Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.

REFERENCES

Amano
M.
,
Ito
M.
,
Kimura
K.
,
Fukata
Y.
,
Chihara
K.
,
Nakano
T.
,
Matsuura
Y.
,
Kaibuchi
K.
(
1996
).
Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).
J. Biol. Chem
271
,
20246
20249
Bell
G. I.
(
1978
).
Models for the specific adhesion of cells to cells.
Science
200
,
618
627
Bi
G.-Q.
,
Morris
R. L.
,
Liao
G.
,
Alderton
J. M.
,
Scholey
J. M.
,
Steinhardt
R. A.
(
1997
).
Kinesin-and Myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.
J. Cell Biol
138
,
999
1008
Buck
C. A.
,
Horwitz
A. F.
(
1987
).
Cell surface receptors for extracellular matrix molecules.
Annu. Rev. Cell Biol
3
,
179
205
Chew
T. L.
,
Masaracchia
R. A.
,
Goeckeler
Z. M.
,
Wysolmerski
R. B.
(
1998
).
Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK).
J. Muscle Res. Cell Motil
19
,
839
854
Ciano
P. S.
,
Colvin
R. B.
,
Dvorak
A. M.
,
McDonagh
J.
,
Dvorak
H. F.
(
1986
).
Macrophage migration in fibrin gel matrices.
Lab. Invest
54
,
62
70
Citi
S.
,
Kendrick-Jones
J.
(
1987
).
Regulation of non-muscle myosin structure and function.
BioEssays
7
,
155
159
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
Cramer
L. P.
,
Mitchison
T. J.
,
Theriot
J. A.
(
1994
).
Actin-dependent motile forces and cell motility.
Curr. Opin. Cell Biol
6
,
82
86
Cramer
L. P.
,
Mitchison
T. J.
(
1995
).
Myosin is involved in postmitotic cell spreading.
J. Cell Biol
131
,
179
189
Crowley
E.
,
Horwitz
A. F.
(
1995
).
Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions.
J. Cell Biol
131
,
525
537
Devreotes
P. N.
,
Zigmond
S. H.
(
1988
).
Chemotaxis in Eukaryotic Cells: A focus on leukocytes and Dictyostelium.
Annu. Rev. Cell Biol
8
,
649
686
Giancotti
F. G.
,
Ruoslahti
E.
(
1990
).
Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells.
Cell
60
,
849
859
Gillespie
G. Y.
,
Soroceanu
L.
,
Manning
T. J. J.
,
Gladson
C. L.
,
Rosenfeld
S. S.
(
1999
).
Glioma migration can be blocked by nontoxic inhibitors of myosin II.
Cancer Res
59
,
2076
2082
Grinnell
F.
(
1994
).
Fibroblasts, myofibroblasts, and wound contraction.
J. Cell Biol
124
,
401
404
Hashimoto
Y.
,
Nakayama
T.
,
Teramoto
T.
,
Kato
H.
,
Watanabe
T.
,
Kinoshita
M.
,
Tsukamoto
K.
,
Tokunaga
K.
,
Kurokawa
K.
,
Nakanishi
S.
,
Matsuda
Y.
,
Nonomura
Y.
(
1991
).
Potent and preferential inhibition of Ca2+/calmodulin-dependent protein kinase II by K252a and its derivative, KT5926.
Biochem. Biophys. Res. Commun
181
,
423
429
Hendey
B.
,
Klee
C. B.
,
Maxfield
F. R.
(
1992
).
Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin.
Science
258
,
296
299
Hendey
B.
,
Maxfield
F. R.
(
1993
).
Regulation of neutrophil motility and adhesion by intracellular calcium transients.
Blood Cells
19
,
143
161
Hendey
B.
,
Lawson
M.
,
Marcantonio
E. E.
,
Maxfield
F. R.
(
1996
).
Intracellular calcium and calcineurin regulate neutrophil motility onvitronectin through a receptor identified by antibodies to integrinsv and 3.
Blood
87
,
2038
2048
Hibbs
M. L.
,
Jakes
S.
,
Stacker
S. A.
,
Wallace
R. W.
,
Springer
T. A.
(
1991
).
The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.
J. Exp. Med
174
,
1227
1238
Huttenlocher
A.
,
Sandborg
R. R.
,
Horwitz
A. F.
(
1995
).
Adhesion in cell migration.
Curr. Opin. Cell Biol
7
,
697
706
Huttenlocher
A.
,
Palecek
S. P.
,
Lu
Q.
,
Zhang
W.
,
Mellgren
R. L.
,
Lauffenburger
D. A.
,
Ginsberg
M. H.
,
Horwitz
A. F.
(
1997
).
Regulation of cell migration by the calcium-dependent protease calpain.
J. Biol. Chem
272
,
32719
32722
Ingber
D.
(
1991
).
Integrins as mechanochemical transducers.
Curr. Opin. Cell Biol
3
,
841
848
Ingber
D. E.
(
1993
).
Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton.
J. Cell Sci
104
,
613
627
Isemura
M.
,
Mita
T.
,
Satoh
K.
,
Narumi
K.
,
Motomiya
M.
(
1991
).
Myosin light chain kinase inhibitors ML-7 and ML-9 inhibit mouse lung carcinoma cell attachment to the fibronectin substratum.
Cell Biol. Int. Rep
15
,
965
972
Jay
P. Y.
,
Pham
P. A.
,
Wong
S. A.
,
Elson
E. L.
(
1995
).
A mechanical function of myosin II in cell motility.
J. Cell Sci
108
,
387
393
Kuo
S. C.
,
Lauffenburger
D. A.
(
1993
).
Relationship between receptor/ligand binding affinity and adhesion strength.
Biophys. J
65
,
2191
2200
Kureishi
Y.
,
Kobayashi
S.
,
Amano
M.
,
Kimura
K.
,
Kanaide
H.
,
Nakano
T.
,
Kaibuchi
K.
,
Ito
M.
(
1997
).
Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.
J. Biol. Chem
272
,
12257
12260
Lawson
M. A.
,
Maxfield
F. R.
(
1995
).
Ca2+-and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils.
Nature
377
,
75
79
Lee
J.
,
Leonard
M.
,
Oliver
T.
,
Ishihara
A.
,
Jacobson
K.
(
1994
).
Traction forces generated by locomoting keratocytes.
J. Cell Biol
127
,
1957
1964
Mandeville
J. T.
,
Ghosh
R. N.
,
Maxfield
F. R.
(
1995
).
Intracellular calcium levels correlate with speed and persistent forward motion in migrating neutrophils.
Biophys. J
68
,
1207
1217
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
Marks
P. W.
,
Hendey
B.
,
Maxfield
F. R.
(
1991
).
Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration.
J. Cell Biol
112
,
149
158
Matsumura
F.
,
Ono
S.
,
Yamakita
Y.
,
Totsukawa
G.
,
Yamashiro
S.
(
1998
).
Specific localization of serine 19 phosphorylated myosin II duringcell locomotion and mitosis of cultured cells.
J. Cell Biol
140
,
119
129
Mizuno
T.
,
Kagami
O.
,
Sakai
T.
,
Kawasaki
K.
(
1996
).
Locomotion of neutrophil fragments occurs by graded radial extension.
Cell Motil. Cytoskel
35
,
289
297
Nakanishi
S.
,
Yamada
K.
,
Iwahashi
K.
,
Kuroda
K.
,
Kase
H.
(
1990
).
KT5926, a potent and selective inhibitor of myosin light chain kinase.
Mol. Pharmacol
37
,
482
488
Niggli
V.
(
1999
).
Rho-kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration.
FEBS Lett
445
,
69
72
Regen
C. M.
,
Horwitz
A. F.
(
1992
).
Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.
J. Cell Biol
119
,
1347
1359
Ruchhoeft
M. L.
,
Harris
W. A.
(
1997
).
Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo.
J. Neurobiol
32
,
567
578
Saitoh
M.
,
Naka
M.
,
Hidaka
H.
(
1986
).
The modulatory role of myosin light chain phosphorylation in human platelet activation.
Biochem. Biophys. Res. Commun
140
,
280
287
Saitoh
M.
,
Ishikawa
T.
,
Matsushima
S.
,
Naka
M.
,
Hidaka
H.
(
1987
).
Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase.
J. Biol. Chem
262
,
7796
7801
Schmidt
C. E.
,
Chen
T.
,
Lauffenburger
D. A.
(
1994
).
Simulation of integrin-cytoskeletal interactions in migrating fibroblasts.
Biophys. J
67
,
461
474
Sellers
J. R.
(
1991
).
Regulation of cytoplasmic and smooth muscle myosin.
Curr. Opin. Cell Biol
3
,
98
104
Smilenov
L.
,
Mikhailov
A.
,
Pelman
R. J. J.
,
Marcantonio
E. E.
,
Gundersen
G. G.
(
1999
).
Focal adhesion motility revealed in stationary fibroblasts.
Science
286
,
1172
1174
Soll
D. R.
,
Voss
E.
,
Varnum-Finney
B.
,
Wessels
D.
(
1988
).
‘Dynamic Morphological System’: A method for quantitating changes in shape, pseudopod formation, and motion in normal and mutant amoebae of Dictyostelium discoideum.
J. Cell. Biochem
37
,
177
192
Spudich
J. A.
(
1989
).
In pursuit of myosin function.
Cell Regul
1
,
1
11
Tranquillo
R. T.
,
Lauffenburger
D. A.
,
Zigmond
S. H.
(
1988
).
Astochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations.
J. Cell Biol
106
,
303
309
Trybus
K. M.
(
1991
).
Regulation of smooth muscle myosin.
Cell Motil. Cytoskel
18
,
81
85
Valerius
N. H.
,
Stendahl
O.
,
Hartwig
J. H.
,
Stossel
T. P.
(
1981
).
Distribution of actin-binding protein and myosin in polymorphonuclear leukocytes during locomotion and phagocytosis.
Cell
24
,
195
202
Welch
M. D.
,
Mallavarapu
A.
,
Rosenblatt
J.
,
Mitchison
T. J.
(
1997
).
Actin dynamics in vivo.
Curr. Opin. Cell Biol
9
,
54
61
Wessels
D.
,
Soll
D. R.
,
Knecht
D.
,
Loomis
W. F.
,
De Lozanne
A.
,
Spudich
J.
(
1988
).
Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain.
Dev. Biol
128
,
164
177
Wilson
A. K.
,
Pollenz
R. S.
,
Chisholm
R. L.
,
de Lanerolle
P.
(
1992
).
The role of myosin I and II in cell motility.
Cancer Metast. Rev.
11
,
79
91
Yumura
S.
,
Mori
H.
,
Fukui
Y.
(
1984
).
Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence.
J. Cell Biol
99
,
894
899
This content is only available via PDF.