NENCKI INSTITUTE OF EXPERIMENTAL BIOLOGY VOLUME 37 NUMBER 4 WARSAWhttp://rcin.org.pl, POLAND 1998 ISSN 0065-1583 Polish Academy of Sciences Nencki Institute of Experimental Biology and Polish Society of Cell Biology ACTA PROTOZOOLOGICA International Journal on Protistology Editor in Chief Jerzy SIKORA Editors Hanna FABCZAK and Anna WASIK Managing Editor Małgorzata WORONOWICZ Editorial Board Andre ADOUTTE, Paris J. I. Ronny LARSSON, Lund Christian F. BARDELE, Tübingen John J. LEE, New York Magdolna Cs. BERECZKY, Göd Jiri LOM, Ćeske Budejovice Y.-Z. CHEN, Beijing Pierangelo LUPORINI, Camerino Jean COHEN, Gif-Sur-Yvette Hans MACHEMER, Bochum John O. COREISS, Albuquerque Jean-Pierre MIGNOT, Aubiere Gyorgy CSABA, Budapest Yutaka NAITOH, Tsukuba Isabelle DESPORTES-LIVAGE, Paris Jytte R. NILSSON, Copenhagen Tom FENCHEL, Helsingor Eduardo ORIAS, Santa Barbara Wilhelm FOISSNER, Salsburg , Dimitrii V. OS SIPO V, St. Petersburg Vassil GOLEMANSKY, Sofia Igor B. RAIKOV, St. Petersburg Andrzej GRĘBECKI, Warszawa, Vice-Chairman Leif RASMUSSEN, Odense Lucyna GRĘBECKA, Warszawa Michael SLEIGH, Southampton Donat-Peter HÄDER, Erlangen Ksenia M. SUKHANOVA, St. Petersburg Janina KACZANOWSKA, Warszawa Jiri VÄVRA, Praha Stanisław L. KAZUBSKI, Warszawa Patricia L. WALNE, Knoxville Leszek KUZNICKI, Warszawa, Chairman ACTA PROTOZOOLOGICA appears quarterly. The price (including Air Mail postage) of subscription to ACTA PROTOZOOLOGICA at 1999 is: US $ 180,- by institutions and US $ 120.- by individual subscribers. Limited number of back volumes at reduced rate are available. TERMS OF PAYMENT: Cheque, money oder or payment to be made to the Nencki Institute of Experimental Biology. Account Number: 11101053-3522-2700-1-34 at Państwowy Bank Kredytowy XIII Oddz. Warszawa, Poland. WITH NOTE: ACTA PROTOZOOLOGICA! For matters regarding ACTA PROTOZOOLOGICA, contact Managing Editor, Nencki Institute of Experimental Biology, ul. Pasteura 3, 02-093 Warszawa, Poland; Fax: (4822)' 8225342; E-mail: [email protected] (for more information see web page http://www.nencki.gov.pl/public.htm). Front cover: Polychaos dubium. In: A. A. Schaeffer (1916) Notes on the specific and other characters of Amoeba proteus Pallas (Leidy), A. discoides spec, no v. and A. dubia spec. nov. Arch. Protistenk. 37: 204 - 228 ©Nencki Institute of Experimental Biology Desktop processing: Justyna Osmulska, Data Processing Polish Academy of Sciences Laboratory of the Nencki Institute This publication is supported by the State Committee for Printed at the MARBIS, ul. Kombatantów 60, Scientific Research 05-070 Sulejówek, Poland http://rcin.org.pl ACTA Acta Protozoologica (1998) 37: 191-199 PROTOZOOLOGICA Participation of Myosin I, Spectrin Analogue and Tyrosine-Phosphory- lated Proteins at Early Stages of Phagocytosis in Acanthamoeba castellanii Katarzyna KWIATKOWSKAand Andrzej SOBOTA Department of Cell Biology, Nencki Institute of Experimental Biology, Warszawa, Poland Summary. Uptake of particles during phagocytosis is driven by the actin-based cytoskeleton. We analyzed the localization of actin filaments, myosin I and II, spectrin analogue and phosphotyrosine-bearing proteins at various stages of yeast phagocytosis in Acanthamoeba in order to examine an involvement of these proteins in alterations of the cytoskeleton. Concentration of microfilaments at forming phagosomes was accompanied by accumulation of the spectrin-like protein and myosin I, but not myosin II. During phagosome maturation, these proteins were no longer detectable in the periphagosomal area. The onset of the phagocytosis was correlated with an intense and transient tyrosine- phosphorylation of 130, 120 and 60 kDa polypeptides which were also enriched at the sites of phagosome formation and disappeared at later stages of particle digestion. Inhibition of protein tyrosine kinase activity by genistein blocked particle uptake indicating the regulatory function played by the phosphorylated proteins in the process. Key words: myosin I, phagocytosis, spectrin, tyrosine phosphorylation. INTRODUCTION It has been established that the phagocytic abilities of the "professional" phagocytes are determined by the expres- Phagocytosis is an actin-dependent process of inter- sion of specific plasma membrane receptors, which bind nalization of large particles, >0.5 |um in diameter, by cells. various ligands localized on the surface of the particles to It is a common phenomenon in Eukaryota, although in be internalized. Among the receptors that promote phago- higher organisms the ability of efficient particle uptake is cytosis are: mannose receptor, complement receptor C3 attributed to specialized cells, so-called "professional" and Fey receptors (for review see: Brown 1994, phagocytes, to which monocytes, neutrophils and mac- Kwiatkowska and Sobota 1999). Clustering of the recep- rophages belong (Rabinovitch 1995). These cells remove tors is thought to occur upon ligand binding which triggers microorganisms, pollution debris and senescent cells of signal transduction events leading eventually to a local the host, as a key element of the immune defense system. polymerization of actin (Griffin et al. 1975, Sheterline etal. 1984, Detmers etal. 1987, Greenberg et al. 1991). Microscopic studies revealed that the actin-based cytosk- Address for correspondence: Andrzej Sobota, Department of eleton provides scaffolding for pseudopods which em- Cell Biology, Nencki Institute of Experimental Biology, ul. Pasteura 3, 02-093 Warszawa, Poland; Fax: (4822) 8225342; E-mail: brace particles, firstly in phagocytic cups and, after [email protected] closure, in nascent phagosomes (Axline and Reaven 1974, http://rcin.org.pl 192 K. Kwiatkowska and A. Sobota Greenberg et al. 1990, Baines et al. 1992, Maniak et al. 4 x 107ml density and grown overnight as described earlier (Kwiatkowska 1995, Allen and Aderem 1996). Direct implication of the and Sobota 1997). After rinsing with 100 mM NaCl, 2 mM MgCl,, active engagement of several actin-binding proteins like 20 mM Hepes, pH 7.0 (NaCl/MgCl2/Hepes medium) the cells were chilled on ice and exposed to lipid-extracted bakeryeast applied at ratio talin, coronin, ABP-120, myosin IB and IC in particle of about 20 yeast per amoeba in the ice-cold buffer. To promote the uptake came from the examination of phagocytosis- binding of the yeast particles to the cells, the incubation proceeded for defective mutants of Dictyosteliuin discoideum which, as 10 min at 0°C after which the excess of particles was washed out. a haploid organism, provides a useful model system for Subsequently, the cells were warmed to 28°C for 3 min to induce the gene targeting and anti-sense RN A studies (Maniak etal. uptake of the bound yeast. The process was terminated by fixation of the cells with 3% formaldehyde for 30 min. 1995, Cox et al. 1996, Jung et al. 1996, Niewohner In experiments using suspended cells, 5-day-old cultures were et al. 1997). shifted to NaCl/MgCl/Hepes medium (3 x 106 cells in 0.5 ml per sample) The last decade has witnessed extensive attempts to and supplemented with yeast-derived zymosan A particles (5 mg in elucidate the signaling pathways leading from ligand- 0.1 ml of medium per sample). Phagocytosis was carried out at room receptor interactions to actin rearrangements during temperature. At various time points of the incubation the samples were phagocytosis, though these studies are almost exclusively mixed with 0.6 ml of ice-cold solution containing 6 mM pervanadate and 200 pM phenylarsine oxide, pelleted and processed for immunoblotting confined to mammalian phagocytes. An increasing interest analysis. To examine the effect of genistein (GIBCO, Gaithersburg, is especially focused on the role of protein tyrosine MD) on phagocytic activity, the suspended cells were preincubated in phosphorylation which controls phagocytosis mediated by NaCl/MgCl2/Hepes medium containing various concentrations of the Fey receptors and bacterial invasions of epithelial cells drug (10 min. room temperature). Subsequently, yeast particles were representing "nonprofessional" phagocytes (Dehio et al. added to the suspension at a ratio of 20 yeast particles per amoeba. Phagocytosis was conducted for 10 min at room temperature in the 1995, Strzelecka et al. 1997a, Hauck et al. 1998). In constant presence of genistein. The cells were next washed twice with contrast, data on the mechanisms which govern phagocy- the NaCl/MgCl2/Hepes medium and fixed with 3% formaldehyde in tosis in protozoa are random, despite the fact that phago- PBS for 10 min. The uptake of yeast was estimated by counting the cytosis being a way of feeding for these organisms. In number of amoeba cells with and without yeast particles under a Nikon Acanathamoeba castellanii, two actin-binding proteins, microscope in the phase contrast mode. myosin I and a a-spectrin immunoanalogue, were local- Immunofluorescence microscopy ized at sites of uptake of distinct particles (Baines et al. 1992, Kwiatkowska and Sobota 1997). Accordingly, mi- Microscopic observations were performed on cells attached to coverslips and undergoing phagocytosis of yeast for 3 min. The cells croinjection of an anti-spectrin antibody inhibited phago- were fixed for 30 min with 3% formaldehyde in PHEM buffer (60 mM cytosis in Amoebaproteus (Choi and Jeon 1992). On the Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl,. pH 6.9; protease other hand, our preliminary studies indicated that tyrosine inhibitors: 2 mM PMSF. 100 pg/ml leupeptin. 10 pg/pepstatin A. 2 pg/ml phosphorylation of proteins may be important