May 2009
Volume 50, Issue 5
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Retinal Cell Biology  |   May 2009
Mertk Drives Myosin II Redistribution during Retinal Pigment Epithelial Phagocytosis
Author Affiliations
  • David J. Strick
    From the Departments of Genetics and
  • Wei Feng
    From the Departments of Genetics and
  • Douglas Vollrath
    From the Departments of Genetics and
    Ophthalmology, Stanford University, Stanford, California.
Investigative Ophthalmology & Visual Science May 2009, Vol.50, 2427-2435. doi:10.1167/iovs.08-3058
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      David J. Strick, Wei Feng, Douglas Vollrath; Mertk Drives Myosin II Redistribution during Retinal Pigment Epithelial Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2009;50(5):2427-2435. doi: 10.1167/iovs.08-3058.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis.

methods. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells.

results. Myh9, the non–muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect.

conclusions. Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Phagocytosis is a receptor-mediated form of endocytosis essential for the removal and destruction of large particles from the extracellular environment. Engulfed particles include pathogens, apoptotic cells, and cellular debris. 1 Phagocytosis is a function performed by multiple cell types, and the intracellular signaling cascades regulating particle internalization are heterogeneous. 2 3 4 5 A common aspect of all phagocytosis pathways is the requirement for cytoskeletal modification to effect the formation and closure of a phagocytic cup, which results in particle engulfment and a nascent phagosome. 1 6 7 The Fc receptor (FcR) mediates phagocytosis of immunoglobulin G–opsonized foreign cells by a process that involves the protrusion of lamellipodia and the activation of Rac and CDC42 to recruit actin to the phagocytic cup. 2 3 8 9 10 In contrast, complement receptor 3 (CR3) binds C3bi-opsonized particles that seem to sink into the cell during internalization. 2 3 7 CR3 receptor activation triggers a signaling cascade involving Rho GTPase that results in the phosphorylation of myosin regulatory light chain (RLC), increased myosin activity, and ultimately actin recruitment to the phagocytic cup. 8 11 A study of bone marrow–derived macrophages from various mouse knockout lines suggests that Fc and complement receptor signaling recruit Rho, Rac, and CDC42 to nascent phagosomes and that Vav GEFs are required to activate Rac only in complement receptor phagocytosis. 12 These results illustrate that a particular cell type, receptor, and engulfed particle determine the downstream signaling pathways regulating actin nucleation and that multiple mechanisms have evolved within mammalian cells to achieve a common biological response: formation of a phagocytic cup. 
The retinal pigment epithelium experiences one of the largest phagocytic burdens of any vertebrate cell type. 13 It is a polarized epithelial cell layer that ingests adjacent photoreceptor outer segment (OS) tips daily as part of a process of renewal of the light-sensing portion of these neurons. 14 The consequences of impairment of retinal pigment epithelial (RPE) phagocytosis are best represented by Royal College of Surgeons (RCS) rats, in which a defect in OS phagocytosis results in the accumulation of a debris layer between photoreceptors and the retinal pigment epithelium and leads to retinal degeneration. 15 16 17 This severe retinal dystrophy phenotype results from a null mutation of the gene encoding the receptor tyrosine kinase Mertk, 18 which was conclusively demonstrated by genetic complementation of the phenotype through viral gene transfer to the RCS retinal pigment epithelium in vivo. 19 20 MERTK mutations also cause retinal degenerative disease in humans. 21 22 23 Studies of wild-type and RCS RPE cells in culture have shown that Mertk acts directly in the phagocytic process and is required for ingestion, but not binding, of OS. 24 F-actin localization to the phagocytic cup appears normal in cultured RCS RPE cells challenged with OS, suggesting that Mertk does not function in actin recruitment. 25 However, the stimulation of Mertk intracellular signaling in a murine leukemic cell line causes rapid and profound alterations in cell shape and adherence. 26 Taken together, these results suggest that signaling through Mertk coordinates the cytoskeletal rearrangements necessary for RPE phagocytic ingestion of OS. 
We wanted to elucidate the mechanism by which Mertk coordinates cytoskeletal rearrangements during RPE ingestion of OS. Our results demonstrate an important role for non–muscle myosin II (NMMII) in this process. 
Materials and Methods
Reagents
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. The Mertk polyclonal antibody was previously described. 24 Other antibodies used in this study include anti–Myh9 (Sigma), anti–Myh10 (Covance, Denver, CO), anti–rhodopsin (a generous gift from Robert Molday, University of British Columbia, BC, Canada), goat anti–mouse and anti–rabbit horseradish peroxidase conjugates (Jackson ImmunoResearch, West Grove, PA), and goat anti–mouse and anti–rabbit conjugates (Oregon Green and Texas Red; Invitrogen, Carlsbad, CA). The ZO-1 (R26.4C) antibody developed by Daniel A. Goodenough was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA. (±)-Blebbistatin was obtained from EMD Chemicals (Gibbstown, NJ) and diluted in 90% dimethyl sulfoxide (DMSO) according to the manufacturer’s directions. The construction and preparation of recombinant adenovirus expressing Mertk (Ad-Mertk) was previously described. 20 Long Evans and Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Pigmented RCS rats were a generous gift from Machelle Pardue (Emory University, Atlanta, GA). The ethical treatment of our laboratory animals conforms to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Cell Culture
RPE-J cells (ATCC, Manassas, VA) were maintained in DMEM (Mediatech, Manassas, VA) with 4% FBS (Gemini Bio-Products, West Sacramento, CA), MEM nonessential amino acids, and antibiotic/antimycotic (Invitrogen) at 33°C with 5% CO2. ARPE-19 cells were a generous gift from Leonard Hjelmeland (University of California, Davis, CA) and were cultured as described. 27 Primary RPE cells from RCS and Long Evans rats were cultured as previously described 24 28 in an eight-well chamber slide system (LabTek II; Nalge Nunc, Rochester, NY). Bovine eyes were obtained from Animal Technologies (Tyler, TX). Bovine OS and rat OS were isolated as previously described 16 and stored in Hanks’ balanced salt solution with 5% sucrose at −80°C. 
Immunoprecipitation
RPE-J cells were transduced at a multiplicity of infection (MOI) of 2 with Ad-Mertk. 20 ARPE-19 cells were transduced with Ad-Mertk at an MOI of 5. After 48 hours, the cells were challenged with 2 × 107 bovine OS/mL or medium alone for the indicated times. Cells were washed three times with Tris-buffered saline, pH 7.6, and lysed with 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% NP-40 (Igepal; Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaVO3, and protease inhibitors (Roche, Indianapolis, IN). Lysates were centrifuged at 16,000g for 10 minutes at 4°C. Supernatants were immunoprecipitated with anti-Mertk or anti–Myh9 antibodies overnight at 4°C. Antibody-target complexes were captured with agarose (Protein A/G; Pierce Biotechnology, Rockford, IL) for 4 hours at 4°C. Beads were washed three times with lysis buffer and then twice with 50 mM Tris-Cl, pH 8.0. Agarose (Protein A/G; Pierce Biotechnology) beads were resuspended in Laemmli sample buffer and boiled for 5 minutes before immunoblotting. 
Mass Spectrometry
RPE-J cells were immunoprecipitated as described, fractionated on a 7.5% acrylamide/Bis-acrylamide (37.5:1) gel, and stained with Coomassie G250 stain (Bio-Rad, Hercules, CA). Bands were excised and destained for 2 hours with 10% acetic acid and 50% methanol, followed by dH2O washes to neutralize the pH. Gel slices were treated with 4.5 mM dithiothreitol (Sigma) in 100 mM Tris, pH 7.8, at 55°C for 30 minutes, followed by a wash with 10 mM iodoacetamide in 100 mM Tris, pH 7.7, for 1 hour in the dark. Slices were washed again in 500 μL of 50 mM Tris (pH 7.8)/50% acetonitrile for 30 minutes. The solution was removed, and the gel was dried. Then 5 pM trypsin (Promega, Madison, WI) was added in 25 mM Tris, pH 7.8, plus a minimal amount of 25 mM Tris, pH 7.8, to allow slices to rehydrate. After incubation overnight at 37°C, the solution phase was removed and injected into a mass spectrometer (LCQ Deca XP LC; Thermo Scientific, Waltham, MA). Peptide fingerprint data were analyzed with protein analysis software (Mascot; Matrix Science, Boston, MA). 
RPE-Phagocytic Assays and Blebbistatin Treatment
RPE cells were cultured for 6 days before blebbistatin treatment. Blebbistatin (100 μM) was applied 30 minutes before the addition of OS and again during incubation with 1 × 107 OS/mL for 4 hours at 37°C. Unbound OS was removed by three washes with PBS, and the cells were fixed in 3.5% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 30 minutes at 37°C. Bound and total OS were differentially labeled with anti–rhodopsin antibody, as previously described. 16 24 Slides were mounted with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and visualized with an inverted epifluorescent microscope (DMB IRBl Leica, Wetzlar, Germany) using a 40× (0.55 NA) objective. Images were captured with a charge-coupled device camera and imaging software (SimplePCI; Hamamatsu Corp, Sewickley, PA). Ten images per treatment of confluent RPE monolayers were captured and quantified with ImageJ (National Institutes of Health, Bethesda, MD). Ingested OS were quantified by measuring all the OS particles in a given image and subtracting cell surface–bound OS. Statistical differences were calculated from three independent experiments with statistical software (GraphPad Prism 4.0; San Diego, CA) and measured with a Student’s t-test. 
siRNA and Transfection
Myh9 and Myh10 siRNAs were obtained as mixtures of four different duplexes to minimize off-target effects (Smartpools; Thermo Scientific/Dharmacon, Layafette, CO). Nontargeting control siRNA was obtained from Qiagen (Valencia, CA). Primary RPE cells were cultured in eight-well chamber slides for 6 days before transfection. siRNA liposome complexes were formed by incubating 0.5 μg of the indicated siRNAs with 3.9 μL transfection reagent (RNAiFect; Qiagen) diluted in 100 μL growth medium for 15 minutes. siRNA liposome complexes were incubated with cells in a 150-μL total volume for 24 hours. After 72 hours, transfection was repeated to ensure that any cells that proliferated during that time would receive a dose of the siRNA. Phagocytic assays were performed on day 5 after initial transfection, and statistical differences of three independent experiments were quantified as described earlier. 
Confocal Microscopy, Virus Transduction, and Image Analysis
Primary RPE cells were incubated with 1 × 107 rat OS/mL for various times. Unbound OS underwent three washes with PBS, and cells were fixed with 3.5% paraformaldehyde in PBS (Electron Microscopy Sciences) for 30 minutes at room temperature. Cells were washed and permeabilized with 0.2% Triton X-100, blocked with 5% normal goat serum in PBS, and incubated with the appropriate primary and secondary antibodies. Slides were mounted with mounting medium (Vectashield; Vector Laboratories) and viewed under a confocal microscope (LSM510; Zeiss, Oberkochen, Germany) with a Plan-Neo 40× (1.3 NA) or a Plan-Neo 63× (1.4 NA) objective. Images from a Z-series were assembled in a Z-projection, and ImageJ was used to view all the myosin for a given field. Brightness and contrast were adjusted with imaging software (Photoshop CS2; Adobe Systems, San Jose, CA). All images are representative of three independent experiments. For virus complementation experiments, RCS RPE cells were isolated from 6-day-old pups and cultured for 5 to 6 days. Ten MOI Ad-Mertk was added for 2 hours at 37°C and washed away. The cells were incubated in normal growth medium and assayed for 48 hours, as described. For the measurement of NMMII-A on the lateral cell membranes, images were acquired with an epifluorescent microscope (DMB IRB; Leica) with a 40× (0.55 NA) objective. With the use of ImageJ, boxes were drawn around random cell borders, and the maximum pixel intensity was recorded for 30 areas per treatment. 
Retinal Pigment Epithelium/Choroid/Sclera Immunostaining
Wholemounts of retinal pigment epithelium/choroid/sclera were dissected from postnatal day 40 C57BL/Ka mice (wild-type for Mertk), fixed in 4% paraformaldehyde for 2 hours, and permeabilized in 0.2% Triton X-100 for 1 hour. The mounts were blocked for 1 hour with 5% goat serum in PBS, incubated with the appropriate primary and secondary antibodies, and imaged with confocal microscopy, as described. To ensure that this method measured ingested phagosomes, we confirmed that the rhodopsin-positive puncta localized to the same Z-axis as ZO-1. Confocal micrographs were assembled in a Z-projection using ImageJ to show all the myosin in a given field. 
Results
Association of Mertk with NMMII-A and Actin during OS Ingestion
We used a proteomics approach to identify proteins that interact with Mertk in an OS-dependent manner. RPE-J cells were transduced with recombinant adenovirus-expressing rat Mertk (Ad-Mertk) to ensure adequate Mertk expression and then challenged with OS or culture medium alone for the indicated times. Cell lysates were immunoprecipitated with an antibody directed against the C-terminal 100 amino acids of the cytoplasmic domain of Mertk. A 220-kDa protein and a 47-kDa protein associate at a high molar ratio with Mertk and were enriched in OS-treated samples (Fig. 1A) . These bands were excised and analyzed by mass spectrometry. The peptide fingerprints for the 220-kDa and 47-kDa proteins revealed high sequence similarity with the NMMII-A heavy chain Myh9 (accession number NP037326) and actin (accession number NP112406), respectively. 
To verify the interaction between NMMII-A and Mertk, RPE-J cells were challenged with OS, and protein lysates were immunoprecipitated as described and were analyzed by immunoblotting. Initially, NMMII-A constitutively associated with Mertk at low levels (Fig. 1B) . At 120 minutes after the addition of OS, but not at later time points, high levels of NMMII-A associated with Mertk in an OS-dependent manner. We confirmed these results by performing reciprocal immunoprecipitation in human ARPE-19 cells. Protein lysates from cells challenged with OS were immunoprecipitated with an anti–Myh9 antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-Mertk. Again, Mertk associated with NMMII-A in these samples, though with slower kinetics than in RPE-J (Fig. 1C) , consistent with our studies of the kinetics of OS phagocytosis in ARPE-19 cells (Strick and Vollrath, unpublished observations, 2007). In both immunoprecipitation experiments, the beads-only control was negative, demonstrating that the immunoprecipitation reactions were specific for antibody-bound protein complexes. These results show that NMMII-A and actin associate with the Mertk receptor during OS ingestion and suggest that Mertk promotes the assembly and activation of the actinomyosin complex necessary for OS phagocytosis by the retinal pigment epithelium. 
Redistribution of NMMII-A and NMMII-B in Response to OS Ingestion in RPE Cells
NMMII isoforms are hexamers composed of two heavy chains, two essential light chains, and two regulatory light chains, with the heavy chain composition conferring the identity of the isoform. 29 30 MYH9 and MYH10 genes encode the heavy chains for NMMII-A and NMMII-B, respectively. 31 32 33 To determine whether myosin II functions in OS phagocytosis, we assessed the localization of the NMMII-A and NMMII-B isoforms in the retinal pigment epithelium during OS ingestion. Cultured rat primary RPE cells were immunostained with antibodies directed against NMMII-A or NMMII-B and rhodopsin to visualize OS phagosomes. In cells challenged with medium alone, NMMII-A localized to the boundaries of confluent RPE cells (Fig. 2A [180 minutes (-) OS]), similar to the localization of F-actin and myosin I. 24 34 In cells challenged with medium plus OS, NMMII-A redistributed from lateral cell membranes to punctate structures resembling phagosomes localized within the cytoplasm (Fig. 2A) . After 60 minutes of incubation with OS, cells exhibited a mixture of cell boundary staining and positive puncta within the cytoplasm. After 120 minutes (data not shown) and 180 minutes (Fig. 2A) , NMMII-A was localized to cytoplasmic puncta, colocalizing with OS phagosomes labeled by rhodopsin. Similar results were observed when cells were stained for NMMII-B (Fig. 2B) , which also redistributed from the cell membrane to distinct puncta colocalizing with ingested OS phagosomes at 180 minutes These data demonstrate that NMMII-A and NMMII-B initially localize to the lateral membranes of RPE cells and redistribute to phagosomes during OS engulfment in cultured RPE cells. 
To assess the possibility of NMMII-A redistribution in vivo, we stained wholemounts of mouse retinal pigment epithelium/choroid/sclera and examined the localization of NMMII-A in areas with many or few phagosomes. Consistent with our cell culture experiments (Fig. 2) , in areas with few rhodopsin-positive phagosomes myosin II-A localized primarily to lateral cell membranes (Fig. 3 , upper panel), whereas in areas with abundant rhodopsin staining, NMMII-A was present primarily in puncta that colocalized with OS phagosomes (Fig. 3 , lower panel). These results indicate that our cell culture findings accurately replicated the RPE phagocytic process in vivo. 
Mertk-Dependent NMMII-A Redistribution from Lateral Cell Membranes
We next examined whether Mertk is required for NMMII redistribution in response to OS. We assessed NMMII-A localization by confocal microscopy on primary RPE cells cultured from RCS rats, which have a null mutation in Mertk. When RCS cells were cultured in medium lacking OS, NMMII-A localization to cell boundaries was prominent, a pattern similar to that observed in RPE cells that expressed functional Mertk (compare Fig. 2A[180 minutes (-) OS] and Fig. 4A [180 minutes (-) OS]). After 180 minutes of OS incubation with RCS RPE cells, the bulk (81%) of NMMII-A remained localized to the lateral cell membranes, whereas a small amount localized to punctate structures containing rhodopsin (Figs. 4A[middle], 4B). In contrast, significantly less (35%) NMMII-A localized to the cell boundaries in wild-type cells treated for 180 minutes with OS (Fig. 4B) . These data demonstrated that myosin II redistribution in response to OS was largely absent in cells that lacked a functional Mertk receptor. Furthermore, adding functional Mertk to RCS RPE cells through viral transduction restored the redistribution response of NMMII-A to OS challenge to that observed in wild-type cells (Figs. 4A[bottom], 4B). Thus, Mertk is required for the proper localization of NMMII-A to OS phagosomes during OS phagocytosis. 
Inhibition of OS Phagocytosis by Pharmacologic Inhibition and siRNA Knockdown of NMMII-A and NMMII-B
Colocalization of NMMII-A and NMMII-B to rhodopsin-positive phagosomes suggests a functional requirement for NMMII in RPE phagocytosis. To test this, we used pharmacologic inhibition of NMMII and siRNA knockdowns of the individual NMMII-A or NMMII-B heavy chains. Rat primary RPE cells were treated with blebbistatin, a small molecule that specifically inhibits myosin II ATPase activity, and were challenged with OS. The results clearly demonstrate that blebbistatin inhibited RPE phagocytosis of OS (Fig. 5A , Total). Blebbistatin-treated cells exhibited a 66% decrease in the amount of ingested phagosomes compared with control cells treated with DMSO (Fig. 5B) . Blebbistatin treatment also resulted in a modest increase (11%) in surface-bound OS (Fig. 5A[bound], 5B), suggesting that OS binds to the RPE cell surface but is not ingested when NMMII function is inhibited. 
Blebbistatin inhibits multiple NMMII isoforms, including skeletal myosin II, NMMII-A, and NMMII-B. 35 To determine whether NMMII-A and NMMII-B are both required for OS ingestion, we used siRNAs targeted to the NMMII-A or NMMII-B heavy chain. Rat primary RPE cells were cultured for 6 days and transfected with siRNAs targeted against rat NMMII-A or NMMII-B. To verify knockdown and specificity for a given myosin isoform, we immunostained siRNA-transfected cells for NMMII-A or NMMII-B. Cells transfected with NMMII-A siRNA showed a significant decrease in NMMII-A, but not NMMII-B, immunostaining (Fig. 6A) . Conversely, cells transfected with NMMII-B siRNA showed efficient knockdown of NMMII-B but not NMMII-A (Fig. 6A) , thus demonstrating the specificity of the siRNA duplex mixtures. Knockdown of NMMII-A and NMMII-B siRNA diminished phagocytosis by 61% and 39%, respectively, compared with cells treated with control siRNA (Fig. 6B) . Taken together, these results demonstrate that NMMII-A and NMMII-B function in the phagocytic trafficking of OS. 
Discussion
We have shown that Mertk functions by recruiting NMMII to the site of OS engulfment and that NMMII activity is required for the normal ingestion of OS by RPE cells. The timing of the association of Mertk with NMMII-A is consistent with a role in the ingestion phase of OS phagocytosis. Furthermore, NMMII is positioned to the correct subcellular location for phagocytosis, as evidenced by movement of a substantial fraction of NMMII from the cell periphery to sites of OS ingestion. RCS retinal pigment epithelium has a remarkable defect in the redistribution of NMMII-A, and this defect is rescued by the addition of Mertk to these cells, demonstrating NMMII-A localization to OS phagosomes is dependent on Mertk. Finally, inhibition of NMMII function by two distinct approaches attenuates the phagocytosis of OS, demonstrating the necessity of NMMII in this process. Our results link two key molecules whose functions are coordinated to achieve OS ingestion during RPE phagocytosis: a cell membrane receptor capable of transducing an external signal and a cytoplasmic molecular motor capable of effecting changes in cell shape. 
Further studies are needed to determine the specific nature of the association between Mertk and NMMII-A. Mertk may bind directly to NMMII-A because the NMMII-A heavy chain binds directly to the TRPM7 cation channel 36 and myosin RLC binds directly to the NMDA receptor. 37 Alternatively, Mertk may interact indirectly with NMMII-A and actin as part of a large multisubunit protein complex at the site of OS engulfment. The latter model is consistent with the disproportionate stoichiometry we observed in which a small molar amount of Mertk associates with a large amount of NMMII-A heavy chain and actin (Fig. 1A) . The Mertk/NMMII-A interaction we detected by immunoprecipitation also appeared to occur at a discrete time point (Figs. 1B 1C)that correlated with the peak of OS ingestion in two different RPE cell lines. Mertk is activated and functions specifically during the ingestion phase of RPE phagocytosis. 24 Thus, the activation of Mertk coincides with an increased interaction between the receptor and NMMII-A. Our data suggest that Mertk serves as a nexus for the assembly of a multiprotein ingestisome complex that promotes phagocytic engulfment of OS by the retinal pigment epithelium. 
NMMII-A and NMMII-B can exhibit distinct patterns of localization in cells 38 39 and distinct enzymatic activities in vitro. 40 41 42 However, NMMII-A can compensate for the loss of NMMII-B function in a specific tissue, 43 and NMMII-B and NMMII-C are expressed in tissues minimally affected by the loss of NMMII-A function in human MYH9 disease. 44 These results suggest that NMMII-A and NMMII-B (and perhaps NMMII-C) are functionally redundant in certain settings. Our observations that NMMII-A and NMMII-B redistribute from the cell periphery to sites of OS phagocytosis and that siRNA-mediated knockdown of either protein leads to partial inhibition of phagocytosis indicate that both isoforms function in RPE engulfment of OS. Consistent with this notion is the lack of reported retinal dystrophy in humans with MYH9 mutations, 45 but these phenotypic data are difficult to interpret because the mutations are likely hypomorphic and preserve some NMMII-A function. Although the degree to which NMMII-A and NMMII-B carry out overlapping or distinct tasks in RPE phagocytosis remains to be determined, to our knowledge this is the first report demonstrating a functional role for NMMII in RPE phagocytosis and the first time NMMII-B has been implicated in any phagocytic pathway. 
NMMII functions in multiple membrane reorganization pathways, such as cytokinesis, cell migration, and vesicle trafficking. 46 47 In intestinal epithelial cells, NMMII-A localizes to the perijunctional F-actin belt, where it regulates apical tight junction assembly and disassembly. 48 49 50 Our results suggest that NMMII-A and NMMII-B also localize to the perijunctional F-actin belt in cultured primary RPE cells not challenged with OS (Figs. 2A[180 minutes (-) OS] and 2B [180 minutes (-) O]). The mechanism by which Mertk marshals NMMII from the cell periphery to sites of OS ingestion is unknown. Recruitment of NMMII to the cleavage furrow during cytokinesis is dependent on the expression RhoGEF, Rho kinase, and RhoI 51 and on the activation of NMMII though phosphorylation of the RLC. 52 Mertk associates with rhodopsin-positive phagosomes and the kinetics 24 are similar to those we observed for NMMII (Fig. 2) . Although a physical interaction between Mertk and NMMII could affect NMMII activity and recruitment to the phagosome, Mertk may also alter NMMII function by activating one or more downstream signaling partners, including Vav, 53 PKC, 54 and FAK. 55 In either case, our results demonstrate that Mertk is required for the spatial localization of NMMII to the phagosome during OS engulfment. 
Myosin motors have multifaceted roles in the different stages of phagocytosis. For instance, myosins I and X function in phagocytic cup closure and extension of pseudopodia in macrophages, 56 57 whereas myosin VII functions in the postengulfment trafficking of phagosomes in the retinal pigment epithelium. 58 In macrophages, NMMII-A appears to be bifunctional, recruiting actin to the ingestion site in CR3-mediated phagocytosis 11 and extending and squeezing pseudopodia (phagocytic cup) around the particle in FcR-mediated phagocytosis. 59 Although there are obvious differences between highly motile macrophages and nonmotile polarized RPE cells, there may be mechanistic parallels in selected aspects of phagocytosis. In a manner similar to FcR-mediated phagocytosis, the retinal pigment epithelium extends a de novo pseudopodium that is independent of the RPE apical microvilli, and these pseudopodial membranes pinch off the degraded OS disc tip. 60 61 Although actin localizes to the RPE phagocytic cup in the absence of Mertk, 25 the loss of Mertk function severely disrupts proper NMMII localization. Furthermore, the inhibition of NMMII by blebbistatin significantly attenuates OS ingestion. Thus, analogous to FcR-mediated phagocytosis, NMMII may function in RPE phagocytosis by regulating the extension or closure of pseudopodia to form or close the phagocytic cup around ingested OS. 
In summary, our data demonstrate that Mertk alters the cytoskeleton during RPE phagocytosis of OS through the recruitment of NMMII. Future studies will help define the regulatory mechanisms governed by Mertk that are necessary for the redistribution of NMMII to the site of OS engulfment and will determine whether Mertk activates NMMII directly or indirectly. Elucidation of these mechanisms will further define the role of Mertk in plasticity of the cytoskeleton during RPE phagocytosis and will elucidate its role in other systems. 
 
Figure 1.
 
NMMII-A and actin associate with Mertk during OS ingestion. (A) RPE-J cell proteins were immunoprecipitated with anti-Mertk at various time points after the addition of OS. We observed a concomitant increase in 200-kDa (arrowhead) and 47.5-kDa (arrow) bands in OS-treated samples. These proteins were identified as Myh9 and actin, respectively. (B) RPE-J cells were treated as described, and the lysates were immunoprecipitated (IP) with anti-Mertk, fractionated by SDS-PAGE, and immunoblotted (IB) with the indicated antibodies. Equal amounts of total protein (8% of input) were fractionated, as demonstrated by the expression of Mertk, Myh9, and actin in all samples (bottom). (C) ARPE-19 cell lysates immunoprecipitated with anti–Myh9 (NMMII-A) were fractionated by SDS-PAGE and immunoblotted with the indicated antibodies (IB). Total denotes 8% of protein lysate before immunoprecipitation.
Figure 1.
 
NMMII-A and actin associate with Mertk during OS ingestion. (A) RPE-J cell proteins were immunoprecipitated with anti-Mertk at various time points after the addition of OS. We observed a concomitant increase in 200-kDa (arrowhead) and 47.5-kDa (arrow) bands in OS-treated samples. These proteins were identified as Myh9 and actin, respectively. (B) RPE-J cells were treated as described, and the lysates were immunoprecipitated (IP) with anti-Mertk, fractionated by SDS-PAGE, and immunoblotted (IB) with the indicated antibodies. Equal amounts of total protein (8% of input) were fractionated, as demonstrated by the expression of Mertk, Myh9, and actin in all samples (bottom). (C) ARPE-19 cell lysates immunoprecipitated with anti–Myh9 (NMMII-A) were fractionated by SDS-PAGE and immunoblotted with the indicated antibodies (IB). Total denotes 8% of protein lysate before immunoprecipitation.
Figure 2.
 
NMMII-A and NMMII-B colocalize with ingested OS. Primary RPE cells were isolated from 6-day-old Long Evans rats and were cultured for 6 days before incubation with OS or control medium. Preparations were immunostained with antibodies for NMMII-A (A) or NMMII-B (B) and rhodopsin to visualize phagosomes. Regions were selected based on the resemblance to retinal pigment epithelium in vivo (i.e., binucleate cuboidal cells) and imaged using confocal microscopy. Arrows denote selected areas of colocalization.
Figure 2.
 
NMMII-A and NMMII-B colocalize with ingested OS. Primary RPE cells were isolated from 6-day-old Long Evans rats and were cultured for 6 days before incubation with OS or control medium. Preparations were immunostained with antibodies for NMMII-A (A) or NMMII-B (B) and rhodopsin to visualize phagosomes. Regions were selected based on the resemblance to retinal pigment epithelium in vivo (i.e., binucleate cuboidal cells) and imaged using confocal microscopy. Arrows denote selected areas of colocalization.
Figure 3.
 
NMMII-A redistribution occurs in vivo. A C57BL/Ka mouse retinal pigment epithelium/choroid/sclera wholemount was stained with antibodies for NMMII-A and rhodopsin and was imaged by confocal microscopy. Areas were selected in the same wholemount for low (upper) and high (lower) numbers of rhodopsin-positive phagosomes. In cells with abundant phagosomes, NMMII-A colocalizes with rhodopsin (arrows), and little NMMII-A is present at cell boundaries. In cells with few phagosomes, NMMII-A remained at lateral cell boundaries (arrowheads). Examples of both types of cells are seen in the lower merged image. Red-appearing puncta in the upper merged image presumably indicate mature phagosomes in which NMMII-A is no longer present.
Figure 3.
 
NMMII-A redistribution occurs in vivo. A C57BL/Ka mouse retinal pigment epithelium/choroid/sclera wholemount was stained with antibodies for NMMII-A and rhodopsin and was imaged by confocal microscopy. Areas were selected in the same wholemount for low (upper) and high (lower) numbers of rhodopsin-positive phagosomes. In cells with abundant phagosomes, NMMII-A colocalizes with rhodopsin (arrows), and little NMMII-A is present at cell boundaries. In cells with few phagosomes, NMMII-A remained at lateral cell boundaries (arrowheads). Examples of both types of cells are seen in the lower merged image. Red-appearing puncta in the upper merged image presumably indicate mature phagosomes in which NMMII-A is no longer present.
Figure 4.
 
NMMII-A redistribution requires Mertk. (A) Primary RPE cells from RCS rats were isolated and cultured as in Figure 2 . Cells were treated with Ad-Mertk or no virus and were incubated with OS or control medium for 180 minutes and immunostained for the NMMII-A and rhodopsin to visualize phagosomes. Images were acquired as described for Figure 2 . Arrows highlight selected areas in which myosin II failed to redistribute in response to OS treatment. (B) Amounts of NMMII-A remaining on lateral cell membranes were measured and were graphed as arbitrary units ± SE. Statistical differences were measured using Student’s t-test. ***P < 0.0001. We observed significant differences between wild-type (Long-Evans) and RCS RPE cells in the amount of NMMII-A remaining on the lateral cell membranes 180 minutes after OS challenge.
Figure 4.
 
NMMII-A redistribution requires Mertk. (A) Primary RPE cells from RCS rats were isolated and cultured as in Figure 2 . Cells were treated with Ad-Mertk or no virus and were incubated with OS or control medium for 180 minutes and immunostained for the NMMII-A and rhodopsin to visualize phagosomes. Images were acquired as described for Figure 2 . Arrows highlight selected areas in which myosin II failed to redistribute in response to OS treatment. (B) Amounts of NMMII-A remaining on lateral cell membranes were measured and were graphed as arbitrary units ± SE. Statistical differences were measured using Student’s t-test. ***P < 0.0001. We observed significant differences between wild-type (Long-Evans) and RCS RPE cells in the amount of NMMII-A remaining on the lateral cell membranes 180 minutes after OS challenge.
Figure 5.
 
Blebbistatin treatment of RPE cells inhibits OS ingestion. Primary RPE cells from Long-Evans rats treated with blebbistatin or vehicle (DMSO) were challenged with OS and assayed for phagocytosis. (A) Images depict total and bound OS in blebbistatin and vehicle-treated cells from a representative phagocytic assay. There is an obvious difference between total and bound samples in the DMSO control (left), whereas no such difference is seen in blebbistatin-treated cells (right). (B) Quantification of three independent phagocytic assays of blebbistatin compared with vehicle-treated cells. Phagocytosis is graphed as a percentage of control ± SE. Percentage ingested is derived by subtracting bound from total. Statistical differences were measured with a Student’s t-test. *P < 0.05.
Figure 5.
 
Blebbistatin treatment of RPE cells inhibits OS ingestion. Primary RPE cells from Long-Evans rats treated with blebbistatin or vehicle (DMSO) were challenged with OS and assayed for phagocytosis. (A) Images depict total and bound OS in blebbistatin and vehicle-treated cells from a representative phagocytic assay. There is an obvious difference between total and bound samples in the DMSO control (left), whereas no such difference is seen in blebbistatin-treated cells (right). (B) Quantification of three independent phagocytic assays of blebbistatin compared with vehicle-treated cells. Phagocytosis is graphed as a percentage of control ± SE. Percentage ingested is derived by subtracting bound from total. Statistical differences were measured with a Student’s t-test. *P < 0.05.
Figure 6.
 
NMMII-A and NMMII-B siRNA inhibits OS phagocytosis by RPE cells. (A) Primary RPE cells from Long Evans rats were transfected with the NMMII-A, NMMII-B, and control siRNAs and were assayed for NMMII-A and NMMII-B knockdown with the use of immunofluorescence microscopy, which indicated that the NMMII-A and NMMII-B siRNAs were specific for their respective targets. Nuclei were stained using DAPI. (B) Cells transfected with NMMII-A, NMMII-B, or control siRNAs were assayed for phagocytic activity. Phagosome ingestion was quantified for three independent assays and graphed as a percentage of control ± SE. Statistical differences were measured with a Student’s t-test. **P < 0.01;***P < 0.001.
Figure 6.
 
NMMII-A and NMMII-B siRNA inhibits OS phagocytosis by RPE cells. (A) Primary RPE cells from Long Evans rats were transfected with the NMMII-A, NMMII-B, and control siRNAs and were assayed for NMMII-A and NMMII-B knockdown with the use of immunofluorescence microscopy, which indicated that the NMMII-A and NMMII-B siRNAs were specific for their respective targets. Nuclei were stained using DAPI. (B) Cells transfected with NMMII-A, NMMII-B, or control siRNAs were assayed for phagocytic activity. Phagosome ingestion was quantified for three independent assays and graphed as a percentage of control ± SE. Statistical differences were measured with a Student’s t-test. **P < 0.01;***P < 0.001.
The authors thank Aaron Straight for helpful advice with blebbistatin, Kitty Lee at the Stanford Cell Sciences Imaging Facility for confocal assistance, Andy Fire for advice concerning image quantification, members of the Vollrath laboratory for helpful discussions, and Dean Bok and Boris Stanzel for critically reading the manuscript. 
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Figure 1.
 
NMMII-A and actin associate with Mertk during OS ingestion. (A) RPE-J cell proteins were immunoprecipitated with anti-Mertk at various time points after the addition of OS. We observed a concomitant increase in 200-kDa (arrowhead) and 47.5-kDa (arrow) bands in OS-treated samples. These proteins were identified as Myh9 and actin, respectively. (B) RPE-J cells were treated as described, and the lysates were immunoprecipitated (IP) with anti-Mertk, fractionated by SDS-PAGE, and immunoblotted (IB) with the indicated antibodies. Equal amounts of total protein (8% of input) were fractionated, as demonstrated by the expression of Mertk, Myh9, and actin in all samples (bottom). (C) ARPE-19 cell lysates immunoprecipitated with anti–Myh9 (NMMII-A) were fractionated by SDS-PAGE and immunoblotted with the indicated antibodies (IB). Total denotes 8% of protein lysate before immunoprecipitation.
Figure 1.
 
NMMII-A and actin associate with Mertk during OS ingestion. (A) RPE-J cell proteins were immunoprecipitated with anti-Mertk at various time points after the addition of OS. We observed a concomitant increase in 200-kDa (arrowhead) and 47.5-kDa (arrow) bands in OS-treated samples. These proteins were identified as Myh9 and actin, respectively. (B) RPE-J cells were treated as described, and the lysates were immunoprecipitated (IP) with anti-Mertk, fractionated by SDS-PAGE, and immunoblotted (IB) with the indicated antibodies. Equal amounts of total protein (8% of input) were fractionated, as demonstrated by the expression of Mertk, Myh9, and actin in all samples (bottom). (C) ARPE-19 cell lysates immunoprecipitated with anti–Myh9 (NMMII-A) were fractionated by SDS-PAGE and immunoblotted with the indicated antibodies (IB). Total denotes 8% of protein lysate before immunoprecipitation.
Figure 2.
 
NMMII-A and NMMII-B colocalize with ingested OS. Primary RPE cells were isolated from 6-day-old Long Evans rats and were cultured for 6 days before incubation with OS or control medium. Preparations were immunostained with antibodies for NMMII-A (A) or NMMII-B (B) and rhodopsin to visualize phagosomes. Regions were selected based on the resemblance to retinal pigment epithelium in vivo (i.e., binucleate cuboidal cells) and imaged using confocal microscopy. Arrows denote selected areas of colocalization.
Figure 2.
 
NMMII-A and NMMII-B colocalize with ingested OS. Primary RPE cells were isolated from 6-day-old Long Evans rats and were cultured for 6 days before incubation with OS or control medium. Preparations were immunostained with antibodies for NMMII-A (A) or NMMII-B (B) and rhodopsin to visualize phagosomes. Regions were selected based on the resemblance to retinal pigment epithelium in vivo (i.e., binucleate cuboidal cells) and imaged using confocal microscopy. Arrows denote selected areas of colocalization.
Figure 3.
 
NMMII-A redistribution occurs in vivo. A C57BL/Ka mouse retinal pigment epithelium/choroid/sclera wholemount was stained with antibodies for NMMII-A and rhodopsin and was imaged by confocal microscopy. Areas were selected in the same wholemount for low (upper) and high (lower) numbers of rhodopsin-positive phagosomes. In cells with abundant phagosomes, NMMII-A colocalizes with rhodopsin (arrows), and little NMMII-A is present at cell boundaries. In cells with few phagosomes, NMMII-A remained at lateral cell boundaries (arrowheads). Examples of both types of cells are seen in the lower merged image. Red-appearing puncta in the upper merged image presumably indicate mature phagosomes in which NMMII-A is no longer present.
Figure 3.
 
NMMII-A redistribution occurs in vivo. A C57BL/Ka mouse retinal pigment epithelium/choroid/sclera wholemount was stained with antibodies for NMMII-A and rhodopsin and was imaged by confocal microscopy. Areas were selected in the same wholemount for low (upper) and high (lower) numbers of rhodopsin-positive phagosomes. In cells with abundant phagosomes, NMMII-A colocalizes with rhodopsin (arrows), and little NMMII-A is present at cell boundaries. In cells with few phagosomes, NMMII-A remained at lateral cell boundaries (arrowheads). Examples of both types of cells are seen in the lower merged image. Red-appearing puncta in the upper merged image presumably indicate mature phagosomes in which NMMII-A is no longer present.
Figure 4.
 
NMMII-A redistribution requires Mertk. (A) Primary RPE cells from RCS rats were isolated and cultured as in Figure 2 . Cells were treated with Ad-Mertk or no virus and were incubated with OS or control medium for 180 minutes and immunostained for the NMMII-A and rhodopsin to visualize phagosomes. Images were acquired as described for Figure 2 . Arrows highlight selected areas in which myosin II failed to redistribute in response to OS treatment. (B) Amounts of NMMII-A remaining on lateral cell membranes were measured and were graphed as arbitrary units ± SE. Statistical differences were measured using Student’s t-test. ***P < 0.0001. We observed significant differences between wild-type (Long-Evans) and RCS RPE cells in the amount of NMMII-A remaining on the lateral cell membranes 180 minutes after OS challenge.
Figure 4.
 
NMMII-A redistribution requires Mertk. (A) Primary RPE cells from RCS rats were isolated and cultured as in Figure 2 . Cells were treated with Ad-Mertk or no virus and were incubated with OS or control medium for 180 minutes and immunostained for the NMMII-A and rhodopsin to visualize phagosomes. Images were acquired as described for Figure 2 . Arrows highlight selected areas in which myosin II failed to redistribute in response to OS treatment. (B) Amounts of NMMII-A remaining on lateral cell membranes were measured and were graphed as arbitrary units ± SE. Statistical differences were measured using Student’s t-test. ***P < 0.0001. We observed significant differences between wild-type (Long-Evans) and RCS RPE cells in the amount of NMMII-A remaining on the lateral cell membranes 180 minutes after OS challenge.
Figure 5.
 
Blebbistatin treatment of RPE cells inhibits OS ingestion. Primary RPE cells from Long-Evans rats treated with blebbistatin or vehicle (DMSO) were challenged with OS and assayed for phagocytosis. (A) Images depict total and bound OS in blebbistatin and vehicle-treated cells from a representative phagocytic assay. There is an obvious difference between total and bound samples in the DMSO control (left), whereas no such difference is seen in blebbistatin-treated cells (right). (B) Quantification of three independent phagocytic assays of blebbistatin compared with vehicle-treated cells. Phagocytosis is graphed as a percentage of control ± SE. Percentage ingested is derived by subtracting bound from total. Statistical differences were measured with a Student’s t-test. *P < 0.05.
Figure 5.
 
Blebbistatin treatment of RPE cells inhibits OS ingestion. Primary RPE cells from Long-Evans rats treated with blebbistatin or vehicle (DMSO) were challenged with OS and assayed for phagocytosis. (A) Images depict total and bound OS in blebbistatin and vehicle-treated cells from a representative phagocytic assay. There is an obvious difference between total and bound samples in the DMSO control (left), whereas no such difference is seen in blebbistatin-treated cells (right). (B) Quantification of three independent phagocytic assays of blebbistatin compared with vehicle-treated cells. Phagocytosis is graphed as a percentage of control ± SE. Percentage ingested is derived by subtracting bound from total. Statistical differences were measured with a Student’s t-test. *P < 0.05.
Figure 6.
 
NMMII-A and NMMII-B siRNA inhibits OS phagocytosis by RPE cells. (A) Primary RPE cells from Long Evans rats were transfected with the NMMII-A, NMMII-B, and control siRNAs and were assayed for NMMII-A and NMMII-B knockdown with the use of immunofluorescence microscopy, which indicated that the NMMII-A and NMMII-B siRNAs were specific for their respective targets. Nuclei were stained using DAPI. (B) Cells transfected with NMMII-A, NMMII-B, or control siRNAs were assayed for phagocytic activity. Phagosome ingestion was quantified for three independent assays and graphed as a percentage of control ± SE. Statistical differences were measured with a Student’s t-test. **P < 0.01;***P < 0.001.
Figure 6.
 
NMMII-A and NMMII-B siRNA inhibits OS phagocytosis by RPE cells. (A) Primary RPE cells from Long Evans rats were transfected with the NMMII-A, NMMII-B, and control siRNAs and were assayed for NMMII-A and NMMII-B knockdown with the use of immunofluorescence microscopy, which indicated that the NMMII-A and NMMII-B siRNAs were specific for their respective targets. Nuclei were stained using DAPI. (B) Cells transfected with NMMII-A, NMMII-B, or control siRNAs were assayed for phagocytic activity. Phagosome ingestion was quantified for three independent assays and graphed as a percentage of control ± SE. Statistical differences were measured with a Student’s t-test. **P < 0.01;***P < 0.001.
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