Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

REFERENCES

Brix
K.
,
Stockem
W.
(
1989
).
Functional analysis of actin fibrils in Physarum polycephalum. A morphological and quantitative study.
Cell Tissue Res
257
,
115
122
Brock
A. M.
,
Pardee
J. D.
(
1988
).
Cytoimmunofluorescent localization of severin in Dictyostelium amoebae.
Dev. Biol
128
,
30
39
Cooper
J. A.
(
1987
).
Effects of cytochalasin and phalloidin on actin.
J. Cell Biol
105
,
1473
1478
Cooper
J. A.
,
Bryan
J.
,
Schwab
B.
III
,
Frieden
C.
,
Loftus
D. J.
,
Elson
E. L.
(
1987
).
Microinjection of gelsolin into living cells.
J. Cell Biol
104
,
491
501
Croop
J.
,
Holtzer
H.
(
1975
).
Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid.
J. Cell Biol
65
,
271
285
Cunningham
C. C.
,
Stossel
T. P.
,
Kwiatkowski
D. J.
(
1991
).
Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin.
Science
251
,
1233
1236
De Corte
V.
,
Gettemans
J.
,
Waelkens
E.
,
Vandekerckhove
J.
(
1996
).
In vivo phosphorylation of actin in Physarum polycephalum. Study of the substrate specificity of the actin-fragmin kinase.
Eur. J. Biochem
241
,
901
908
Eichinger
L.
,
Bomblies
L.
,
Vandekerckhove
J.
,
Schleicher
M.
,
Gettemans
J.
(
1996
).
A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.
EMBOJ
15
,
5547
5556
Finidori
J.
,
Friederich
E.
,
Kwiatkowski
D. J.
,
Louvard
D.
(
1992
).
In vivo analysis of functional domains from villin and gelsolin.
J. Cell Biol
116
,
1145
1155
Fuchtbauer
A.
,
Jockush
B. M.
,
Maruta
H.
,
Kilimann
M. W.
,
Isenberg
G.
(
1983
).
Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells.
Nature
304
,
361
364
Gettemans
J.
,
De Ville
Y.
,
Vandekerckhove
J.
,
Waelkens
E.
(
1992
).
Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase.
EMBO J
11
,
3185
3191
Gettemans
J.
,
De Ville
Y.
,
Vandekerckhove
J.
,
Waelkens
E.
(
1993
).
Purification and partial amino acid sequence of the actin-fragmin kinase from Physarum polycephalum.
Eur. J. Biochem
214
,
111
119
Gettemans
J.
,
De Ville
Y.
,
Vandekerckhove
J.
,
Waelkens
E.
(
1995
).
The actin-binding properties of the actin-fragmin complex. Regulation bycalcium, phospholipids and phosphorylation.
J. Biol. Chem
270
,
2644
2651
Hasegawa
T.
,
Takahashi
S.
,
Hayashi
H.
,
Hatano
S.
(
1980
).
Fragmin: A calcium ion sensitive regulatory factor on the formation of actin filaments.
Biochemistry
19
,
2677
2683
Helmchen
F.
,
Imoto
K.
,
Sackmann
B.
(
1996
).
Ca2+-buffering and action potential-evoked Ca2+-signaling in dendrites of pyramidal neurons.
Biophys. J
70
,
1069
1081
Hinssen
H.
(
1981
).
An actin modulating protein from Physarum polycephalum. I. Isolation and purification.
Eur. J. Cell. Biol
23
,
225
233
Hinssen
H.
(
1981
).
An actin modulating protein from Physarum polycephalum. II. Ca2+-dependence and other properties.
Eur. J. Cell Biol
23
,
234
240
Huckriede
A.
,
Hinssen
H.
,
Fuchtbauer
A.
,
Chaponnier
C.
,
Weeds
A.
(
1990
).
Differential effects of gelsolins on tissue culture cells.
Cell Motil. Cytoskel
16
,
229
238
Jalink
K.
,
van Corven
E. J.
,
Moolenaar
W. H.
(
1990
).
Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2+-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action.
J. Biol. Chem
265
,
12232
12239
Janmey
P.
(
1994
).
Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.
Annu. Rev. Physiol
56
,
169
191
Klauke
N.
,
Plattner
H.
(
1997
).
Imaging of Ca2+transients induced in Paramecium cells by a polyamine secretagogue.
J. Cell Sci
110
,
975
983
Kukulies
J.
,
Brix
K.
,
Stockem
W.
(
1985
).
Fluorescent analog cytochemistry of the actin system and cell surface morphology in Physarum microplasmodia.
Eur. J. Cell Biol
39
,
62
69
Kukulies
J.
,
Brix
K.
,
Stockem
W.
(
1987
).
Studies on microplasmodia of Physarum polycephalum. VI. Functional analysis of a cortical and fibrillar actin system by use of fluorescent-analog cytochemistry.
Cell Tissue Res
250
,
125
134
Mertens
N.
,
Remaut
E.
,
Fiers
W.
(
1995
).
Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines.
Gene
164
,
9
15
Mitchison
T. J.
,
Cramer
L. P.
(
1996
).
Actin-based cell motility and cell locomotion.
Cell
84
,
371
379
Neher
E.
(
1995
).
The use of Fura-2 for estimating Ca buffer and Ca fluxes.
Neuropharmacology
34
,
1423
1442
Ridley
A.
,
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
Sampath
P.
,
Pollard
T. D.
(
1991
).
Effects of cytochalasin, phalloidin and pH on the elongation of actin filaments.
Biochemistry
30
,
1973
1980
Schafer
D. A.
,
Cooper
J. A.
(
1995
).
Control of actin assembly at filament ends.
Annu. Rev. Cell. Dev. Biol
11
,
497
518
Schliwa
M.
(
1982
).
Action of cytochalasin D on cytoskeletal networks.
J. Cell Biol
92
,
79
91
Stockem
W.
,
Brix
K.
(
1994
).
Analysis of microfilament organization and contractile activities in Physarum.
Int. Rev. Cytol
149
,
145
215
Stossel
T. P.
(
1989
).
From signal to pseudopod. How cells control cytoplasmic actin assembly.
J. Biol. Chem
264
,
18261
18264
Stossel
T. P.
(
1993
).
On the crawling of animal cells.
Science
260
,
1086
1094
T'Jampens
D.
,
Meerschaert
K.
,
Constantin
B.
,
Bailey
J.
,
Cook
L. J.
,
De Corte
V.
,
Gevaert
K.
,
De Mol
H.
,
Goethals
M.
,
van Damme
J.
,
Vandekerckhove
J.
,
Gettemans
J.
(
1997
).
Molecular cloning, over-expression, development regulation and immunolocalization of fragmin, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia.
J. Cell Sci
110
,
1215
1226
Waelkens
E.
,
Gettemans
J.
,
De Corte
V.
,
De Ville
Y.
,
Goris
J.
,
Vandekerckhove
J.
,
Merlevede
W.
(
1995
).
Microfilament dynamics: Regulation of actin polymerization by actin-fragmin kinase and phosphatases.
Advan. Enzyme Regul
35
,
199
227
Wodnicka
M.
,
Pierzchalska
M.
,
Bereiter-Hahn
J.
,
Kajstura
J.
(
1992
).
Comparative study on effects of cytochalasins B and D on F-actin content in different cell lines and different culture conditions.
Folia Histochem. Cytobiol
30
,
107
112
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