Polarization of Myosin II Heavy Chain-Protein Kinase C in Chemotaxing Dictyostelium Cells

Abstract

Eukaryotic cells need morphological polarity to carry out chemotaxis (Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes, P. N. (1998) Cell 95, 81–91; Jin, T., Zhang, N., Long, Y., Parent, C., and Devreotes, P. N. (2000) Science 287, 1034–1036; Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W., and Bourne, H. R. (2000) Science 287, 1037–1040), but sensing direction does not require polarization of chemoattractant receptors. When cells are exposed to a gradient of chemoattractant, activation occurs selectively at the stimulated edge. Such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems. Here we show that in Dictyostelium discoideum cells exposed to a cAMP gradient the myosin II heavy chain kinase (MHC-PKC) and myosin II translocate to opposite ends of the cell. We further show that MHC-PKC C1 domain is responsible for the localization of MHC-PKC to the cell leading edge, but it is not sufficient to promote cell polarization. Our findings suggest a mechanism by which MHC-PKC regulates myosin II, allowing cell polarization and movement in the direction of the cAMP source. Eukaryotic cells need morphological polarity to carry out chemotaxis (Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes, P. N. (1998) Cell 95, 81–91; Jin, T., Zhang, N., Long, Y., Parent, C., and Devreotes, P. N. (2000) Science 287, 1034–1036; Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W., and Bourne, H. R. (2000) Science 287, 1037–1040), but sensing direction does not require polarization of chemoattractant receptors. When cells are exposed to a gradient of chemoattractant, activation occurs selectively at the stimulated edge. Such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems. Here we show that in Dictyostelium discoideum cells exposed to a cAMP gradient the myosin II heavy chain kinase (MHC-PKC) and myosin II translocate to opposite ends of the cell. We further show that MHC-PKC C1 domain is responsible for the localization of MHC-PKC to the cell leading edge, but it is not sufficient to promote cell polarization. Our findings suggest a mechanism by which MHC-PKC regulates myosin II, allowing cell polarization and movement in the direction of the cAMP source. myosin II heavy chain protein kinase C green fluorescent protein 4-morpholineethanesulfonic acid Chemotaxis in eukaryotic cells is mediated by changes in the organization and function of cytoskeletal structures containing actin and myosin II (6Trinkaus J.P. Cell into Organs: The Force That Shapes the Embryo. Prentice-Hall Inc., Englewood Cliffs, NJ1984Google Scholar, 7Spudich J.A. Cell Regul. 1989; 1: 1-11Crossref PubMed Scopus (185) Google Scholar). Studies on the role of myosin II inDictyostelium chemotaxis suggest that myosin II monomers undergo transient assembly into bipolar filaments that may precede recruitment into the cytoskeleton and that the cycles of myosin II assembly and disassembly may be regulated by phosphorylation of myosin II heavy chain (MHC)1 (8Malchow D. Bohme R. Rahmsdorf H.J. Eur. J. Biochem. 1981; 17: 213-218Google Scholar, 9Berlot C.H. Spudich J.A. Devreotes P.N. Cell. 1985; 43: 307-314Abstract Full Text PDF PubMed Scopus (114) Google Scholar). Indeed, cAMP stimulation causes myosin II that exists as thick filaments to translocate to the cell cortex. This translocation is correlated with a transient increase in the rate of MHC as well as light chain phosphorylation (8Malchow D. Bohme R. Rahmsdorf H.J. Eur. J. Biochem. 1981; 17: 213-218Google Scholar, 9Berlot C.H. Spudich J.A. Devreotes P.N. Cell. 1985; 43: 307-314Abstract Full Text PDF PubMed Scopus (114) Google Scholar). In addition, a novel protein kinase C (MHC-PKC), which we purified and cloned from chemotactically competent Dictyostelium cells, phosphorylates MHC in response to cAMP stimulation (10Ravid S. Spudich J.A. J. Biol. Chem. 1989; 264: 15144-15150Abstract Full Text PDF PubMed Google Scholar, 11Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar, 12Abu-Elneel K. Karchi M. Ravid S. J. Biol. Chem. 1996; 271: 977-984Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In vitrophosphorylation of MHC by this kinase results in inhibition of myosin II thick filament formation (11Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar, 13Pasternak C. Flicker P.F. Ravid S. Spudich J.A. J. Cell Biol. 1989; 109: 203-210Crossref PubMed Scopus (59) Google Scholar). Morphological polarity is necessary for chemotaxis by eukaryotic cells, but it does not require receptor polarization (5Wang M. Van-Haastert P.J. Devreotes P.N. Schaap P. Dev. Biol. 1988; 128: 72-77Crossref PubMed Scopus (26) Google Scholar).Dictyostelium chemotaxis is accompanied by asymmetric recruitment to the cell surface of signal transduction proteins (2Jin T. Zhang N. Long Y. Parent C. Devreotes P.N. Science. 2000; 287: 1034-1036Crossref PubMed Scopus (244) Google Scholar, 4Janetopoulos C. Jin T. Devreotes P.N. Science. 2001; 291: 2408-2411Crossref PubMed Scopus (374) Google Scholar), the cytosolic regulator of adenylyl cyclase (1Parent C.A. Blacklock B.J. Froehlich W.M. Murphy D.B. Devreotes P.N. Cell. 1998; 95: 81-91Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 14Parent C.A. Devreotes P.N. Science. 1999; 284: 765-770Crossref PubMed Scopus (715) Google Scholar), and the pleckstrin homology domain of the AKT protein kinase (15Meili R. Ellsworth C. Lee S. Reddy T.B., Ma, H. Firtel R.A. EMBO J. 1999; 18: 2092-2105Crossref PubMed Scopus (374) Google Scholar). Neutrophils also show such asymmetry when exposed to chemoattractant; the pleckstrin homology domain of the AKT protein kinase is recruited selectively to the membrane at the leading edge of the cell (3Servant G. Weiner O.D. Herzmark P. Balla T. Sedat J.W. Bourne H.R. Science. 2000; 287: 1037-1040Crossref PubMed Scopus (727) Google Scholar). In addition, cytoskeletal proteins such as actin and several actin-binding proteins have been shown to transiently accumulate at the leading edge of chemotaxing cells (16Westphal M. Jungbluth A. Heidecker M. Muhlbauer B. Heizer C. Schwartz J.M. Marriott G. Gerisch G. Curr. Biol. 1997; 7: 176-183Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17Gerisch G. Albrecht R. Heizer G. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18Kreitmeier M. Gerisch G. Heizer C. Muller-Taubenberger A. J. Cell Biol. 1995; 129: 179-188Crossref PubMed Scopus (89) Google Scholar, 19Aizawa H. Fukui Y. Yahara I. J. Cell Sci. 1997; 110: 2333-2344PubMed Google Scholar). To define the spatiotemporal dynamics of MHC-PKC and myosin II that may lead to cell polarization and directed movement inDictyostelium we expressed GFP-tagged MHC-PKC and myosin II in mhc-pkc null cells and myosin II null cells, respectively. We found that in cells exposed to a cAMP gradient, the MHC-PKC-GFP localized to the leading edge of the cell, whereas myosin II translocated to its posterior part. These findings indicate a mechanism whereby MHC-PKC and myosin II contribute to cell polarization and chemotaxis. All DNA manipulations followed standard methods (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A MHC-PKC-GFP expression plasmid was constructed as follows. A 1.8-kb fragment of MHC-PKC was isolated from pBluescript-MHC-PKC (12Abu-Elneel K. Karchi M. Ravid S. J. Biol. Chem. 1996; 271: 977-984Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) by digestion withBglII-BamHI and cloned into pDXA-Hy (21Manstein D.J. Schuster H.P. Morandini P. Hunt D.M. Gene (Amst.). 1995; 162: 129-134Crossref PubMed Scopus (192) Google Scholar) to create pDXA-MHC-PKC. To isolate the GFP, the plasmid pGFP10.1 containing the GFP cDNA (22Chalfie M., Tu, Y. Euskirchen G. Ward W.W. Prasher D.C. Science. 1994; 263: 802-805Crossref PubMed Scopus (5409) Google Scholar) was digested with BamHI and cloned in frame in front of the MHC-PKC to create the expression vector pDXA-MHC-PKC-GFP. The pDXA-C1-GFP expression plasmid was created by restricting the pDXA-MHC-PKC-GFP withBsaBI and rendered blunt. This was followed by restriction with XhoI, resulting in the deletion of a 1.4-kb fragment from the 3 prime. The expression plasmid was then ligated resulting in GFP fused in frame to a 0.4-kb fragment from the 5 prime of MHC-PKC encoding the C1 domain. The expression plasmid pDXA-MHC-PKCΔC1-GFP was created by restriction of pDXA-MHC-PKC-GFP with BsaBI, and EcoRI rendered it blunt. This plasmid was ligated. The restriction resulted in the deletion of the C1 domain. Dictyosteliumcells were grown in HL-5 medium (23Spudich J.A. Methods Cell Biol. 1987; 28: 3-8Crossref PubMed Scopus (6) Google Scholar), supplemented with 60 units of penicillin and 60 μg of streptomycin/ml at 21 °C. Transformations were performed using the calcium phosphate procedure as described previously (24Egelhoff T.T. Titus M.A. Manstein D.J. Ruppel K.M. Spudich J.A. Methods Enzymol. 1991; 196: 319-335Crossref PubMed Scopus (71) Google Scholar). The mhc-pkc null cells were transformed with either pDXA-MHC-PKC-GFP, pDXA-C1-GFP, or pDXA-MHC-PKCΔC1-GFP to create mhc-pkc-gfp, c1-gfp, andΔc1-gfp cell lines respectively. The myosin IInull cell line HS1 (25Ruppel K.M. Uyeda T.Q.P. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar) was transformed with pBigGFPmyo to createmyosin II-gfp cells (26Moores S.L. Sabry J.H. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 443-446Crossref PubMed Scopus (172) Google Scholar). Transformed cells were selected and grown in the presence of G418 at 5 μg/ml (Geneticin, Invitrogen). Amoeba of Dictyostelium discoideum strains were grown in HL-5 medium (23Spudich J.A. Methods Cell Biol. 1987; 28: 3-8Crossref PubMed Scopus (6) Google Scholar), harvested at a density of 2 × 106 cells/ml, washed twice in MES buffer (20 mm MES, pH 6.8, 0.2 mmCaCl2, 2 mm MgSO4), and resuspended in MES buffer at a density of 2 × 107 cells/ml. Cells were shaken at 100 rpm at 22 °C for 3.5 h. 5 mmcaffeine was added to the suspension 30 min prior to the addition of cAMP. Cells were developed for 4 h as described above, washed in 10 mm Tris-HCl (pH 8.0) and 150 mm KCl, and lysed in 50 mm Tris-HCl (pH 8.0), 20 mm sodium pyrophosphate (pH 6.8), 5 mm EDTA, 5 mm EGTA, 0.5% Triton X-100, and protease inhibitor mix (Sigma). Protein concentration was determined by the method of Peterson (27Peterson G. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7066) Google Scholar), and the lysates were electrophoresed on SDS-PAGE (28Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Western blots were probed with MHC-PKC polyclonal antibody (11Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar) or with GFP antibody (Santa Cruz Biotechnology, Inc.). The blots were developed using a horseradish peroxidase-coupled secondary antibody (Bio-Rad). ECL was performed using a kit from Amersham Biosciences. 4-h developed Dictyostelium amoebae expressing MHC-PKC-GFP, C1-GFP, ΔC1-GFP, or myosin II-GFP (26Moores S.L. Sabry J.H. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 443-446Crossref PubMed Scopus (172) Google Scholar) were placed on a coverslip mounted on a Zigmod chamber (29Zigmond S.H. Methods Enzymol. 1988; 162: 65-72Crossref PubMed Scopus (46) Google Scholar), in which one well was filled with MES buffer and the other with MES buffer containing 1 μm cAMP. After waiting 10 min to allow development of a gradient, the cells were observed with a Zeiss Axiovert microscope using a Planapochromat 100y1.4 objective and a basement port connected to a cooled charge-coupled device (CCD) camera. Images were taken every 5 s with the shutter set at a 20-ms exposure with a 100-watt mercury lamp attenuated by a 50% neutral density filter to prevent light-induced damage to the cells. Images were collected and analyzed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD). To define the spatiotemporal dynamics of MHC-PKC that may lead to cell polarization and directed movement we expressed GFP-tagged MHC-PKC (MHC-PKC-GFP; Fig. 1) inmhc-pkc null cells (12Abu-Elneel K. Karchi M. Ravid S. J. Biol. Chem. 1996; 271: 977-984Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). MHC-PKC-GFP rescued allmhc-pkc null cell defects. mhc-pkc null cells exhibit substantial myosin II overassembly in vivo, as well as aberrant cell polarization, chemotaxis, and morphological differentiation (12Abu-Elneel K. Karchi M. Ravid S. J. Biol. Chem. 1996; 271: 977-984Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, expression of MHC-PKC-GFP resulted in cells showing similar chemotaxis and differentiation properties to wild-type cells (data not shown). These results indicate that tagging the MHC-PKC with GFP did not affect its properties. To study the localization properties of MHC-PKC during chemotaxis toward cAMP, mhc-pkc-gfp cells were subjected to a cAMP gradient using a Zigmond chamber (29Zigmond S.H. Methods Enzymol. 1988; 162: 65-72Crossref PubMed Scopus (46) Google Scholar). The chemotactic response ofmhc-pkc-gfp cells was observed at 5-s intervals 10 min after placing the coverslip containing cells in the chamber (Fig.2). Cells freshly placed in the chamber (time, 0 s) were round, and the MHC-PKC-GFP was distributed evenly throughout the cytoplasm. As time progressed the cell sent lamellipodia toward the region of high cAMP concentration and became polarized. The localization of MHC-PKC was also altered together with the changes in cell morphology. In a cell first subjected to a cAMP gradient, the MHC-PKC-GFP was distributed evenly throughout the cytoplasm (Fig. 2, 0–20 s), but after 25–30 s it was concentrated at the leading edge of the cell. These results indicate that a cell moving up a cAMP gradient achieves cell polarization as well as polarization of MHC-PKC. To study the localization properties of myosin II in chemotaxing cells, we expressed myosin II-GFP in myosin II null cells (Fig. 1). Moores et al. (26Moores S.L. Sabry J.H. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 443-446Crossref PubMed Scopus (172) Google Scholar) have shown that purified myosin II-GFP protein displays wild-type myosin II properties in vitro. Furthermore, expression of myosin II-GFP fully complements the myosin II null cell phenotype. To follow myosin II dynamics, myosin II-gfp cells were subjected to a cAMP gradient similar to that described above. As shown in Fig. 3, 30 s after exposing the cell to the cAMP gradient, the cell became polarized sending out lamellipodia along with thin filopodia toward the region of high cAMP concentration. At the same time most of the myosin II-GFP became localized to the posterior part of the cell. These results indicate that, in a cAMP gradient, Dictyosteliumcells send their MHC-PKC-GFP and myosin II-GFP to opposite ends of the cell. These findings are consistent with experiments in polarizedDictyostelium cells in which immunofluorescent stained myosin II was found to be localized at the posterior part of the cell (30Yumura S. Fukui Y. Nature. 1985; 314: 194-196Crossref PubMed Scopus (175) Google Scholar, 31Nachmias V.T. Fukui Y. Spudich J.A. Cell Motil. Cytoskeleton. 1989; 13: 158-169Crossref PubMed Scopus (39) Google Scholar, 32Chu Q. Fukui Y. Cell. Motil. Cytoskeleton. 1996; 35: 254-268Crossref PubMed Scopus (12) Google Scholar). Expression of C1 from mammalian PKC results in translocation of this domain to the cell membrane (33Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). This observation together with biochemical evidence indicates that PKC binds to the cell membrane through its C1 domain (for reviews see Refs. 34Kikkawa U. Kishimoto A. Nishizuka Y. Annu. Rev. Biochem. 1989; 58: 31-44Crossref PubMed Scopus (580) Google Scholar and 35Nakamura S.-i. Nishizuka Y. J. Biochem. (Tokyo). 1994; 115: 1029-1034Crossref PubMed Scopus (137) Google Scholar). To define the MHC-PKC domain responsible here for binding MHC-PKC to the cell membrane and thus enabling it to concentrate at the anterior part of chemotaxing cells, we expressed two MHC-PKC truncation proteins tagged with GFP in mhc-pkc null cells. The first protein was the C1 domain of MHC-PKC, and the second was MHC-PKC in which the C1 domain had been deleted (ΔC1-GFP) (Fig. 1). Both cell lines were subjected to a cAMP gradient in the Zigmond chamber (29Zigmond S.H. Methods Enzymol. 1988; 162: 65-72Crossref PubMed Scopus (46) Google Scholar) as described above. In contrast to MHC-PKC-GFP, which at first appeared diffusely distributed throughout the cytoplasm and then gradually concentrated at the leading edge of the cell (Fig. 2), the C1-GFP appeared as small spherical aggregates that concentrated at the cell region facing the cAMP high concentration (Fig. 4). These results suggest that similar to mammalian PKC, MHC-PKC binds to the cell membrane through its C1 domain. The ability of C1 domain to localize to the cell leading edge indicates that C1 domain possessed the information required for the localization of MHC-PKC to this region in a cAMP gradient. Even though C1 domain localized to the cell leading edge, the c1-gfp cells did not undergo cell polarization or chemotaxis; this may be because of the absence of MHC-PKC catalytic domain that is required for cell polarization and chemotaxis (12Abu-Elneel K. Karchi M. Ravid S. J. Biol. Chem. 1996; 271: 977-984Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Deletion of the C1 domain (i.e. ΔC1-GFP) also resulted in spherically shaped proteins, but in contrast to C1-GFP, the ΔC1-G distributed evenly throughout the cytoplasm regardless of cAMP stimulation (Fig. 5). These results further indicate that MHC-PKC concentrates at the cell membrane in response to cAMP stimulation using its C1 domain, and when the C1 domain is absent, the protein remains in the cytoplasm. Similar toc1-gfp cells, Δc1-gfp cells did not undergo cell polarization and chemotaxis; this can be because of the absence of C1 that is required for MHC-PKC binding to the cell membrane, a step necessary for MHC-PKC activation (36Dembinsky A. Rubin H. Ravid S. J. Cell Biol. 1996; 134: 911-921Crossref PubMed Scopus (29) Google Scholar). The appearance of C1-GFP and ΔC1-GFP in aggregates may be because of the expression of truncated MHC-PKC proteins. The results described above indicate that exposure ofDictyostelium cells to the cAMP gradient results in cell polarization and localization of MHC-PKC and myosin II to opposite sites of the cell. Furthermore, the MHC-PKC C1 domain is required for the localization of MHC-PKC to the cell leading edge of chemotaxing cell; however the C1 protein is not sufficient for cell polarization and chemotaxis. These results fit with our previous observations in which we biochemically followed the localization and activation of MHC-PKC and of myosin II stimulated with cAMP (36Dembinsky A. Rubin H. Ravid S. J. Cell Biol. 1996; 134: 911-921Crossref PubMed Scopus (29) Google Scholar). Based both on these previous results and the current results, we suggest the following model for the involvement of MHC-PKC and myosin II in cell polarization and chemotaxis. The unstimulated cell is rounder in shape because of a contractile shell formed by an actin-myosin II network in the cortex. This network presumably inhibits events necessary for pseudopodial projection. cAMP stimulation of one edge of the cell results in activation of the cytosolic MHC-PKC, causing it to translocate to the membrane. Here it concentrates in an active form at the site of cAMP stimulation (i.e. the cell's leading edge). Active MHC-PKC phosphorylates the cortical MHC at the anterior part of the cell, causing disassembly of myosin II thick filaments. The disassembled myosin II molecules reassemble at the posterior part of the cell, thus providing a force that moves the cell forward. We thank Dr. J. A. Spudich for providing GFP-myosin II expression vector and Dictyostelium myosin IInull cells.