or expressed at very low levels in peripheral depending on the substrate being internalized and receptor types blood monocytes, but is a constitutively expressed factor in nu- involved, but the clustering of occupied receptors to enhance merous types of vertebrate tissue cells. PALLD is significantly substrate-binding affinity in the phagosome is a common feature upregulated during the differentiation of monocytes into dendritic (20, 22). cells (16). PALLD upregulation during all-trans-retinoic acid Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P ] is essential for (ATRA)–induced myeloid differentiation of acute promyelocytic 2 the initial actin polymerization that drives pseudopod formation, leukemia cells implies that PALLD has a role in the differentiation whereas its hydrolysis is a prerequisite for actin depolymerization that and/or function of mature myeloid cells (17). allows complete phagosome closure. In mammalian cells, PI(4,5)P2 is metabolized by conversion to PI(3,4,5)P3 via class I PI3Ks; cleavaged into inositol 3,4,5-trisphosphate and diacylglycerol by State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui- phospholipase C; and dephosphorylated by inositol phosphatases Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shang- hai 200025, China such as inositol polyphosphate-5-phosphatase and/or oculocere- 1H.-M.S., X.-L.C., and X.-J.C. contributed equally to this work. brorenal syndrome of Lowe (OCRL) (23, 24). Efficient phagocytosis relies on the focal exocytosis of intra- Received for publication December 2, 2016. Accepted for publication June 12, 2017. cellular compartments that contributes to the release of membrane This work was supported by National Key Basic Research Program of China Grant 2013CB966800, Ministry of Health Grant 201202003, Mega-Projects of Scientific tension, allowing efficient phagosome formation around large par- Research for the 12th Five-Year Plan (2013ZX09303302), National Natural Science ticles (25, 26). Phagosomes undergo a maturation process following Foundation of China Grant 81123005, and the Samuel Waxman Cancer Research Foundation Co-Principal Investigator Program. scission from the plasma membrane, sequentially fusing with endo- somes and lysosomes, and ultimately becoming phagolysosomes, Address correspondence and reprint requests to Prof. Sai-Juan Chen, Prof. Zhu Chen, or Prof. Jiang Zhu, State Key Laboratory of Medical Genomics, Shanghai Institute of which are highly acidic and hydrolase-rich organelles that degrade Hematology, RuiJin Hospital Affiliated to Shanghai Jiao Tong University School of internalized particles (20). Medicine, 197 RuiJin Road II, Shanghai 200025, China. E-mail address: sjchen@stn. sh.cn (S.-J.C.), [email protected] (Z.C.), or [email protected] (J.Z.) In this study, we demonstrate a new function for PALLD in the The online version of this article contains supplemental material. promotion of phagocytic cup extension and closure. Our data identified PALLD as a crucial factor involved in the regulation of Abbreviations used in this article: ATRA, all-trans-retinoic acid; BM, bone marrow; BMDC, BM-derived dendritic cell; KD, knockdown; NC, negative control; OCRL, F-actin dynamics throughout multiple steps of phagocytosis, in- oculocerebrorenal syndrome of Lowe; PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate; cluding serum-mediated particle binding via modulation of FcgRIIA sh, short hairpin; siRNA, small interfering RNA. clustering, and delivery of PI(4,5)P2 phosphatase OCRL in nascent Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 phagosomes. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602018 1818 PALLD REGULATES ACTIN DYNAMICS IN PHAGOCYTOSIS

Materials and Methods Fluor 546 phalloidin and Alexa Fluor 635 phalloidin (Invitrogen). Src Reagents and cell culture family kinase inhibitor PP2 was purchased from Selleckchem; cytochalasin D and ATRA were purchased from Sigma-Aldrich. In addition, 1.75 mm The following Abs were used in the experiments: anti-PALLD (Proteintech Fluoresbrite Bright Blue Microspheres were purchased from Poly- and Novus Biologicals); anti-FLAG M2 and anti–b-actin (Sigma-Aldrich); sciences; unlabeled zymosan and ﬂuorescein-conjugated zymosan were anti-FcgIIA IV.3 (Stem Cell); anti-Src (Cell Signaling and Proteintech); purchased from Invitrogen. Murine GM-CSF and IL-4 were purchased anti-OCRL (Cell Signaling and Abcam); anti-PI(4,5)P2, anti-ARP3, and from PeproTech. anti-GFP (Abcam); anti-RAC1 and anti-CDC42 (Thermo Fisher); anti– The leukemia cell lines used in this study included human acute mye- phospho-Src Tyr416, anti-EEA1, anti-RAB7, and anti-LAMP1 (Cell Sig- loblastic leukemia HL60, SKNO-1, and Kasumi-1; human acute promyelocytic naling); anti–Gr-1-PE, CD11c-FITC, and anti–CD16-PE (eBioscience); leukemia NB4; human acute myelomonocytic leukemia OCI-AML3; human CD11b-PE (BioLegend); anti-CD18 (Proteintech); Alexa Fluor 488 anti- acute monocytic leukemia U937 and THP1; and human acute erythroid leu- rabbit IgG and Alexa Fluor 594 anti-mouse IgG (Jackson); and Alexa kemia HEL. The cell lines were cultured in RPMI 1640 medium containing Downloaded from http://www.jimmunol.org/

FIGURE 1. ATRA-induced PALLD upregulation is correlated with enhanced phagocytosis in myeloid cells. (A) NB4 cells were treated with 1 mM ATRA for the indicated durations. Induced PALLD expression was analyzed by Northern blot, and total RNA was determined by 28s rRNA. (B) Immunoblot of PALLD in the lysates from NB4 cells treated with 1 mM ATRA for the indicated durations. b-actin was used as by guest on September 29, 2021 a loading control. (C) Immunoblot of PALLD in the lysates from various cell lines and mouse primary bone marrow (BM) cells untreated or treated with 1 mM ATRA for 48 h. (D) The cells were treated as in (C), and their phagocytic abilities were analyzed by flow cytometric assay. Upper, representative flow cytometric analysis of phagocytosis; lower, graph showing the quantification of phagocytosis of three independent experiments. (E) Effect of ATRA treatment on the phagocytic ability of different cell populations in BM cells examined by flow cytometry. Left, representative flow cytometric analysis of phagocytosis; right, graph showing the quantification of phagocytosis of three independent experiments. Data are expressed as mean 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001. The Journal of Immunology 1819

10% FBS. HEK 293T cells and primary mouse bone marrow (BM) cells were PLV-mcherry vector. OCRL and its truncated mutants were cloned into grown in DMEM supplemented with 10% FBS. pFLAG-CMV4 and pLVX-BFP, and PALLD and its truncated sequences Mouse BM-derived dendritic cells (BMDCs) were obtained from healthy were cloned into pBCAG-FLAG-HA-Myc and pEGFP-C1 vectors. pLVX- C57BL/6 mice. Briefly, mouse BM cells differentiated into dendritic cells when IRES-puro was constructed by replacing IRES-puro with BFP. D499A, being cultured in the presence of 0.02 ng/ml IL-4 and 0.02 ng/mlGM-CSFin a mutant without phosphatase activity of OCRL, was cloned into the RPMI 1640 medium supplemented with 10% FBS for 6 d at 37˚C (27). pLVX-GFP vector. These plasmids were transfected into HEK293T cells by the calcium phosphate method. Transfected cells were analyzed 48 h Northern blot analysis posttransfection. The PALLD gene was amplified by the following primer templates: 59-TT- GGAGCTTCTCAATAAACA-39 and 59-TTGGAGCTTCTCAATAAACA-39. Generation of stable lentiviral cell lines 32 Then the DNA fragments were labeled with [a- P]dCTP by the random- Lentiviral particles were generated by cotransfection of HEK293T cells primed DNA-labeling method. Total RNA was extracted from ATRA-treated with transfer plasmid, and psPAX2 and pMD2G helper plasmids by the leukemia cells harvested at the indicated time points. Samples containing calcium phosphate method. Cells were infected by incubating the cells with equal amounts of total RNA were separated by denatured RNA electropho- lentiviruses for 24 h at 37˚C, in the presence of 8 mg/ml polybrene (Sigma- resis according to m.w., and the RNA was transferred from the gel to a nylon Aldrich). After 48 h, cells were selected with puromycin (1 mg/ml, final membrane by the upward capillary transfer method. The nylon membrane concentration) or sorted, based on fluorescent protein. For BMDCs, lentivi- was then cross-linked by UV and incubated overnight with hybridization ruses were added at day 2, and cells were selected with 1 mg/ml puromycin 3 probes. The membrane was washed thrice with 2 SSC containing 0.1% on day 4 and thereafter. SDS at 56˚C and autoradiographed at 280˚C for another 2 d. Migration assays Plasmids and transfection Cell migration was performed in 8-mm Corning Transwell porous cham- pLVX-NC-puro (negative control [NC]) and pLVX-KD-puro (PALLD bers. Cells (∼2.5 3 105) in RPMI 1640 medium containing 10% FBS were Downloaded from knockdown [KD]) plasmids were modified from pLVX-short hairpin (sh) added to the upper chambers in triplicate. Then RPMI 1640 medium was RNA2 (Clontech) by replacing the zsGreen with puromyocin. The NC small added to the lower chambers. Nonmigrated cells in the upper chambers interfering RNA (siRNA) sequence was 59-GCGTGTAGCTAGCAGAGG- were removed after 2-h incubation at 37˚C. Cells in the lower chambers 39. The following PALLD-targeting siRNA (KD) sequences were used as were counted. follows: in NB4 cells (59-CATGTAGAGTGGCTGGAAA-39); in U937 and THP1 cells (59-CAGGGACTAGACATCAAAG-39);andinmouseBMcells Detection of chemokine levels (59-AGCCAAAGATCTATTGGTTTA-39). The following OCRL-targeting siRNA sequences were used: sh-1, 59-GAAAGGATCAGTGTCGATA-39; sh-2, The chemokines secreted by differentiated NB4 cells were detected by http://www.jimmunol.org/ 59-CCGAATTCTTTGGAGAGGAACAAAT-39. VAMP3 was cloned into Human Chemokine Array Kit (catalog ARY017; R&D Systems). Equivalent

FIGURE 2. PALLD is involved in serum- opsonized phagocytosis. (A and B) NB4 cells stably transduced, with control (NC) or shRNA against PALLD (KD), were treated with 1 mM ATRA for the indicated durations. (A) Immu- noblot of PALLD in NB4 NC and KD cell by guest on September 29, 2021 lysates. (B)Flowcytometricassayofthe phagocytosis of the serum-opsonized fluorescent microspheres by ATRA-treated NC and KD cells. Left, representative flow cytometric analysis of phagocytosis; right, graph showing the quantification of phagocytosis of three independent experiments. (C) ATRA-treated NC and KD cells were incubated for 1 h with only IgG or IgM plus fresh serum-opsonized fluorescent microspheres. Then phagocytosis efficiency was measured by flow cytometry. Left, representative flow cytometric analysis of phagocytosis; right, graph showing the quantification of phagocytosis of three independent experiments. (D) ATRA-treated NB4 NC and KD cells were incubated with serum-opsonized zymosan-FITC for 1 h, then fixed and labeled with phalloidin (red). Phagocytosis percentage (left chart) and phagocytic index (right chart) were derived from the measurements of at least 50 cells per experiment. Left, representative images; right, quantification of phagocytosis of three independent experiments. Original magnification 363. (E) Immunoblot of PALLD in lysates from the BMDCs. (F) Phagocytic ability of CD11c- positive BMDC-NC and BMDC-KD cells was examined by flow cytometry. Left, representative flow cytometric analysis; right, graph showing the quantification of three independent experiments is provided. Data are expressed as mean 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001. 1820 PALLD REGULATES ACTIN DYNAMICS IN PHAGOCYTOSIS numbers of NB4 NC or KD cells were treated with ATRA for 72 h. The collected and analyzed by Leica SP2. Colocalization efficiency was analyzed supernatant was collected and assayed according to the instruction manual. by the Image-pro plus version 6.0. Phagocytosis assays Statistical analysis Suspended cells were washed with PBS buffer and resuspended in HBSS Statistical analyses of data were conducted by Student t test. The p values buffer (125 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 1 mM KH2PO4,1mM ,0.05 were considered statistically significant. CaCl2, 1 mM MgCl2, and 20 mM HEPES [pH 7.4]). Microspheres or zymosan was opsonized by fresh serum or specific opsonins at 37˚C for 1 h. After incubation with microspheres or zymosan (ratio of cells to Results microspheres = 1:100; ratio of cells to zymosan particles = 1:5) at 37˚C for PALLD upregulation is correlated with the induced phagocytic the indicated durations, the cells were washed with precooled PBS to stop activity during ATRA-induced differentiation of myeloid cells phagocytosis. Then the cells were analyzed by flow cytometry or fixed for immunofluorescence inspection. Human IgG (100 mg/ml, final concen- ATRA induced PALLD expression in NB4 cells at the mRNA and tration) alone or IgM (20 mg/ml, final concentration) plus fresh serum were protein levels in a time-dependent manner; PALLD expression used as two kinds of opsonins. The phagocytic index is the number of could be detected 24–48 h poststimulation (Fig. 1A, 1B). PALLD phagocytic particles per cell. For the binding experiments, the above was also induced in other ATRA-treated leukemia cell lines, such procedures were performed at 4˚C. as OCI-AML3, U937, and THP1, as well as in mouse primary BM Western blots and immunoprecipitation cells (Fig. 1C). These results demonstrated that ATRA upregulate PALLD in normal and malignant myeloid cells. Cells were washed twice with cold PBS and lysed with radioimmunoprecipi- tation assay or 1% Nonidet P-40 buffer. The crude lysates were centrifuged at NB4 cell differentiation was only slightly affected by PALLD, as 12,000 rpm for 15 min at 4˚C to remove large particulates. The supernatant evidenced by the insignificant differences in cell morphology and Downloaded from was collected for further experiments. The primary Ab was added to the lysis CD11b expression detected between the ATRA-treated control supernatant and incubated at 4˚C overnight for coimmunoprecipitation NB4 cells (NC) and PALLD KD NB4 cells (KD) (Supplemental studies. Protein A beads (GE) were added and incubated for 4 h. The beads Fig. 1A, 1B). PALLD KD evidently affected neutrophil-associated were then washed thrice with lysis buffer and eluted by boiling in SDS- loading buffer. functions, such as migration, cell adhesion, and chemokine secretion (Supplemental Fig. 1C–E). Phagocytic capability sig- Immunofluorescent microscopy

nificantly increased in ATRA-treated OCI-AML3, U937, THP1, http://www.jimmunol.org/ Cells were resuspended in HBSS buffer at 37˚C for 30 min to fix cells on and primary mouse BM cells. Meanwhile, the other cell lines, slides. After phagocytosis, the cells were washed with precooled PBS such as SKNO-1, Kasumi-1, and HEL cells, showed relatively high buffer, fixed, and stained with Abs. For FcgR clustering, anti-FcgIIA IV.3 PALLD expression, but did not display upregulation and increased Ab (5 mg/ml, final concentration) was incubated with cells for 20 min at 4˚C and washed with PBS. Then the fluorescent secondary Ab (10 mg/ml, final phagocytic capability after ATRA treatment (Fig. 1D). Besides, we concentration) was added to stimulate FcgRIIA clustering (28). Cells were found that ATRA treatment enhanced the phagocytic ability of both imaged with the oil immersion 363 objective of microscope. Images were myeloid cells and professional phagocytes such as dendritic cells as by guest on September 29, 2021

FIGURE 3. Palladin regulates particle binding, pseudopod extension, and phagosome closure. (A) Schematic diagram of phagocytosis progress. (B) ATRA-treated NB4 NC or KD cells were incubated with fluorescent particles at 4˚C to prevent internalization, and their particulate binding ability was then examined. Left, representative flow cytometry result; right, graph showing the quantification of binding of three independent experiments. (C) ATRA-treated NC and KD cells were incubated with serum-coated zymosan (green) at 37˚C for the indicated durations, then fixed and labeled with phalloidin (red) and DAPI (blue). Representative images of different phagocytic events, as indicated by different numbers, are shown. (D) The pseudopod length of NC and KD cells, as shown in (C), was measured (n = 15 for each condition) in three independent experiments (right). Pseudopod length was measured along the lines drawn at the phagocytic site (left). Original magnification 363. (E–G) Different phagocytic events were measured per 20 cells as in (C)(n . 60 for each condition) from three independent experiments. Data are expressed as mean 6 SEM. *p # 0.05, **p # 0.01. The Journal of Immunology 1821 marked by Gr-1 and CD11c, respectively (Fig. 1E). These results maturation (20, 21). Phagosome formation is further divided into the indicated that induced PALLD expression is correlated with ATRA- particle binding, pseudopod extension, and internalization stages. induced phagocytic capability in granulocytes, monocytes, and During the phagosome maturation, actin molecules are removed to dendritic cells, although PALLD alone cannot induce phagocytosis. facilitate vesicle fusion with other compartments (Fig. 3A) (29, 30). PALLD is indispensable for serum-opsonized phagocytosis Given that phagocytic internalization is a temperature-dependent process (with the optimum temperature at 37˚C), we examined the To study the role of PALLD in phagocytosis, PALLD KD NB4, binding level of serum-coated particles at 4˚C. After 1 h of incuba- U937, or THP1 cells were constructed by stably transfecting tion, ∼49% of the control cells were bound with beads, whereas only shRNA against PALLD (Fig. 2A; Supplemental Fig. 2B, 2D). All ∼28% of the PALLD KD cells remained bound with beads (Fig. 3B), of these cell lines were treated with ATRA for indicated durations. suggesting that PALLD is involved in particle binding (25). Phagocytosis was triggered by fresh human serum- or no serum- Serum-opsonized zymosan-FITC was used as a phagocytic opsonized fluorescent microspheres. The phagocytic ability of target, and phagocytosis was traced at different time points by each cell type was evaluated. Interestingly, PALLD KD had minor phalloidin dyeing. Fig. 3C shows several representative images of effects on non–serum-mediated phagocytosis and significantly cells captured at different stages of phagocytosis, such as early reduced the fresh serum-mediated phagocytosis (Fig. 2B; Supple- phagocytic cup formation (marked by 1), cup closure before mental Fig. 2A, 2C, 2E). PALLD silencing reduced phagocytic abil- scission (marked by 2), and phagosome maturation (marked by 3). ity when IgG or complement was used as an opsonin (Fig. 2C). After 5 min of incubation, pseudopods were shortened in PALLD Serum-opsonized zymosan, a 3-mm–diameter particle prepared from KD cells (Fig. 3D), and, at the same time, ∼25% of the control the yeast cell wall, was also used as a phagocytic target. In addition, Downloaded from cells formed phagocytic cups, whereas ,5% of the PALLD KD the phagocytosis percentage and phagocytosis index were reduced cells formed phagocytic cups (Fig. 3E). After 10 min of incubation, when PALLD was knocked down (Fig. 2D). Similarly, PALLD KD in ∼ , CD11c+ BMDCs reduced their phagocytic ability approximately by 25% of the control cells formed phagosomes, whereas 10% of half (Fig. 2E, 2F). These results suggested that PALLD is indis- the PALLD-defective cells formed phagosomes (Fig. 3F). After pensable to fresh serum-, IgG-, or complement-opsonized phagocy- 20 min of incubation, the percentage of zymosan-positive cells was ∼35% in the control group, whereas it was ∼20% in the PALLD KD

tosis in multiple types of myeloid cells. http://www.jimmunol.org/ group. However, the percentage of cells remaining at the cup closure PALLD regulates particle binding, pseudopod extension, and before scission was ∼30 and ∼15% in the PALLD KD and control phagosome closure cells, respectively, suggesting that the closure of phagocytic cups To study PALLD’s participation in phagocytosis, we examined the was blocked when PALLD is defective (Fig. 3G). These results stages with potential PALLD involvement. Fig. 3A delineates the two suggested that PALLD KD reduces pseudopod extension and delays main phases of phagocytosis: phagosome formation and phagosome phagosome closure (30). by guest on September 29, 2021

FIGURE 4. PALLD promotes particle binding by regulating FcgRIIA clustering. (A) NB4 NC and KD cells pretreated with ATRA for 72 h were incubated with anti-FcgRIIA Ab at 4˚C for 30 min, followed by incubation with Alexa Fluor 594 secondary Ab (red) for 20 min. The cells were then fixed and labeled with DAPI (blue). FcgRIIA clustering was observed under a microscope. The images are representative of three experiments. Original magnification 363. (B) The expression levels of FcgRIIA, FcgRIIIA, and CD11b/CD18 in NC and KD cells, as shown in (A), were evaluated by flow cytometry. (C) NB4 cells were pretreated with ATRA for 72 h, and then treated with DMSO, 5 mM PP2, or 10 nM cytochalasin D for another 15 min; FcgRIIA clustering was then observed under a microscope. Images from two representative experiments are provided. (D) NC and KD cells were pretreated for 72 h with ATRA and then treated with DMSO, 5 mM PP2, or 10 nM cytochalasin D for another 15 min, and the cell’s particulate-binding ability was then examined by flow cytometry. Quantification of three independent experiments was shown. (E) NB4 cells pretreated with ATRA were incubated with serum-opsonized zymosan for 15 min, fixed, labeled with c-Src (red) and PALLD (green), and observed under microscopy. (F) Lysates of NB4 cells pretreated with ATRA for 72 h were immunoprecipitated with normal rabbit IgG or PALLD Ab. The lysates were then immunoblotted with c-Src Ab. (G) NC and KD cells pretreated with ATRA were incubated with serum-opsonized E. coli for the indicated durations. The lysates were analyzed by Western blot for phosphorylated c-Src Tyr416 and b-actin. Scale bar, 5 mm. Original magnification 363. Data are expressed as mean 6 SEM. **p # 0.01. n.s., not significant. 1822 PALLD REGULATES ACTIN DYNAMICS IN PHAGOCYTOSIS

FIGURE 5. PALLD promotes ARP3 recruitment onto F-actin. (A, B, and E) The recruitment of RAC1 (red) (A), CDC42 (red) (B), and ARP3(red) (E) in phagocytic cups was quantiﬁed in NC and KD cells pretreated with ATRA by measuring the relative ﬂuorescent in- tensities in the cups and the cortical region of the cell, as indicated by two short lines. Left, one representative image; right, quantitative data changes were showing for RAC1 (A), CDC42 (B), and ARP3 (E) of three independent experiments, n . 10 cups per experiment. (C) NB4 cells pretreated with ATRA were incubated

with serum-opsonized zymosan for 15 min, fixed, la- Downloaded from beled with ARP3 (green) and PALLD (red), and observed under microscopy (right). (D) Lysate of NB4 cells pretreated with ATRA for 72 h was immunoprecipitated with normal rabbit IgG or PALLD Ab. The lysates were then immunoblotted with ARP3 Ab. Scale bar, 5 mm. Original magnification 363. Data are expressed as mean 6 SEM. *p # 0.05. n.s, not http://www.jimmunol.org/ significant. by guest on September 29, 2021

PALLD KD does not affect focal exocytosis and of phagocytic substrates in mature myeloid cells by regulating phagosome maturation substrate-elicited clustering and the affinity of phagocytic recep- Pseudopod extension and phagosome closure are facilitated by the tors (22). Thus, we examined the relationship between PALLD recruitment of intracellular vesicles to the phagocytic cup in a and FcgR clustering during the binding process. FcgR clustering process called focal exocytosis (25). To study the role of PALLD was induced and detected by the FcgRIIA Ab plus fluorescent in focal exocytosis, we analyzed the recruitment of VAMP3-mCherry, secondary Ab. FcgR clusters in control cells became evident after a recycling endosome maker, at the phagocytic cups (26). PALLD 20 min of incubation at 4˚C, whereas, in contrast, immune complex– KD did not affect the recruitment of VAMP3-mcherry–positive stimulated FcgR clustering was impeded in PALLD-deficient cells vesicles to the plasma membrane, suggesting that PALLD may not at the same time points (Fig. 4A). Thus, PALLD is required for affect focal exocytosis during serum-mediated phagocytosis (Supple- efficient FcgRclustering. mental Fig. 3A). Several studies have reported that the actin cytoskeleton and After phagosome formation, the phagosome fuses with intra- Src kinase may be involved in FcgR clustering. The cortical actin cellular organelles, such as early endosome, late endosome, and cytoskeleton under the cell membrane may restrict receptor mo- lysosome, in the process of phagosome maturation (31). We in- bility and clustering (32). Besides, Sobota et al. (33) proposed a vestigated the recruitment of EEA1 (early endosome maker), positive feedback loop wherein FcgR phosphorylation by Src kinase RAB7 (late endosome maker), and LAMP1 (lysosome maker) facilitates clustering, further increasing the efficiency of particle between control and PALLD KD cells at different time points. No binding. Therefore, we studied whether PALLD affected FcgR difference was observed in the recruitment of these intracellular clustering via the regulation of actin polymerization or c-Src acti- organelle makers between PALLD KD and control cells (Supplemental vation. Cytochalasin D, an actin polymerization inhibitor, and Fig. 3B, 3C). Therefore, PALLD might not be involved in phago- PP2, a Src kinase inhibitor, reduced FcgR clustering (Fig. 4C). PP2 some maturation. and cytochalasin D appeared to reduce the differences in binding levels between PALLD-deficient and control cells (Fig. 4D). Yeast PALLD regulates FcgR clustering to promote particle binding two-hybrid analysis had suggested that PALLD physically associ- Phagocytic particles bind to the cell surface through membrane- ated with c-Src (8). The present study used coimmunoprecipitation bound receptors. We excluded the possibility that the binding and colocalization assays to confirm that PALLD and c-Src indeed defect in PALLD-deficient cells resulted from a plausible alteration interacted during phagocytosis (Fig. 4E, 4F). Moreover, c-Src showed of expression of the main Fcg receptor (FcgRIIA and FcgRIIIA) dynamic phosphorylation during phagocytosis of serum-opsonized or complement receptor subtypes C3b (CD11b/CD18) on the cell Escherichia coli. However, the phosphorylation of c-Src Y416 in surface (Fig. 4B). Alternatively, PALLD may reinforce the binding PALLD KD cells was considerably lower than in control cells The Journal of Immunology 1823

FIGURE 6. PALLD interacts with PI(4,5)P2 phosphatase OCRL. (A) NB4 cells were pretreated with ATRA for 72 h, incubated with serum-opsonized zymosan for 20 min, fixed, labeled with OCRL (green) and PALLD (red), and observed under microscopy (left). Colocalization efficiency of PALLD and OCRL was analyzed in three independent experiments (right). Original magnification 363. (B) NB4 cells pretreated with ATRA for 72 h. The lysates were then immunoprecipitated with IgG or anti-OCRL Ab and immunoblotted with PALLD Ab. (C and D) Schematic Downloaded from representation of different OCRL truncations (C)or PALLD truncations (D) used in the transfection experiments. (E and F) 293T cells transduced with the indicated constructs. The coimmunoprecipitation experiments were performed as indicated. Scale bar, 5 mm. http://www.jimmunol.org/ by guest on September 29, 2021

(Fig. 4G). In conclusion, PALLD may be involved in FcgRclus- PALLD recruits OCRL to phagosomes tering both by actin cytoskeleton regulation and c-Src kinase acti- To obtain further insight into the molecular mechanisms respon- vation, thereby promoting particle binding. sible for PALLD activity in phagocytosis, we conducted coimmunoprecipitation experiments with anti-PALLD Ab and performed The role of PALLD-mediated actin dynamics in phagocytic mass spectrometry analysis on lysates from ATRA-treated NB4 cells cup formation (data not shown). We found that OCRL physically associated with PALLD is an actin-associated protein. Thus, it may affect F-actin PALLD. This specific interaction was confirmed by Western blotting dynamics during phagocytosis. F-actin remodeling during phago- (Fig. 6B). OCRL and PALLD partially colocalized at phagosomes cytosis can be divided into at least five stages (Supplemental Fig. 4A) (Fig. 6A). A series of truncated OCRL and PALLD forms were (30). Actin remodeling in PALLD-deficient cells was blocked at the constructed, in the domain-mapping experiment, which showed that pseudopod extension and actin depolymerization stages (indicated as the C terminus part of PALLD containing the third IgC2 domain stages 2 and 5) (Supplemental Fig. 4B), which may partially explain interacted with 5-phosphatase domain of OCRL (Fig. 6C–F). their reduced phagocytic ability. We next analyzed the effect of PALLD KD on the OCRL re- Previous studies have shown that, during pseudopod extension, cruitment at phagosome formation sites. PALLD KD decreased Rho GTPases such as RAC1 and CDC42 are activated early to OCRL recruitment by ∼30% (Fig. 7A). Moreover, OCRL KD also recruit the N-WASP/WASP actin nucleation-promoting factors, reduced phagocytic capability in differentiated NB4 cells (Fig. 7B, which in turn stimulate the ARP2/3 complex to promote actin 7C), but did not significantly affect particle binding (Fig. 7D). nucleation and polymerization (12). Compared with the phago- Given that OCRL regulates PI(4,5)P2, we hypothesized that cytic cups in control cells, we found higher RAC1 accumulation in OCRL locally participates in the hydrolysis of PI(4,5)P2 and PI the blocked cups formed in PALLD KD cells (Fig. 5A). We did not (3,4,5)P3 to complete phagosome closure. We found that PALLD find any significant changes in CDC42 recruitment by PALLD KD KD caused the retention of PI(4,5)P2 and increased F-actin at the cells (Fig. 5B). ARP3, the downstream effector of Rho GTPases, phagosome (Fig. 7E). Compared with OCRL KD cells, the cells coimmunoprecipitated and colocalized with PALLD (Fig. 5C, with both genes knocked down did not exhibit a reduced phago- 5D), suggesting that PALLD may stimulate the ARP2/3 complex cytic phenotype, suggesting that the effect of PALLD in phago- to promote actin nucleation and polymerization (12). Moreover, cytosis is OCRL dependent (Fig. 7F). We also established a cell ARP3 recruitment was more intense in control cells than in line carrying the phosphatase-inactive D499A mutation of OCRL PALLD KD cells (Fig. 5E). Collectively, these results suggested (34). KD of PALLD in this cell line did not further reduce that PALLD regulates actin dynamics during phagocytic cup phagocytic capability, indicating that the role of PALLD in formation. phagocytosis depends on the catalytic activity of OCRL (Fig. 7G). 1824 PALLD REGULATES ACTIN DYNAMICS IN PHAGOCYTOSIS

FIGURE 7. OCRL is recruited downstream of PALLD in phagocytosis. (A)NC and KD cells pretreated with ATRA were incubated with serum-opsonized zymosan- FITC for 15 min, fixed, labeled with OCRL (red) and phalloidin (purple), and observed under microscopy (left). The percentages of OCRL-positive phagosome indicated in (A) were shown from three independent experiments (n . 100 for each condition) (right). Original magnification 363. (B) Immunoblot of OCRL in the lysates from NB4 cells stably transduced with control (NC) or shRNA sequences targeting OCRL. (C and D) ATRA-treated NB4 cells were transfected with NC or OCRL-targeting shRNA sequences and incubated with serum-opsonized microspheres for 1 h; Downloaded from phagocytosis (C) or binding level (D) was measured by flow cytometry. Measurement was performed in three independent experiments. (E) NB4 NC and KD cells pretreated with ATRA were incubated with serum-opsonized zymosan for 20 min, http://www.jimmunol.org/ fixed, labeled with PI(4,5)P2 (red) and phalloidin (purple), and observed under microscopy. Left, one representative image; right, quantitative data showing changes in

fluorescence intensity of PI(4,5)P2 (n =22 for NC, and n = 23 for KD group) of three independent experiments. Original magnification 363. (F) NB4 cells treated with NC or OCRL-targeting shRNA alone were further exposed to PALLD-targeting shRNA. by guest on September 29, 2021 The cells’ phagocytic abilities were assessed by flow cytometry. Measurement was performed in three independent experiments. (G) NB4 cells were transfected with phosphatase-inactivate, D499A mutant of OCRL, and infected with PALLD shRNA or control. Phagocytosis was measured by flow cytometry. Measurement was performed in three independent experiments. (H) Sche- matic diagram of the interaction between PALLD and OCRL coordinated during phagosome closure. Scale bar, 5 mm. Data are expressed as mean 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001. n.s, not significant.

These results suggested that OCRL could be delivered at phag- closure. PALLD mechanistically promoted FcgRIIA clustering osome sites via PALLD recruitment, which might play an essential role during the particle-binding step, regulated actin polymerization in the regulation of PI(4,5)P2 hydrolysis and actin depolymerization, a during the pseudopod extension stage, and recruited OCRL for the step necessary to complete phagosome closure (Fig. 7H) (35). hydrolysis of PI(4,5)P2, which led to actin depolymerization during phagosome closure. Discussion It seems that PALLD is essential for embryogenesis, and palld2/2 In this study, we determined that PALLD was highly induced by mice die perinatally (36). Therefore, our work mainly relied on the ATRA during the maturation of myeloid leukemia cells. PALLD analyses of PALLD KD cell lines when PALLD was induced by plays an important role in the development of phagocytic capacity, ATRA. ATRA-induced PALLD upregulation was parallel to in- although it is unessential for ATRA-induced neutrophil differen- creased phagocytic ability in primary BM cells, indicating that the tiation. PALLD expression facilitates the key steps of phagosome regulation of phagocytosis by PALLD also occurs in nonmalignant formation: particle binding, pseudopod extension, and phagosome myeloid cells. The Journal of Immunology 1825

Receptor clustering is much more complex than simple protein– Disclosures protein contact across juxtaposed membranes. It involves the large- The authors have no financial conflicts of interest. scale reorganization of many membrane proteins into a highly structured and dynamic micrometer-scale region at the cell-substrate interface, which has been termed as supramolecular activation References 1. Parast, M. M., and C. A. Otey. 2000. Characterization of palladin, a novel protein cluster or immunological synapse (37). Single phagocytic recep- localized to stress fibers and cell adhesions. J. Cell Biol. 150: 643–656. tors, like FcgRs, have comparatively low affinity for their ligands, 2. Goicoechea, S. M., D. Arneman, and C. A. Otey. 2008. The role of palladin in thus requiring the simultaneous engagement of multiple receptors actin organization and cell motility. Eur. J. Cell Biol. 87: 517–525. 3. Rachlin, A. S., and C. A. Otey. 2006. Identification of palladin isoforms and to securely capture a particle, which in turn generates a phagocytic characterization of an isoform-specific interaction between Lasp-1 and palladin. signal (22, 33, 38). FcgRIIA mobility is regulated by Src-family J. Cell Sci. 119: 995–1004. kinases and the actin cytoskeleton (38, 39). PALLD KD cells had a 4. Boukhelifa, M., M. M. Parast, J. E. Bear, F. B. Gertler, and C. A. Otey. 2004. Palladin is a novel binding partner for Ena/VASP family members. Cell Motil. reduced phosphorylation level of c-Src Y416 during phagocytosis, Cytoskeleton 58: 17–29. implying a new activity for PALLD in receptor clustering by pro- 5. Ro¨nty, M., A. Taivainen, M. Moza, C. A. Otey, and O. Carpeń. 2004. Molecular analysis of the interaction between palladin and alpha-actinin. FEBS Lett. 566: moting Src-family kinase activation (38, 39). The mechanisms link- 30–34. ing numerous membrane proteins to the cytoskeleton are still unclear, 6. Boukhelifa, M., M. Moza, T. Johansson, A. Rachlin, M. Parast, S. Huttelmaier, although it is widely accepted that the actin cytoskeleton influences P. Roy, B. M. Jockusch, O. Carpen, R. Karlsson, and C. A. Otey. 2006. The proline- rich protein palladin is a binding partner for profilin. FEBS J. 273: 26–33. the receptor clustering; thus, a full understanding of membrane pro- 7. Goicoechea, S., D. Arneman, A. Disanza, R. Garcia-Mata, G. Scita, and tein organization is required (32, 40). In this regard, the results of the C. A. Otey. 2006. Palladin binds to Eps8 and enhances the formation of dorsal current study suggested that PALLD may be an important member of ruffles and podosomes in vascular smooth muscle cells. J. Cell Sci. 119: 3316– Downloaded from 3324. actin components that regulate receptor clustering. The possibility 8. Ro¨nty, M., A. Taivainen, L. Heiska, C. Otey, E. Ehler, W. K. Song, and that PALLD may affect the mobility of other membrane proteins will O. Carpen. 2007. Palladin interacts with SH3 domains of SPIN90 and Src and is be an interesting topic for future studies. required for Src-induced cytoskeletal remodeling. Exp. Cell Res. 313: 2575– 2585. PI(4,5)P2 is predominantly found in the plasma membrane and 9. Dixon, R. D., D. K. Arneman, A. S. Rachlin, N. R. Sundaresan, M. J. Costello, is a major determinant of the actin cytoskeleton structures and S. L. Campbell, and C. A. Otey. 2008. Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin. J. Biol. Chem. dynamics (41). PI(4,5)P2 undergoes a biphasic change at the phago- 283: 6222–6231. http://www.jimmunol.org/ cytic cup: PI(4,5)P2 initially accumulates and is then eliminated from 10. Maeda, M., E. Asano, D. Ito, S. Ito, Y. Hasegawa, M. Hamaguchi, and T. Senga. the nascent phagosome prior to its scission from the plasma mem- 2009. Characterization of interaction between CLP36 and palladin. FEBS J. 276: 2775–2785. brane (23). Emerging evidence suggests that phosphatidylinositol 11. Chin, Y. R., and A. Toker. 2010. The actin-bundling protein palladin is an Akt1- 5-phosphatases contribute to the elimination of PI(4,5)P2 by sealing specific substrate that regulates breast cancer cell migration. Mol. Cell 38: 333– phagosomes (24). PALLD physically interacted with OCRL to 344. 12. Qian, X., D. D. Mruk, E. W. Wong, P. P. Lie, and C. Y. Cheng. 2013. Palladin is a promote OCRL recruitment at the phagosome, thus participating in regulator of actin filament bundles at the ectoplasmic specialization in adult rat the hydrolysis of PI(4,5)P2 and PI(3,4,5)P3 to complete phagosome testes. Endocrinology 154: 1907–1920. 13. Garcia-Palmero, I., S. Torres, R. A. Bartolome, A. Pelaez-Garcia, M. J. Larriba, closure. Collectively, our observations showed that PALLD acts in M. Lopez-Lucendo, C. Pena, B. Escudero-Paniagua, A. Munoz, and J. I. Casal. phagocytosis by switching actin polymerization on or off in a phase- 2016. Twist1-induced activation of human fibroblasts promotes matrix stiffness by guest on September 29, 2021 dependent manner during phagosome formation. by upregulating palladin and collagen a1(VI). Oncogene 35: 5224–5236. 14. Yadav, R., R. Vattepu, and M. R. Beck. 2016. Phosphoinositide binding inhibits Rho GTPases such as RAC1 and CDC42 stimulate actin assembly actin crosslinking and polymerization by palladin. J. Mol. Biol. 428: 4031–4047. by acting as upstream factors (21). However, our observations did not 15. Gurung, R., R. Yadav, J. G. Brungardt, A. Orlova, E. H. Egelman, and imply a regulatory role for PALLD on these two factors. Surpris- M. R. Beck. 2016. Actin polymerization is stimulated by actin cross-linking protein palladin. Biochem. J. 473: 383–396. ingly, Rac1 accumulation was higher in the blocked cups formed in 16. Mykka¨nen, O. M., M. Gro¨nholm, M. Ro¨nty, M. Lalowski, P. Salmikangas, PALLD KD cells, whereas the intensity of ARP3 recruitment was H. Suila, and O. Carpeń. 2001. Characterization of human palladin, a reduced in PALLD KD cells. This phenomenon may reflect a neg- microfilament-associated protein. Mol. Biol. Cell 12: 3060–3073. 17. Liu, T. X., J. W. Zhang, J. Tao, R. B. Zhang, Q. H. Zhang, C. J. Zhao, J. H. Tong, ative feedback effect. Previous studies suggested that PALLD dra- M. Lanotte, S. Waxman, S. J. Chen, et al. 2000. Gene expression networks matically reduces depolymerization by eliminating the lag phase underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells. Blood 96: 1496–1504. characteristic of the slow nucleation step of actin polymerization. 18. Woodfin, A., M. Beyrau, M. B. Voisin, B. Ma, J. R. Whiteford, P. L. Hordijk, The results of the current study suggested that PALLD can also N. Hogg, and S. Nourshargh. 2016. ICAM-1-expressing neutrophils exhibit promote actin depolymerization by locally recruiting OCRL to hy- enhanced effector functions in murine models of endotoxemia. Blood 127: 898– 907. drolyze PI(4,5)P2 during the phagosome closure stage. 19. Liu, G., X. Hu, B. Sun, T. Yang, J. Shi, L. Zhang, and Y. Zhao. 2013. Phos- Expression of PALLD is regulated by multiple factors, and it phatase Wip1 negatively regulates neutrophil development through p38 MAPK- seems that PALLD is usually upregulated in the situations in- STAT1. Blood 121: 519–529. 20. Garcıá-Garcıá, E., and C. Rosales. 2002. Signal transduction during Fc receptor- volving an organized actin cytoskeleton. For example, PALLD is mediated phagocytosis. J. Leukoc. Biol. 72: 1092–1108. highly upregulated in tumor-associated fibroblasts and in kidney 21. Freeman, S. A., and S. Grinstein. 2014. Phagocytosis: receptors, signal inte- gration, and the cytoskeleton. Immunol. Rev. 262: 193–215. disease, as well as contributing to epithelial cell migration after 22. Jaumouille´, V., and S. Grinstein. 2011. Receptor mobility, the cytoskeleton, and injury. However, few studies have investigated the function of particle binding during phagocytosis. Curr. Opin. Cell Biol. 23: 22–29. PALLD in the phagocytosis of myeloid cells. It is interesting to 23. Scott, C. C., W. Dobson, R. J. Botelho, N. Coady-Osberg, P. Chavrier, D. A. Knecht, C. Heath, P. Stahl, and S. Grinstein. 2005. Phosphatidylinositol- explore in the future studies whether certain phagocytic defect- 4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J. associated diseases such as primary immune deficiency diseases Cell Biol. 169: 139–149. are attributed to the expressional and/or functional deficiency of 24. Bohdanowicz, M., D. M. Balkin, P. De Camilli, and S. Grinstein. 2012. Re- cruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes PALLD (42). phosphoinositides and attenuates Akt signaling. Mol. Biol. Cell 23: 176–187. 25. Huynh, K. K., J. G. Kay, J. L. Stow, and S. Grinstein. 2007. Fusion, fission, and secretion during phagocytosis. Physiology (Bethesda) 22: 366–372. Acknowledgments 26. Bajno, L., X. R. Peng, A. D. Schreiber, H. P. Moore, W. S. Trimble, and We thank Xiang Zhang for providing FLAG-PALLD and its truncated plas- S. Grinstein. 2000. Focal exocytosis of VAMP3-containing vesicles at sites of mids, and all colleagues at the Shanghai Institute of Hematology for con- phagosome formation. J. Cell Biol. 149: 697–706. 27. Inaba, K., W. J. Swiggard, R. M. Steinman, N. Romani, G. Schuler, and structive discussion and technical assistance. We thank Wenda Xi for C. Brinster. 2009. Isolation of dendritic cells. Curr.Protoc.Immunol. Chapter 3: writing and revising the paper. Unit 3.7. 1826 PALLD REGULATES ACTIN DYNAMICS IN PHAGOCYTOSIS

28. Mao, Y. S., M. Yamaga, X. Zhu, Y. Wei, H. Q. Sun, J. Wang, M. Yun, Y. Wang, 35. Marion, S., J. Mazzolini, F. Herit, P. Bourdoncle, N. Kambou-Pene, G. Di Paolo, M. Bennett, et al. 2009. Essential and unique roles of PIP5K- S. Hailfinger, M. Sachse, J. Ruland, A. Benmerah, A. Echard, et al. 2012. The gamma and -alpha in Fcgamma receptor-mediated phagocytosis. J. Cell Biol. NF-kB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and 184: 281–296. OCRL-bearing vesicles. Dev. Cell 23: 954–967. 29. Link, T. M., U. Park, B. M. Vonakis, D. M. Raben, M. J. Soloski, and 36. Liu, X. S., X. H. Li, Y. Wang, R. Z. Shu, L. Wang, S. Y. Lu, H. Kong, Y. E. Jin, M. J. Caterina. 2010. TRPV2 has a pivotal role in macrophage particle binding L. J. Zhang, J. Fei, et al. 2007. Disruption of palladin leads to defects in de- and phagocytosis. Nat. Immunol. 11: 232–239. finitive erythropoiesis by interfering with erythroblastic island formation in 30. Babuta, M., M. S. Mansuri, S. Bhattacharya, and A. Bhattacharya. 2015. The mouse fetal liver. Blood 110: 870–876. Entamoeba histolytica, Arp2/3 complex is recruited to phagocytic cups through 37. Cochran, J. R., D. Aivazian, T. O. Cameron, and L. J. Stern. 2001. Receptor an atypical kinase EhAK1. PLoS Pathog. 11: e1005310. clustering and transmembrane signaling in T cells. Trends Biochem. Sci. 26: 31. Huynh, K. K., E. L. Eskelinen, C. C. Scott, A. Malevanets, P. Saftig, and 304–310. S. Grinstein. 2007. LAMP proteins are required for fusion of lysosomes with 38. Kwiatkowska, K., J. Frey, and A. Sobota. 2003. Phosphorylation of Fcgam- phagosomes. EMBO J. 26: 313–324. maRIIA is required for the receptor-induced actin rearrangement and capping: 32. Andrews, N. L., K. A. Lidke, J. R. Pfeiffer, A. R. Burns, B. S. Wilson, the role of membrane rafts. J. Cell Sci. 116: 537–550. J. M. Oliver, and D. S. Lidke. 2008. Actin restricts FcepsilonRI diffusion and 39. Jaumouille´, V., Y. Farkash, K. Jaqaman, R. Das, C. A. Lowell, and S. Grinstein. facilitates antigen-induced receptor immobilization. Nat. Cell Biol. 10: 955–963. 2014. Actin cytoskeleton reorganization by Syk regulates Fcg receptor respon- 33. Sobota, A., A. Strzelecka-Kiliszek, E. Gładkowska, K. Yoshida, K. Mrozinska, siveness by increasing its lateral mobility and clustering. Dev. Cell 29: 534–546. and K. Kwiatkowska. 2005. Binding of IgG-opsonized particles to Fc gamma R 40. Jaqaman, K., and S. Grinstein. 2012. Regulation from within: the cytoskeleton in is an active stage of phagocytosis that involves receptor clustering and phos- transmembrane signaling. Trends Cell Biol. 22: 515–526. phorylation. J. Immunol. 175: 4450–4457. 41. Szymanska, E., A. Sobota, E. Czuryło, and K. Kwiatkowska. 2008. Expression of 34. Oltrabella, F., G. Pietka, I. B. Ramirez, A. Mironov, T. Starborg, PI(4,5)P2-binding proteins lowers the PI(4,5)P2 level and inhibits FcgammaRIIA- I. A. Drummond, K. A. Hinchliffe, and M. Lowe. 2015. The Lowe syndrome mediated cell spreading and phagocytosis. Eur. J. Immunol. 38: 260–272. protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule. 42. Blander, J. M. 2017. The many ways tissue phagocytes respond to dying cells. PLoS Genet. 11: e1005058. Immunol. Rev. 277: 158–173. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021