Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.

REFERENCES

Bright
G. R.
,
Rogowska
J.
,
Fisher
G. W.
,
Taylor
D. L.
(
1987
).
Fluorescence ratio imaging microscopy: temporal and spatial measurements in single living cells.
Biotechniques
5
,
556
563
Brundage
R. A.
,
Fogarty
K. E.
,
Tuft
R. A.
,
Fay
F. S.
(
1991
).
Calcium gradients underlying polarization and chemotaxis of eosinophils.
Science
254
,
703
7706
Cheng
T. P. O.
,
Murakami
N.
,
Elzinga
M.
(
1992
).
Localization of myosin IIB at the leading edge of growth cones from rat dorsal ganglionic cells.
FEBS Lett
311
,
91
94
Condeelis
J. S.
,
Taylor
D. L.
(
1977
).
The contractile basis of amoeboid movement. V. The control of gelation, solation and contraction in extracts from Dictyostelium discoideum.
J. Cell Biol
74
,
901
927
Conrad
P. A.
,
Giuliano
K. A.
,
Fisher
G.
,
Collins
K.
,
Matsudaira
P. T.
,
Taylor
D. L.
(
1993
).
Relative distribution of actin, myosin I and myosin II during the wound healing response of fibroblasts.
J. Cell Biol
120
,
1381
1391
Conrad
A. H.
,
Jaffredo
T.
,
Conrad
G. W.
(
1995
).
Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro.
Cell Motil. Cytoskel
31
,
93
112
Cuda
G.
,
Pate
E.
,
Cooke
R.
,
Sellers
J. R.
(
1997
).
In vitro actin filament sliding velocities produced by mixtures of different types of myosin.
Biophys. J
72
,
1767
1779
Daniel
J. L.
,
Sellers
J. R.
(
1992
).
Purification and characterization of platelet myosin.
Meth. Enzymol
215
,
78
88
DeBiasio
R. L.
,
Wang
L.-L.
,
Fisher
G. W.
,
Taylor
D. L.
(
1988
).
The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts.
J. Cell Biol
107
,
2631
2645
DeLozanne
A.
,
Spudich
J. A.
(
1987
).
Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.
Science
236
,
1086
1091
Giuliano
K. A.
,
Kolega
J.
,
DeBiasio
R. L.
,
Taylor
D. L.
(
1992
).
Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.
Mol. Biol. Cell
3
,
1037
1048
Giuliano
K. A.
,
Taylor
D. L.
(
1990
).
Formation, transport, contraction, and disassembly of stress fibers in fibroblasts.
Cell Motil. Cytoskel
16
,
14
21
Hahn
K.
,
DeBiasio
R.
,
Taylor
D. L.
(
1992
).
Patterns of elevated free calcium and calmodulin activation in living cells.
Nature
359
,
736
738
Hoock
T. C.
,
Newcomb
P. M.
,
Herman
I. M.
(
1991
).
-Actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during cellular response to injury.
J. Cell Biol
112
,
653
664
Huxley
H. E.
(
1969
).
The mechanism of muscular contraction.
Science
164
,
1356
1366
Katsuragawa
Y.
,
Yanagisawa
M.
,
Inoue
A.
,
Masaki
T.
(
1989
).
Two distinct nonmuscle myosin-heavy-chain mRNAs are differently expressed in various chicken tissues.
Eur. J. Biochem
184
,
611
616
Kelley
C. A.
,
Sellers
J. R.
,
Gard
D. L.
,
Bui
D.
,
Adelstein
R. S.
,
Baines
I. C.
(
1996
).
Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities.
J. Cell Biol
134
,
675
687
Knecht
D. A.
,
Loomis
W. F.
(
1987
).
Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum.
Science
236
,
1081
1086
Kolega
J.
(
1998
).
Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells.
J. Cell. Biochem
68
,
1
13
Kolega
J.
(
1997
).
Asymmetry in the distribution of free versus cytoskeletal myosin II in locomoting microcapillary endothelial cells.
Exp. Cell Res
231
,
66
82
Kolega
J.
,
Janson
L. W.
,
Taylor
D. L.
(
1991
).
The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells.
J. Cell Biol
114
,
993
1003
Kolega
J.
,
Taylor
D. L.
(
1993
).
Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport.
Mol. Biol. Cell
4
,
819
836
Li
D.
,
Miller
M.
,
Chantler
P. D.
(
1994
).
Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane.
Proc. Nat. Acad. Sci. USA
91
,
853
857
Maupin
P.
,
Phillips
C. L.
,
Adelstein
R. S.
,
Pollard
T. D.
(
1994
).
Differential localization of myosin-II isoforms in human cultured cells and blood cells.
J. Cell Sci
107
,
3077
3090
McKenna
N. M.
,
Wang
Y.-l.
,
Konkel
M. E.
(
1989
).
Formation and movement of myosin-containing structures in living fibroblasts.
J. Cell Biol
109
,
1163
1172
Murakami
N.
,
Mehta
P.
,
Elzinga
M.
(
1991
).
Studies on the distribution of cellular myosin with antibodies to isoform-specific synthetic peptides.
FEBS Lett
278
,
23
25
Pasternak
C.
,
Spudich
J. A.
,
Elson
E. L.
(
1989
).
Capping of surface receptors and concomitant cortical tension are generated by conventional myosin.
Nature
341
,
549
551
Phillips
C. L.
,
Yamakawa
K.
,
Adelstein
R. S.
(
1995
).
Cloning of the cDNA encoding human nonmuscle myosin heavy chain B and analysis of human tissues with isoform-specific antibodies.
J. Muscle Res. Cell Motil
16
,
379
389
Rochlin
M. W.
,
Itoh
K.
,
Adelstein
R. S.
,
Bridgman
P. C.
(
1995
).
Localization of myosin II A and B isoforms in cultured neurons.
J. Cell Sci
108
,
3661
3670
Sanger
J. M.
,
Mittal
B.
,
Dome
J. S.
,
Sanger
J. W.
(
1989
).
Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells.
Cell Motil. Cytoskel
14
,
201
219
Sellers
J. R.
(
1991
).
Regulation of cytoplasmic and smooth muscle myosin.
Curr. Opin. Cell Biol
3
,
98
104
Tan
J. L.
,
Ravid
S.
,
Spudich
J. A.
(
1992
).
Control of nonmuscle myosins by phosphorylation.
Annu. Rev. Biochem
61
,
721
759
Taylor
D. L.
,
Blinks
J. R.
,
Reynolds
G.
(
1980
).
Contractile basis of ameboid movement VIII. Aequorin luminescence during ameboid movement, endocytosis and capping.
J. Cell Biol
86
,
599
607
Trybus
K. M.
(
1991
).
Assembly of cytoplasmic and smooth muscle myosins.
Curr. Opin. Cell Biol
3
,
105
111
Verkhovsky
A. B.
,
Svitkina
T. M.
,
Borisy
G. G.
(
1995
).
Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles.
J. Cell Biol
131
,
989
1002
Wessels
D.
,
Soll
D. R.
,
Knecht
D.
,
Loomis
W. F.
,
DeLozanne
A.
,
Spudich
J.
(
1988
).
Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain.
Dev. Biol
128
,
164
177
Wessels
D.
,
Soll
D. R.
(
1990
).
Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement.
J. Cell Biol
111
,
1137
1148
Wiseman
J. W.
,
Glover
L. A.
,
Hesketh
J. E.
(
1997
).
Evidence for a localization signal in the 3untranslated region of myosin heavy chain messenger RNA.
Cell. Biol. Int
21
,
243
248
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