Rac1 is a Ρ-family GTP-binding protein that controls lamellipodia formation and membrane ruffling in fibroblasts. Recently, Rac1 and Cdc42, another member of the Ρ-family, have been shown to regulate Fc receptor-mediated phagocytosis in macrophages by controlling different steps of membrane and actin dynamics leading to particle engulfment. Here, we investigated the function of Rac1 using a membrane recruitment system that mimics phagocytosis. Recruitment of an activated Rac1 protein to the cytoplasmic domain of an engineered membrane receptor by using rapamycin as a bridge induces ingestion of latex beads bound to the receptor. Rac1-mediated bead uptake depends on actin polymerisation since actin filaments accumulate at the bead/membrane binding sites and internalisation is inhibited by cytochalasin D. Internalisation is also abolished upon substitution of Phe37 to Leu in the Rac1 effector region. Our results indicate that by promoting actin polymerisation at particle attachment sites, Rac1 by acting through specific downstream effectors induces plasma membrane remodeling that allows particle internalisation in a membrane-enclosed phagosome.

Allen
L. A.
,
Aderem
A.
(
1996
).
Molecular definition of distinct cytoskeletal structures involved in complement-and Fc receptor-mediated phagocytosis in macrophages.
J. Exp. Med
184
,
627
637
Allen
W. E.
,
Jones
G. E.
,
Pollard
J. W.
,
Ridley
A. J.
(
1997
).
Rho, Rac and Cdc42 regulate actin organisation and cell adhesion in macrophages.
J. Cell Sci
110
,
707
720
Araki
N.
,
Johnson
M. T.
,
Swanson
J. A.
(
1996
).
A role for phosphoinositide 3_-kinase in the completion of macropinocytosis and phagocytosis by macrophages.
J. Cell Biol
135
,
1249
1260
Braun
L.
,
Ohayon
H.
,
Cossart
P.
(
1998
).
The InIB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells.
Mol. Microbiol
27
,
1077
1087
Caron
E.
,
Hall
A.
(
1998
).
Identification of two distinct mechanisms ofphagocytosis controlled by different rho GTPases.
Science
282
,
1717
1721
Castellano
F.
,
Montcourrier
P.
,
Guillemot
J. C.
,
Gouin
E.
,
Machesky
L. M.
,
Cossart
P.
,
Chavrier
P.
(
1999
).
Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.
Curr. Biol
9
,
351
360
Chen
J.
,
Zheng
X. F.
,
Brown
E. J.
,
Schreiber
S. L.
(
1995
).
Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue.
Proc. Nat. Acad. Sci. USA
92
,
4947
4951
Cox
D.
,
Chang
P.
,
Kurosaki
T.
,
Greenberg
S.
(
1996
).
Syk tyrosine kinase is required for immunoreceptor tyrosine activation motif-dependent actin assembly.
J. Biol. Chem
271
,
16597
16602
Cox
D.
,
Chang
P.
,
Zhang
Q.
,
Reddy
P. G.
,
Bokoch
G. M.
,
Greenberg
S.
(
1997
).
Requirements for both rac1 and cdc42 in membrane ruffling and phagocytosis in leukocytes.
J. Exp. Med
186
,
1487
1494
Crespo
P.
,
Schuebel
K. E.
,
Ostrom
A. A.
,
Gutkind
J. S.
,
Bustelo
X. R.
(
1997
).
Phosphotyrosine-dependent activation of RAc1 GDP/GTP exchange by the vav proto-oncogene product.
Nature
285
,
169
172
Crowley
M. T.
,
Costello
P. S.
,
Fitzer-Attas
C. J.
,
Turner
M.
,
Meng
F.
,
Lowell
C.
,
Tybulewicz
V. L. J.
,
DeFranco
A. L.
(
1997
).
A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages.
J. Exp. Med
186
,
1027
1039
Dramsi
S.
,
Cossart
P.
(
1998
).
Intracellular pathogens and the actin cytoskeleton.
Annu. Rev. Cell Dev. Biol
14
,
137
166
Durden
D. L.
,
Liu
Y. B.
(
1994
).
Protein-tyrosine kinase p72syk in Fc gamma RI receptor signaling.
Blood
84
,
2102
2108
Finlay
B. B.
,
Cossart
P.
(
1997
).
Exploitation of mammalian host cell functions by bacterial pathogens.
Science
276
,
718
725
Ghazizadeh
S.
,
Bolen
J. B.
,
Fleit
H. B.
(
1994
).
Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells.
J. Biol Chem
269
,
8878
8884
Greenberg
S.
,
Chang
P.
,
Silverstein
S. C.
(
1994
).
Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages.
J. Biol Chem
269
,
3897
3902
Greenberg
S.
(
1995
).
Signal Transduction of Phagocytosis.
Trends Biochem. Sci
5
,
93
97
Greenberg
S.
,
Chang
P.
,
Wang
D. C.
,
Xavier
R.
,
Seed
B.
(
1996
).
Clustered syk tyrosine kinase domains trigger phagocytosis.
Proc. Nat. Acad. Sci. USA
93
,
1103
1107
Griffin
F. M.
Jr.
,
Griffin
J. A.
,
Leider
J. E.
,
Silverstein
S. C.
(
1975
).
Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.
J. Exp. Med
142
,
1263
1282
Guillemot
J. C.
,
Montcourrier
P.
,
Vivier
E.
,
Davoust
J.
,
Chavrier
P.
(
1997
).
Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in Fc(epsilon)RI-activated rat basophilic leukemia (RBL-2H3) cells.
J. Cell Sci
110
,
2215
2225
Hackam
D. J.
,
Rotstein
O. D.
,
Schreiber
A.
,
Zhang Wj Grinstein
S.
(
1997
).
Rho is required for the initiation of calcium signaling and phagocytosis by fcgamma receptors in macrophages.
J. Exp. Med
186
,
955
966
Han
J.
,
Luby-Phelps
K.
,
Das
B.
,
Shu
X.
,
Xia
Y.
,
Mosteller
R. D.
,
Krishna
U. M.
,
Falck
J. R.
,
White
M. A.
,
Broek
D.
(
1998
).
Role of substrates and products of PI 3-kinase in regulating activation of rac-related guanosine triphosphatases by Vav.
Science
279
,
558
560
Hanke
J. H.
,
Gardner
J. P.
,
Dow
R. L.
,
Changelian
P. S.
,
Brissette
W. H.
,
Weringer
E. J.
,
Pollok
B. A.
,
Connelly
P. A.
(
1996
).
Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-and FynT-dependent T cell activation.
J. Biol Chem
271
,
695
701
Hartwig
J. H.
,
Bokoch
G. M.
,
Carpenter
C. L.
,
Janmey
P. A.
,
Taylor
L. A.
,
Toker
A.
,
Stossel
T. P.
(
1995
).
Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilised human platelets.
Cell
82
,
643
653
Ireton
K.
,
Payrastre
B.
,
Chap
H.
,
Ogawa
W.
,
Sakaue
H.
,
Kasuga
M.
,
Cossart
P.
(
1996
).
A role for phosphoinositide 3-kinase in bacterial invasion.
Science
274
,
780
782
Joneson
T.
,
McDonough
M.
,
Bar-Sagi
D.
,
Van Aelst
L.
(
1996
).
Rac regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase.
Science
274
,
1374
1376
Koval
M.
,
Preiter
K.
,
Adles
C.
,
Stahl
P. D.
,
Steinberg
T. H.
(
1998
).
Size of IgG-opsonized particles determines macrophage response during internalization.
Exp. Cell Res
242
,
265
273
Lamarche
N.
,
Tapon
N.
,
Stowers
L.
,
Burbelo
P. D.
,
Aspenstrom
P.
,
Bridges
T.
,
Chant
J.
,
Hall
A.
(
1996
).
Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.
Cell
87
,
519
529
Lowry
M. B.
,
Duchemin
A. M.
,
Coggeshall
K. M.
,
Robinson
J. M.
,
Anderson
C. L.
(
1998
).
Chimeric receptors composed of phosphoinositide 3-kinase domains and fcgamma receptor ligand-binding domains mediate phagocytosis in COS fibroblasts.
J. Biol Chem
273
,
24513
24520
Machesky
L. M.
,
Hall
A.
(
1997
).
Role of actin polymerization and adhesion to extracellular matrix in Rac-and Rho-induced cytoskeletal reorganization.
J. Cell Biol
138
,
913
926
Machesky
L. M.
,
Insall
R. H.
(
1998
).
Scar1 and the related wiskott-aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex.
Curr. Biol
8
,
1347
1356
Machesky
L. M.
,
Mullins
R. D.
,
Higgs
H. N.
,
Kaiser
D. A.
,
Blanchoin
L.
,
May
R. C.
,
Hall
M. E.
,
Pollard
T. D.
(
1999
).
Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.
Proc. Nat. Acad. Sci. USA
96
,
3739
3744
Massol
P.
,
Montcourrier
P.
,
Guillemot
J. C.
,
Chavrier
P.
(
1998
).
Fc receptor-mediated phagocytosis requires CDC42 and Rac1.
EMBO J
17
,
6219
6229
Miki
H.
,
Suetsugu
S.
,
Takenawa
T.
(
1998
).
WAVE, a novel WASP-family protein involved in actin reorganisation induced by Rac.
EMBO J
17
,
6932
6941
Van Aelst
L.
,
Joneson
T.
,
Bar-Sagi
D.
(
1996
).
Identification of a novel Rac1-interacting protein involved in membrane ruffling.
EMBO J
15
,
3778
3786
Van Aelst
L.
,
D'Souza-Schorey
C.
(
1997
).
Rho GTPases and signaling networks.
Genes Dev
11
,
2295
2322
Zigmond
S. H.
,
Hirsch
J. G.
(
1972
).
Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis.
Exp. Cell Res
73
,
383
393
Zigmond
S. H.
,
Joyce
M.
,
Borleis
J.
,
Bokoch
G. M.
,
Devreotes
P. N.
(
1997
).
Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42.
J. Cell Biol
138
,
363
374
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