Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.

REFERENCES

Abercrombie
M.
,
Heaysman
J. E. M.
,
Pegrun
S. M.
(
1970
).
The locomotion of fibroblasts in culture. I. Movements of the leading edge.
Exp. Cell Res
59
,
393
398
Argraves
W. S.
,
Suzuki
S.
,
Arai
H.
,
Thompson
K.
,
Pierschbacher
M.
,
Ruoslahti
E.
(
1987
).
Amino acid sequence of the human fibronectin receptor.
J. Cell Biol
105
,
1183
1190
Bajt
M. L.
,
Loftus
J. C.
,
Gawaz
M. P.
,
Ginsberg
M. H.
(
1992
).
Characterization of a gain of function mutation of integrinIIb 3 (platelet glycoprotein IIb-IIIa).
J. Biol. Chem
269
,
20913
20919
Bard
J. B. L.
,
Hay
E. D.
(
1975
).
The behavior of fibroblasts from the developing avian cornea.
J. Cell Biol
67
,
400
418
Beckerle
M. C.
,
Burridge
K.
,
DeMartino
G. N.
,
Croall
D. E.
(
1987
).
Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion.
Cell
51
,
569
577
Burridge
K.
,
Faith
K.
,
Kelly
T.
,
Nuckolls
G.
,
Turner
C. E.
(
1988
).
Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.
Annu. Rev. Cell Biol
4
,
487
525
Chen
W.-T.
(
1979
).
Induction of spreading during fibroblast movement.
J. Cell Biol
81
,
684
691
Chen
W.-T.
(
1981
).
Mechanism of retraction of the trailing edge during fibroblast movement.
J. Cell Biol
90
,
187
200
Cooray
P.
,
Yuan
Y.
,
Schoenwaelder
S. M.
,
Mitchell
C. A.
,
Salem
H. H.
,
Jackson
S. P.
(
1996
).
Focal adhesion kinase (pp125Fak) cleavage and regulation by calpain.
Biochem. J
318
,
41
47
Condeelis
J.
(
1993
).
Life at the leading edge.
Annu. Rev. Cell Biol
9
,
411
444
Couchman
J. R.
,
Rees
D. A.
,
Green
M. R.
,
Smith
C. G.
(
1982
).
Fibronectin has a dual role in locomotion and anchorage of primary chick fibroblasts and can promote entry into the division cycle.
J. Cell Biol
93
,
402
410
Crowley
E.
,
Horwitz
A. F.
(
1995
).
Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions.
J. Cell Biol
131
,
525
537
DiMilla
P. A.
,
Barbee
K.
,
Lauffenburger
D. A.
(
1991
).
Mathematical model for the effects of adhesion and mechanics on cell migration speed.
Biophys. J
60
,
15
37
DiMilla
P. A.
,
Stone
J. A.
,
Quinn
J. A.
,
Albelda
S. M.
,
Lauffenburger
D. A.
(
1993
).
Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength.
J. Cell Biol
122
,
729
737
Du
X.
,
Saido
T. C.
,
Tsubuki
S.
,
Indig
F. E.
,
Williams
M. J.
,
Ginsberg
M. H.
(
1995
).
Calpain cleavage of the cytoplasmic domain of the integrin3 subunit.
J. Biol. Chem
270
,
26146
26151
Duband
J. L.
,
Nuckolls
G. H.
,
Ishihara
A.
,
Hasegawa
T.
,
Yamada
K. M.
,
Thiery
J. P.
,
Jacobson
K.
(
1988
).
Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells.
J. Cell Biol
107
,
1385
1396
Duband
J.-L.
,
Dufour
S.
,
Yamada
S. S.
,
Yamada
K. M.
,
Thiery
J. P.
(
1991
).
Neural crest cell locomotion induced by antibodies to1 integrins: a tool for studying the roles of substratum molecular avidity and density in migration.
J. Cell Sci
98
,
517
532
Dunn
A. D.
,
Zicha
D.
(
1995
).
Dynamics of fibroblast spreading.
J. Cell Sci
108
,
1239
1249
Dustin
M. L.
,
Bromley
S. K.
,
Kan
Z.
,
Peterson
D. A.
,
Unanue
E. R.
(
1997
).
Antigen receptor engagement delivers a stop signal to migrating T lymphocytes.
Proc. Nat. Acad. Sci. USA
94
,
3909
3913
Felder
S.
,
Elson
E. L.
(
1990
).
Mechanics of fibroblast locomotion: quantitative analysis of forces and motions at the leading lamellas of fibroblasts.
J. Cell Biol
111
,
2527
2536
Frelinger
A. L.
,
Du
X.
,
Plow
E. F.
,
Ginsberg
M. H.
(
1991
).
Monoclonal antibodies to ligand-occupied conformer of integrinIIb 3 (glycoprotein IIb-IIa) alter receptor affinity, specificity and function.
J. Biol. Chem
266
,
17106
17111
Goodman
S. L.
,
Risse
G.
,
von der Mark
K.
(
1989
).
The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix.
J. Cell Biol
109
,
799
809
Hahn
K.
,
DeBiasio
R.
,
Taylor
D. L.
(
1992
).
Patterns of elevated free calcium and calcineurin activation in living cells.
Nature
359
,
736
738
Hay
E. D.
(
1985
).
Interaction of migrating embryonic cells with extracellular matrix.
Exp. Biol. Med
10
,
174
193
Hendey
B.
,
Klee
C. B.
,
Maxfield
F. R.
(
1992
).
Inhibition of neutrophil chemotaxis on vitronectin by inhibitors of calcineurin.
Science
258
,
296
299
Huttenlocher
A.
,
Sandborg
R. R.
,
Horwitz
A. F.
(
1995
).
Adhesion in cell migration.
Curr. Opin. Cell Biol
7
,
697
706
Huttenlocher
A.
,
Ginsberg
M. H.
,
Horwitz
A. F.
(
1996
).
Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity.
J. Cell Biol
134
,
1551
1562
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
Hynes
R. O.
(
1992
).
Integrins: versatility, modulation and signaling in cell adhesion.
Cell
69
,
11
25
Inomata
M.
,
Hayashi
M.
,
Ohno-Iwashita
Y.
,
Tsubuki
S.
,
Saido
T. C.
,
Kawahima
S.
(
1996
).
Involvement of calpain in integrin-mediated signal transduction.
Arch. Biochem. Biophys
328
,
129
134
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
Keely
P. J.
,
Fong
A. M.
,
Zutter
M. M.
,
Santoro
S. A.
(
1995
).
Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of anti-sense2 integrin mRNA in mammary cells.
J. Cell Sci
108
,
595
607
Kuijpers
T. W.
,
Mul
E. P. J.
,
Blom
M.
,
Kovach
N. L.
,
Gaeta
F. C. A.
,
Tollefson
V.
,
Elices
M. J.
,
Harlan
J. M.
(
1993
).
Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration.
J. Exp. Med
178
,
279
284
Lauffenburger
D. A.
,
Horwitz
A. F.
(
1996
).
Cell migration: a physically integrated molecular progress.
Cell
84
,
359
369
Loftus
J. C.
,
O'Toole
T. E.
,
Plow
E. F.
,
Glass
A.
,
Frelinger
A. L.
,
Ginsberg
M. H.
(
1990
).
A3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation.
Science
249
,
915
918
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
Mitchison
T. J.
,
Cramer
L. P.
(
1996
).
Actin-based cell motility and cell locomotion.
Cell
84
,
371
379
Niggemann
B.
,
Maaser
K.
,
Lu
H.
,
Kroczek
R.
,
Zanker
K. S.
,
Friedl
P.
(
1997
).
Locomotory phenotypes of human tumor cell lines and T lymphocytes in a three-dimensional collagen lattice.
Canc. Lett
118
,
173
–.
O'Toole
T. E.
,
Loftus
J. C.
,
Plow
E. F.
,
Glass
A.
,
Harper
J. R.
,
Ginsberg
M. H.
(
1989
).
Efficient surface expression of platelet GPIIb-IIIa requires both subunits.
Blood
74
,
14
18
O'Toole
T. E.
,
Loftus
J. C.
,
Du
X.
,
Glass
A. A.
,
Ruggeri
Z. M.
,
Shattil
S. J.
,
Plow
E. F.
,
Ginsberg
M. H.
(
1990
).
Affinity modulation of theIIb 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.
Cell Reg
1
,
883
893
O'Toole
T. E.
,
Katagiri
Y.
,
Faull
R. J.
,
Peter
K.
,
Tamura
R.
,
Quaranta
V.
,
Loftus
J.
,
Shattil
S. J.
,
Ginsberg
M. H.
(
1994
).
Integrin cytoplasmic domains mediate inside-out signal transduction.
J. Cell Biol
125
,
447
460
Palecek
S. P.
,
Schmidt
C. E.
,
Lauffenburger
D. A.
,
Horwitz
A. F.
(
1996
).
Integrin dynamics on the tail region of migrating fibroblasts.
J. Cell Sci
109
,
941
952
Palecek
S. P.
,
Loftus
J. C.
,
Ginsberg
M. H.
,
Lauffenburger
D. A.
,
Horwitz
A. F.
(
1997
).
Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.
Nature
385
,
537
540
Regen
C. M.
,
Horwitz
A. F.
(
1992
).
Dynamics of1 integrin-mediated adhesive contacts in motile fibroblasts.
J. Cell Biol
119
,
1347
1359
Reszka
A.
,
Hayashi
Y.
,
Horwitz
A. F.
(
1992
).
Identification of amino acid sequences in the integrin1 cytoplasmic domain implicated in cytoskeletal association.
J. Cell Biol
117
,
1321
1330
Ruoslahti
E.
,
Hayman
E. G.
,
Pierschbacher
M.
,
Engvall
E.
(
1982
).
Fibronectin: purification, immunochemical properties, and biological activities.
Meth. Enzymol
82
,
803
831
Sastry
S.
,
Lakonishok
M.
,
Thomas
D. A.
,
Muschler
J.
,
Horwitz
A. F.
(
1996
).
Integrinsubunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation.
J. Cell Biol
133
,
169
184
Saterbak
A.
,
Lauffenburger
D. A.
(
1996
).
Adhesion mediated by bonds in series.
Biotechnol. Prog
12
,
682
699
Schmidt
C. E.
,
Horwitz
A. F.
,
Lauffenburger
D. A.
,
Sheetz
M. P.
(
1993
).
Integrin/cytoskeleton interactions in migrating fibroblasts are dynamic, asymmetric, and regulated.
J. Cell Biol
123
,
977
991
Sheetz
M. P.
(
1994
).
Cell migration by graded attachment to substrates and contraction.
Semin. Cell Biol
5
,
149
155
Stossel
T. P.
(
1993
).
On the crawling of animal cells.
Science
260
,
1086
1094
Weber
I.
,
Wallraff
E.
,
Albrecht
R.
,
Gerisch
G.
(
1995
).
Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis.
J. Cell Sci
108
,
1519
1530
Werb
Z.
(
1997
).
ECM and cell surface proteolysis: regulating cellular ecology.
Cell
91
,
439
442
Wessels
D.
,
Vawter-Hugart
H.
,
Murray
J.
,
Soll
D. R.
(
1994
).
Three-dimensional dynamics of pseudopod formation and the regulation of turning during the motility cycle of Dictyostelium.
Cell Motil. Cytoskel
27
,
1
12
Wilson
A.
,
Gorge
G.
,
Claypool
W.
,
DeLanerolle
P.
(
1991
).
An increases or a decrease in myosin II phosphorylation inhibits macrophage motility.
J. Cell Biol
114
,
277
283
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