G Protein {beta} Subunit-null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

doi:10.1083/jcb.141.7.1529

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© The Rockefeller University Press, 0021-9525/1998//1529 $5.00
The Journal of Cell Biology, Volume 141, Number 7, , 1998 1529-1537

Articles

G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

Barbara Peracino^*, Jane Borleis, Tian Jin, Monika Westphal, Jean-Marc Schwartz, Lijun Wu^||, Enrico Bracco^*, Günther Gerisch, Peter Devreotes, and Salvatore Bozzaro^*

^* Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S. Luigi, 10043 Orbassano, Italy; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany; and ^|| Leukosite, Inc., Cambridge, Massachusetts 02142

Chemotaxis and phagocytosis are basically similar in cellsof the immune system and in Dictyostelium amebae. Deletion ofthe unique G protein β subunit in D. discoideum impairedphagocytosis but had little effect on fluid-phase endocytosis,cytokinesis, or random motility. Constitutive expression ofwild-type β subunit restored phagocytosis and normal development.Chemoattractants released by cells or bacteria trigger typicaltransient actin polymerization responses in wild-type cells.In β subunit–null cells, and in a series of βsubunit point mutants, these responses were impaired to a degreethat correlated with the defect in phagocytosis. Image analysisof green fluorescent protein–actin transfected cellsshowed that β subunit– null cells were defectivein reshaping the actin network into a phagocytic cup, and eventuallya phagosome, in response to particle attachment. Our resultsindicate that signaling through heterotrimeric G proteins isrequired for regulating the actin cytoskeleton during phagocyticuptake, as previously shown for chemotaxis. Inhibitors of phospholipaseC and intracellular Ca²⁺ mobilization inhibited phagocytosis,suggesting the possible involvement of these effectors in theprocess.

Abbreviations used in this paper: gβ^–, Gβ-null mutants; GFP–actin, green fluorescent protein–actin fusion protein; PLA2, phospholipase A2; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PI3 kinase, phosphoinositide 3-kinase; PI4 kinase, phosphoinositide 4-kinase.

This paper was supported by grants of the European Union andMinistero Universita E Ricerca Scientifica to S. Bozzaro (ERBCHRXCT930250),the National Institutes of Health to P.N. Devreotes (GM 28007), and Deutsche Forschungsgeheinschaft to G. Gerisch (SFB266/C6).

Address all correspondence to Salvatore Bozzaro, Dip. ScienzeCliniche e Biologiche, Università di Torino, OspedaleS. Luigi, 10043 Orbassano (TO), Italy. Tel.: (39) 11-9038661.Fax: (39) 11-9038639. E-mail: sbozzaro @polito.it

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