Ionized calcium binding adaptor molecule 1, Iba1, is an EF hand calcium binding protein whose expression is restricted to macrophages/microglia. In this study, Iba1 was shown to colocalize with F-actin in membrane ruffles induced by macrophage colony-stimulating factor and in phagocytic cups formed during zymosan phagocytosis. Expression of mutant Iba1 carrying either N- or C-terminal deletions or carrying a substitution in the calcium binding domain, suppressed the membrane ruffling and the phagocytosis. These results indicate that Iba1 is a key molecule in membrane ruffling and the phagocytosis of macrophages/microglia. Furthermore, Iba1 colocalized with a small GTPase Rac in the membrane ruffles and the phagocytic cups. The Iba1 mutants also suppressed membrane ruffling induced by dominant active Rac1V12, but do not affect microspikes by Cdc42V12 and stress fibers by RhoAV14. These observations suggest that Iba1 is involved in Rac and calcium signaling pathways.

REFERENCES

Aktories
K.
,
Braun
U.
,
Rösener
S.
,
Just
I.
,
Hall
A.
(
1989
).
The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3.
Biochem. Biophys. Res. Commun
158
,
209
213
Allen
L.-A. H.
,
Aderem
A.
(
1995
).
A role for MARCKS, theisozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages.
J. Exp. Med
182
,
829
840
Allen
W. E.
,
Jones
G. E.
,
Pollard
J. W.
,
Ridley
A. J.
(
1997
).
Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages.
J. Cell Sci
110
,
707
720
Autieri
M. V.
,
Prystowsky
M. B.
,
Ohlstein
E. H.
(
1996
).
Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries.
DNA Cell Biol
15
,
297
304
Burbelo
P. D.
,
Drechsel
D.
,
Hall
A.
(
1995
).
A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases.
J. Biol. Chem
270
,
29071
29074
Caron
E.
,
Hall
A.
(
1998
).
Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
Science
282
,
1717
1721
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
de Rooij
J.
,
Bos
J. L.
(
1997
).
Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.
Oncogene
14
,
623
625
Downey
G. P.
(
1994
).
Mechanisms of leukocyte motility and chemotaxis.
Curr. Opin. Immunol
6
,
113
124
Graeber
M. B.
,
Lopez-Redondo
F.
,
Ikoma
E.
,
Ishikawa
M.
,
Imai
Y.
,
Nakajima
K.
,
Kreutzberg
G. W.
,
Kohsaka
S.
(
1998
).
The microglia/macrophage response in the neonatal rat facial nucleus following axotomy.
Brain Res
813
,
241
253
Greenberg
S.
,
El Khoury
J.
,
Di Virgilio
F.
,
Kaplan
E. M.
,
Silverstein
S. C.
(
1991
).
Ca2+-independent F-actin assembly and disassembly during Fc receptor-mediated phagocytosis in mouse macrophages.
J. Cell Biol
113
,
757
767
Grynkiewicz
G.
,
Poenie
M.
,
Tsien
R. Y.
(
1985
).
A new generation of Ca2+indicators with greatly improved fluorescence properties.
J. Biol. Chem
260
,
3440
3450
Hackam
D. J.
,
Rotstein
O. D.
,
Schreiber
A.
,
Zhang
W.-j.
,
Grinstein
S.
(
1997
).
Rho is required for the initiation of calcium signaling and phagocytosis by Fcreceptors in macrophages.
J. Exp. Med
186
,
955
966
Hall
A.
(
1998
).
Rho GTPases and the actin cytoskeleton.
Science
279
,
509
514
Hartwig
J. H.
,
Yin
H. L.
(
1988
).
The organization and regulation of the macrophage actin skeleton.
Cell Motil. Cytoskel
10
,
117
125
Heizmann
C. W.
,
Hunziker
W.
(
1991
).
Intracellular calcium-binding proteins: more sites than insights.
Trends Biochem. Sci
16
,
98
103
Hishikawa
T.
,
Cheung
J. Y.
,
Yelamarty
R. V.
,
Knutson
D. W.
(
1991
).
Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.
J. Cell Biol
115
,
59
66
Imai
Y.
,
Matsushima
Y.
,
Sugimura
T.
,
Terada
M.
(
1991
).
A simple and rapid method for generating a deletion by PCR.
Nucl. Acids Res
19
,
2785
2785
Imai
Y.
,
Ibata
I.
,
Ito
D.
,
Ohsawa
K.
,
Kohsaka
S.
(
1996
).
A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage.
Biochem. Biophys. Res. Commun
224
,
855
862
Inoue
K.
,
Nakajima
K.
,
Morimoto
T.
,
Kikuchi
Y.
,
Koizumi
S.
,
Illes
P.
,
Kohsaka
S.
(
1998
).
ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia.
Br. J. Pharmacol
123
,
1304
1310
Iris
F. J. M.
,
Bougueleret
L.
,
Prieur
S.
,
Caterina
D.
,
Primas
G.
,
Perrot
V.
,
Jurka
J.
,
Rodriguez-Tome
P.
,
Claverie
J. M.
,
Dausset
J.
,
Cohen
D.
(
1993
).
Dense Alu clustering and a potential new member of the NFB family within a 90 kilobase HLA class III segment.
Nature Genet
3
,
137
145
Ito
D.
,
Imai
Y.
,
Ohsawa
K.
,
Nakajima
K.
,
Fukuuchi
Y.
,
Kohsaka
S.
(
1998
).
Microglia-specific localisation of a novel calcium binding protein, Iba1.
Brain Res. Mol. Brain Res
57
,
1
9
Janmey
P. A.
(
1994
).
Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.
Annu. Rev. Physiol
56
,
169
191
Kozma
R.
,
Ahmed
S.
,
Best
A.
,
Lim
L.
(
1995
).
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
Mol. Cell. Biol
15
,
1942
1952
Kreutzberg
G. W.
(
1996
).
Microglia: a sensor for pathological events in the CNS.
Trends Neurosci
19
,
312
318
Manser
E.
,
Loo
T.-H.
,
Koh
C.-G.
,
Zhao
Z.-S.
,
Chen
X.-Q.
,
Tan
L.
,
Tan
I.
,
Leung
T.
,
Lim
L.
(
1998
).
PAK kinases are directly coupled to the PIX family of nucleotide exchange factors.
Mol. Cell
1
,
183
192
Marks
P. W.
,
Maxfield
F. R.
(
1990
).
Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils.
J. Cell Biol
110
,
43
52
Maruyama
K.
,
Mikawa
T.
,
Ebashi
S.
(
1984
).
Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.
J. Biochem
95
,
511
519
Massol
P.
,
Montcourrier
P.
,
Guillemot
J.-C.
,
Chavrier
P.
(
1998
).
Fc receptor-mediated phagocytosis requires CDC42 and Rac1.
EMBO J
17
,
6219
6229
Meagher
L. C.
,
Moonga
B. S.
,
Haslett
C.
,
Huang
C. L.-H.
,
Zaidi
M.
(
1991
).
Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages.
Biochem. Biophys. Res. Commun
177
,
460
465
Mitchison
T. J.
,
Cramer
L. P.
(
1996
).
Actin-based cell motility and cell locomotion.
Cell
84
,
371
379
Mizushima
S.
,
Nagata
S.
(
1990
).
pEF-BOS, a powerful mammalian expression vector.
Nucl. Acids Res
18
,
5322
5322
Nakajima
K.
,
Kohsaka
S.
(
1993
).
Functional roles of microglia in the brain.
Neurosci. Res
17
,
187
203
Nobes
C. D.
,
Hall
A.
(
1995
).
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81
,
53
62
Ohsawa
K.
,
Imai
Y.
,
Nakajima
K.
,
Kohsaka
S.
(
1997
).
Generation and characterization of a microglial cell line, MG5, derived from a p53-deficient mouse.
Glia
21
,
285
298
Ridley
A. J.
,
Hall
A.
(
1992
).
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70
,
389
399
Ridley
A. J.
,
Paterson
H. F.
,
Johnston
C. L.
,
Diekmann
D.
,
Hall
A.
(
1992
).
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70
,
401
410
Sekine
A.
,
Fujiwara
M.
,
Narumiya
S.
(
1989
).
Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.
J. Biol. Chem
264
,
8602
8605
Stossel
T. P.
(
1993
).
On the crawling of animal cells.
Science
260
,
1086
1094
Tran
C. T.
,
Wolz
P.
,
Egensperger
R.
,
Kösel
S.
,
Imai
Y.
,
Bise
K.
,
Kohsaka
S.
,
Mehraein
P.
,
Graeber
M. B.
(
1998
).
Differential expression of MHC class II molecules by microglia and neoplastic astroglia: relevance for the escape of astrocytoma cells from immune surveillance.
Neuropathol. Appl. Neurobiol
24
,
293
301
Utans
U.
,
Arceci
R. J.
,
Yamashita
Y.
,
Russell
M. E.
(
1995
).
Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection.
J. Clin. Invest
95
,
2954
2962
von Eitzen
U.
,
Egensperger
R.
,
Kösel
S.
,
Grasbon-Frodl
E.
,
Imai
Y.
,
Bise
K.
,
Kohsaka
S.
,
Mehraein
P.
,
Graeber
M. B.
(
1998
).
Microglia and the development of spongiform change in Creutzfeldt-Jakob disease.
J. Neuropathol.Exp. Neurol
57
,
246
256
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