We have studied the distribution of talin in J774 cells and mouse peritoneal macrophages undergoing Fc receptor-mediated phagocytosis. At early stages of phagocytosis, talin accumulates in the cells' cortical cytoplasm adjacent to the forming phagosome and extends into pseudopods that are encircling the particle. Talin colocalizes with F-actin at these sites. After particle ingestion is completed, F-actin and talin are no longer concentrated adjacent to phagosomes. Thus, talin and F-actin undergo dynamic and coordinate changes in their cytoplasmic location during Fc receptor-mediated phagocytosis

Burridge, Keith

Greenberg, Steven

Silverstein, Samuel C.

English

S Greenberg

K Burridge

S C Silverstein

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Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages.

Columbia University Academic Commons

BriefDefinitive ReportColocalization of F-actin and Talin duringFc Receptor-mediated Phagocytosis inMouse MacrophagesBy Steven Greenberg'*f Keith Burridge,lland Samuel C. Silverstein*SFrom the "Rover Laboratory of Cellular Physiology, and the Departments of Wddccine andSPhysiology and Cellular Biophysics, College ofPhysicians and Surgeons, Columbia University,New York, New York 10032; and the Il Department of Cell Biology and Anatomy, UniversityofNorth Carolina, Chapel Hill, North Carolina 27599SummaryWe have studied the distribution oftalin inJ774 cells and mouse peritoneal macrophages undergoingFc receptor-mediated phagocytosis . At early stages ofphagocytosis, talin accumulates in the cells'cortical cytoplasm adjacent to the forming phagosome and extends into pseudopods that areencircling the particle. Talin colocalizes with F-actin at these sites. After particle ingestion iscompleted, F-actin and talin are no longer concentrated adjacent to phagosomes . Thus, talinand F-actin undergo dynamic and coordinate changes in their cytoplasmic location during Fcreceptor-mediated phagocytosis.Engulfment of ligand-coated particles by leukocytes re-quires communication between ligand-activated plasmamembrane receptors and the actin-based cytoskeleton . For Fcreceptor-mediated phagocytosis, the precise signals thataccomplish this are unknown . F-actin is recruited to the regionof cell membrane in contact with the particle during en-gulfinent (1, 2), and cytochalasins block both F-actin assembly(3) and particle engulfment (4, 5) . These data suggest thatF-actin assembly plays a major role in phagocytosis. Whetherthe newly assembled F-actin interacts directly with thesegments of plasma membrane beneath the forming phago-some is unknown . One system that has been used to explorethe molecular basis of interactions between membrane proteinsand the actin-based cytoskeleton is the fibroblast adhesionplaque (6) . When fibroblasts settle onto surfaces coated withextracellular matrix proteins (e.g ., fibronectin), they bind tothese surfaces via specific transmembrane receptor proteinscalled integrins. Integrins provide transmembrane linkagesbetween extracellular matrix proteins and the cytoskeleton(7) . Talin is a 225-kD cytosolic protein that is recruited tosites where integrins bind to the cytoskeleton (6) . To testwhether talin may serve a similar role during Fc receptor-mediated phagocytosis, we studied its distribution in macro-phages ingesting IgG-coated particles. We report here thattalin colocalizes with F-actin in the submembranous areabeneath forming phagosomes in macrophages undergoing Fcreceptor-mediated phagocytosis .Materials and MethodsCells and Reagents.	J774 cells were maintained in spinner cul-ture in DME supplemented with 10% bovine serum, 100 U/mlpenicillin, and 100 ug/ml streptomycin . Thioglycollate-elicitedmouse peritoneal macrophages were harvested 4 d after the in-traperitoneal injection of1 ml ofthioglycollate broth (Difco Labora-tories, Detroit, MI) . Bodipy-phallacidin was from Molecular Probes(Eugene, OR); fluorescein-conjugated goat anti-rabbit IgG andTexas red--conjugated F(ab')2 goat anti-rabbit IgG was from Jack-son Immunoresearch (West Grove, PA) ; alkaline phosphatase-conjugated goat anti-rabbit IgG was from Promega Biotec (Mad-ison, WI) ; and anti-sheep IgG was from Diamedix (Miami, FL) .Immunoblotting. 1.3 x 106 J774 cells were suspended in Hepes-buffered saline in the presence of 1 mM EDTA, 0.5 mM PMSF,and 5 mM diisopropylfluorophosphate. Sample buffer (8) was added,the mixture was boiled, the cell extract was subjected to SDS-PAGEusing a 7.5% polyacrylamide gel, and the gels were blotted ontonitrocellulose and incubated in saline containing 150 mM NaCl,10% goat serum, 0.25% gelatin, 0.2% NP-40, 20 mM Tris-HCL,pH 7.2 (TBS), and further incubated with rabbit anti-talin an-tiserum (1:2,000 in the above buffer), washed in TBS, incubatedwith alkaline phosphatase-conjugated goat anti-rabbit IgG (1:2,500in TBS), and developed with nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.Phagocytosis. For microinjection studies SRBC were opsonizedwith rabbit anti-SRBC IgG. Forconventional immunofluorescencestudies, SRBC were opsonized using U188, a mouse mAb of theIgG2b subclass directed against SRBC, kindly provided by BettyDiamond (Albert Einstein College ofMedicine, New York) . Phago-1853 J . Exp. Med . © The Rockefeller University Press - 0022-1007/90/12/1853/04 $2.00Volume 172 December 1990 1853-1856 on January 12, 2016jem.rupress.orgDownloaded from Published December 1, 1990cytosis was synchronized by pre-incubating a monolayer of macro-phages with opsonized SRBC (IgG-SRBC) in saline containing125 mM NaCl, 5 mM KCI, 10 mM NaHCO3, 1 mM KH2PO4 ,10 mM glucose, 1 mM CaC12, 1 MM MgC12, 20 mM Hepes pH7.45 (HBS) at 0°C for 10 min . The monolayer was washed in FIBSthree times at 0°C, and incubated at 37°C for the times indicated .Cells were fixed in 3.7% formaldehyde in HBS for 10 min andprocessed for immunofluorescence .Immunofluorescence.	The cells were permeabilized in 0.2% TritonX-100 in TBS for 8 min, washed in TBS, and incubated with ei-ther rabbit antiserum directed against mammalian talin or serumfrom an unimmunized rabbit at a 1:100 dilution for 30 min, washedin TBS and incubated with Texas red-F(ab')2 goat anti-rabbit IgGdiluted 1:50 in TBS for 30 min . The cells were then counterstainedwith bodipy-phallacidin, diluted 1 :10 in TBS for 15 min at 37°C,mounted, and observed by phase contrast and fluorescence micros-copy (Photomicroscope III ; Carl Zeiss, Inc ., Thornwood, NY). Thecells were photographed with Tri-X developed at 800 ASA . Con-trol serum showed virtually no staining .Microinjection ofa 19041) Talin Fragment. Adherent J774 cellswere microinjected with an iodoacetamidotetramethylrhodamine-derivatized 190-kD talin fragment (IATR-talin) (9, 10) as describedby Graessmann and Graessmann (11), allowed to recover for 4 h,and incubated with IgG-SRBC as described above . The cells werefixed in 3.7% formaldehyde in PBS, permeabilized as describedabove, and stained with bodipy-phallcidin diluted 1 :10 in PBS for15 min at 37°C . The cells were photographed as described above .Results and DiscussionWe determined that talin is present in J774 cells by Westernblot analysis of an extract of these cells. The antiserum wasprepared by immunization of rabbits with human platelettalin (12) . This antiserum stained a 225-kD protein (Fig. 1),which is the expected size of talin based on the apparent mo-lecular mass of talin from other mammalian cells (6) . Theadditional bands of slightly lower molecular mass probablyrepresent proteolytic fragments of the native molecule . Serumfrom an unimmunized rabbit did not stain the 225-kD pro-tein or the lower molecular weight fragments (not shown) .At 0°C, the IgG-SRBC attached to the plasma membranesof J774 cells but were not phagocytosed (13), and did notFigure 1 . Immunoblot ofJ774 cell extract stainedwith rabbit antitalin antiserum . Positions ofmolec-ular mass markers in kilodaltons are listed .1854 Colocalization of F-actin and Talin during Phagocytosisstimulate the accumulation of F-actin (not shown) or talin(Fig. 2, arrowheads) in the cytoplasm adjacent to these parti-cles. In contrast, 1.5 min after warming theJ774 cells to 37°C,both F-actin and talin were concentrated beneath formingphagosomes (Fig . 3) . Although talin also was identified else-where in the cytoplasm, the greater intensity of staining be-neath membranes offorming phagosomes suggests that talinwas concentrated there. There was excellent correspondencebetween the cytoplasmic location ofF-actin and of talin . Thus,one of the early events in phagosome formation (Fig . 3) in-volves the recruitment of talin and F-actin to the corticalcytoplasm beneath the nascent phagosome . At a slightly laterstage in particle engulfment (5 min), both F-actin and talinwere still apparent beneath incompletely engulfed IgG-SRBC(Fig . 4, arrowhead) . Talin and F-actin largely disappeared fromthe portions of cytoplasm adjacent to IgG-SRBC that werecompletely (Fig. 4, small arrowhead) or nearly completely (Fig.4, arrow) ingested . To confirm the effect of IgG-SRBC en-gulfment on talin localization, we microinjected J774 cellswith a tluorescently labeled 190-kD fragment of talin (IATRtalin) . Previous work showed that this talin fragment, likenative talin, localizes to focal adhesion sites in fibroblasts (10) .In unstimulated J774 cells, the 190-kD talin fragment wasdistributed in the cytosol and in membrane ruffles (not shown) .In J774 cells ingesting IgG-SRBC, the 190-kD fragment oftalin was concentrated in portions of the cortical cytoplasmdirectly beneath forming phagosomes, in pseudopods ad-vancing around the particles (Fig . 5, arrowheads), and in mem-brane ruffles. Analogous to conventional immunofluorescence(Figs . 2-4), there was excellent correspondence between thedistribution of IATRtalin and F-actin in the cortical cytoplasmof J774 cells engulfing IgG-SRBC .We obtained similar results in thio-macrophages. 1 .5 minafter the onset of ingestion at 37°C, talin (Fig. 5) and F-actin(not shown) localized beneath forming phagosomes. At latertime points, neither talin nor F-actin were apparent adjacentto fully ingested particles .The recruitment of talin during Fc receptor-mediatedphagocytosis is surprising in two respects . First, althoughprevious studies have documented the presence of talin at sitesFigure 2 . Immunofluorescent staining of IgG-RBC and talin in J774cells incubated with IgG-RBC for 10 min at 0°C . Note the absence oftalin localization adjacent to IgG-RBC (arrowheads) . (A) Fluorescein-conjugated anti-rabbit IgG (which stains IgG-RBC) ; (B) talin im-munofluorescence . Bar = 10 lAm . on January 12, 2016jem.rupress.orgDownloaded from Published December 1, 1990ofcontact ofmononuclear cells with their substrates, the talinappeared at these sites only after many hours of adherence(14) . In contrast, particle engulfment is a transient and dy-namic process. Second, talin binds to the cytoplasmic domainsofintegrins (15), at sites where stress fibers insert (6) . In con-Figure 6.	Fluorescent staining of talin beneath forming phagosomesin thio-macrophages undergoing phagorytosis for 1 .5 min . (A) Phase con-trast ; (B) talin immunofluorescence. Bar - 10 Etm .1855 Greenberg et al. Brief Definitive ReportFigure 3 . Fluorescent staining of F-actinand talin inJ774 cells undergoing phago-cytosis for 1.5 min . Note the colocaliza-tion of F-actin and talin staining adjacentto nascent phagosomes (small arrowhead) .Although some cytosolic staining is evi-dent, one phagosome (large arrowhead)shows a concentration of talin, while thesurrounding cytosol has almost no detect-able talin . (A) Phase contrast ; (B) talin im-munofluorescence; (C) F-actin fluorescence.Bar - 10 Wm .Figure 4 . Fluorescent staining of talinand F-actin inJ774 cells undergoing phago-cytosis for 5 min. Note that talin and F-actinare not visible surrounding particles thathave been completelyengulfed (small arrou,head), but are associated withparticles thatare being engulfed (large arrowhead) . F-actinand talin are visible only at the distal tipof a pseudopod encircling one particle thatis nearly completely ingested (arrow). (A)Phase contrast; (B) talin immunofluo-rescence ; (C) F-actin fluorescence. Bar =10 ,am .Figure 5 . Localization of F-actin andmicroinjected IATR-talin inJ774 cells un-dergoing phagocytosis for 1 .5 min . Notethe colocalization of F-actin and the fluores-cent talin fragment in pseudopods (arrow-heads) and membrane ruffles . (A) Phase con-trast ; (B) fluorescence of IATR-talin ; (C)F-actin fluorescence. Bar = 10 pm .trast, macrophages exhibit few, ifany, stress fibers . The pres-ence of talin in the cortical cytoplasm adjacent to formingphagosomes suggests that it may have a role in connectingthe microfilament-based cytoskeleton to the plasma membraneduring phagocytosis .Talin redistribution has been demonstrated in helper andcytotoxic T cells during contact with their respective targets(16, 17), as well as during activation ofplatelets by thrombin(18) . Talin also has been reported to localize to the cortexof transformed fibroblasts as they engulf fibronectin-coatedbeads (19) . These results are not totally unexpected, since ineach of the instances cited integrins play a central role . How-ever, Fc receptors bear no sequence homology to the knownintegrins (20) . Whether integrins participate as cofactors inFc receptor-mediated phagocytosis, as has been suggested(21-23), remains to be clarified. In any case, the identificationof talin in the cortex of macrophages during Fc receptor-mediated phagocytosis suggests a new function for talin, andraises the possibility that other talin-associated proteins (e.g.,o-actinin) participate in the engulfment process as well . on January 12, 2016jem.rupress.orgDownloaded from Published December 1, 1990We thank Glen Nuckolls and Andrew Jackson for their generous help and critical reading of the manu-script, and Betty Diamond for providing mAb U188 .This work was supported by Public Health Service grants AI-20516 to S. C. Silverstein, and GM-29860to K. Burridge . S. Greenberg was supported by a fellowship from the Parker B. Francis Foundation forPulmonary Research .Address correspondence to Steven Greenberg, Department of Physiology/BB 1111, Columbia University,630 West 168th St ., New York, NY 10032.Received for publication 13 July 1990 and in revised form 20 September 1990.Biol. Chem . 264:11938.13 . Silverstein, S.C ., R.M . Steinman, and Z.A. Cohn . 1977 . En-1856 Colocalization of F-actin and Talin during Phagocytosisdocytosis. Annu . Reu Biochem . 46:669 .Marchisio, P.C ., D. Chrillo, A. Teti, A. Zambonin-Zallone,andG. Tarone . 1987. Rous sarcoma virus-transformed fibro-blasts and cells ofmonocytic origin display a peculiar dot-likeorganization ofcytoskeletal proteins involved in microfilament-membrane interactions. Exp Cell Res. 169:202 .Tapley, P., A. Horwitz, C. Buck, K. Duggan, andL. Rohr-schneider. 1989 . Integrins isolated from Rous sarcoma virus-transfected chicken embryo fibroblasts. Oncogene 4:325 .Kupfer, A., S.J . Singer, andG. Dennert. 1986 . On the mecha-nism of unidirectional killing in mixtures of two cytotoxic Tlymphocytes. J. Exp Med. 163:489 .Kupfer, A., S.L . Swain, and S.J . Singer. 1987 . The specific di-rect interaction ofhelper T cells and antigen-presenting B cells.J. Exp Med. 165:1565.Beckerle,M.C ., D.E . Miller, M.E. Bertagnolli, and S.J . Locke.1989 . Activation-dependent redistribution of the adhesionplaque protein, talin, in intact human platelets . J. Cell Biol.109:3333.Mueller, S.C ., T Kelly, M. Dai, H. Dai, and WenTien Chen .1989 . Dynamic cytoskeleton-integrin associations induced bycell binding to immobilized fibronectin .J. Cell Biol. 109:3455.Ravetch, JV, and C.L . Anderson. 1990 . Fc,R family : pro-teins, transcripts and genes. In Fc Receptors and the Actionof Antibodies . H. Metzger, editor. American Society forMicrobiology, Washington, DC. 211-238.Arnaout, M.A., R.F. Todd III, N. Dana, J. Melamed, S.F.Schlossman, and H.R . Colten . 1983 . Inhibition of phagocy-tosis of complement C3- or immunoglobulin G-coated parti-cles and of C3bi binding by monoclonal antibodies to amonocyte-granulocyte membrane glycoprotein (Mo 1).J. Clin .Invest. 72:171 .Brown, E.J ., J.F. Bohnsack, andH.D. Gresham. 1988 . Mech-anismof inhibition ofimmunoglobulin Gmediated phagocy-tosis by monoclonal antibodies that recognize the Mac-1 an-tigen . J. Clin. Invest. 81:365 .Graham, I.L., H.D. Gresham, and Eric J. Brown. 1989 . Animmobile subset of plasma membrane CD11b/CD18 (Mac-1)is involved in phagocytosis of targets recognized by multiplereceptors. J. Immunol. 142:2352.References1. Boxer, L.A ., E. Tessa HedleyWhyte, and T.P. Stossel. 1974 .Neutrophil actindysfunction and abnormal neutrophil behavior. 14 .N. Engl. J. Med. 291:1093.2. Wang, E., J. Michl, L.M . Pfeffer, and S.C. Silverstein. 1984.Interferon suppresses pinocytosis but stimulates phagocytosisin mouse peritoneal macrophages : related changes in cytoskeletalorganization . J. Cell Biol. 98:1328. 15 .3. Greenberg, S., F. Di Virgilio, J. El-Khoury and S.C . Silver-stein. 1988 . F-actin dynamics in phagocytosing macrophages.J. Cell Biol. 107:453a. 16 .4. Zigmond, S.H ., and J.G. Hirsch . 1972 . Effects of cytochalasinBon polymorphonuclear leucocyte locomotion, phagocytosisand glycolysis . Exp Cell Res. 73:383 . 17 .5. Axline, S.G., and E.P. Reaven. 1974 . Inhibition of phagocy-tosis and plasma membrane mobility of the cultivated macro-phage by cytochalasin B. J. Cell Biol. 62:647 . 18 .6. Burridge, K., K. Fath, T Kelly, G. Nuckolls, and C. Turner.1988 . Focal adhesions: transmembranejunctions between theextracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol.4:487 . 19 .7. Tamkun, J.W., D.W. DeSimone,D. Fonda, R.S. Patel, C. Buck,A.F. Horwitz, andR.O . Hynes. 1986 . Structure of integrin,a glycoprotein involved in the transmembrane linkagebetween 20 .fibronectin and actin. Cell. 46:271 .8. Laemmli, U.K . 1970 . Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4 . Nature (Lond.).2237:680 . 21 .9. Hock,R.S ., J.M. Sanger, andJ.W. Sanger. 1989 . Talin dynamicsin living microinjected nonmuscle cells . Cell Motil. Cytolskeleton .14:271 .10 . Nuckolls, G.H., C.E . Turner, and K. Burridge. 1990 . Func-tional studies of the domains of talin. J. Cell Biol. 110:1635.11 . Graessmann, M., andA. Graessmann.1976. Early simian virus 22 .40-specific RNA contains information for tumor antigen for-mation and chromatin replication . Proc Natl. Acad. Sci. USA .73:366 .12 . Turner, C.E ., F.M. Pavalko, andK. Burridge. 1989 . The role 23 .of phosphorylation and limited proteolytic cleavage of talinand vinculin in the disruvtion of focal adhesion inteeTit . J . on January 12, 2016jem.rupress.orgDownloaded from Published December 1, 1990

Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages

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Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages

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