To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

Albers
K.
,
Fuchs
E.
(
1989
).
Expression of mutant keratin cDNAs inepithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
J. Cell Biol
108
,
1477
1493
Aubin
J. E.
,
Osborn
M.
,
Franke
W. W.
,
Weber
K.
(
1980
).
Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis.
Exp. Cell Res
129
,
149
165
Bachant
J. B.
,
Klymkowsky
M. W.
(
1996
).
A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates.
J. Cell Biol
132
,
153
165
Bologna
M.
,
Allen
R.
,
Dulbecco
R.
(
1986
).
Organization of cytokeratin bundles by desmosomes in rat mammary cells.
J. Cell Biol
102
,
560
567
Brown
D. T.
,
Anderton
B. H.
,
Wylie
C. C.
(
1983
).
Alterations in the organisation of cytokeratin filaments in normal and malignant human colonic epithelial cells during mitosis.
Cell Tissue Res
233
,
619
628
Celis
J. E.
,
Larsen
P. M.
,
Fey
S. J.
,
Celis
A.
(
1983
).
Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.
J. Cell Biol
97
,
1429
1434
Celis
J. E.
,
Small
J. V.
,
Larsen
P. M.
,
Fey
S. J.
,
De Mey
J.
,
Celis
A.
(
1984
).
Intermediate filaments in monkey kidney TC7 cells: focal centers and interrelationship with other cytoskeletal systems.
Proc. Nat. Acad. Sci. USA
81
,
1117
1121
Chou
C.-F.
,
Riopel
C. L.
,
Rott
L. S.
,
Omary
M. B.
(
1993
).
A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility.
J. Cell Sci
105
,
433
444
Deery
W. J.
(
1993
).
Role of phosphorylation in keratin and vimentin filament integrity in cultured thyroid epithelial cells.
Cell Motil. Cytoskel
26
,
325
339
Eckert
B. S.
,
Daley
R. A.
,
Parysek
L. M.
(
1982
).
Assembly of keratin onto PtK1cytoskeletons: evidence for an intermediate filament organizing center.
J. Cell Biol
92
,
575
578
Eger
A.
,
Stockinger
A.
,
Wiche
G.
,
Foisner
R.
(
1997
).
Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells.
J. Cell Sci
110
,
1307
1316
Eriksson
J. E.
,
Opal
P.
,
Goldman
R. D.
(
1992
).
Intermediate filament dynamics.
Curr. Opin. Cell Biol
4
,
99
104
Foisner
R.
,
Leichtfried
F. E.
,
Herrmann
H.
,
Small
J. V.
,
Lawson
D.
,
Wiche
G.
(
1988
).
Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.
J. Cell Biol
106
,
723
733
Franke
W. W.
,
Schmid
E.
,
Osborn
M.
,
Weber
K.
(
1978
).
Different intermediate-sized filaments distinguished by immunofluorescence microscopy.
Proc. Nat. Acad. Sci. USA
75
,
5034
5038
Franke
W. W.
,
Weber
K.
,
Osborn
M.
,
Schmid
E.
,
Freudenstein
C.
(
1978
).
Antibody to prekeratin. Decoration of tonofilament-like arrays in various cells of epithelial character.
Exp. Cell Res
116
,
429
445
Franke
W. W.
,
Schmid
E.
,
Grund
C.
,
Geiger
B.
(
1982
).
Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates.
Cell
30
,
103
113
Franke
W. W.
,
Schmid
E.
,
Wellsteed
J.
,
Grund
C.
,
Gigi
O.
,
Geiger
B.
(
1983
).
Change of cytokeratin filament organization during the cell cycle: selective masking of an immmunologic determinant in interphase PtK2cells.
J. Cell Biol
97
,
1255
1260
Franke
W. W.
,
Schmid
E.
,
Mittnacht
S.
,
Grund
C.
,
Jorcano
J. L.
(
1984
).
Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells.
Cell
36
,
813
825
Franke
W. W.
,
Winter
S.
,
Schmid
E.
,
Söllner
P.
,
Hämmerling
G.
,
Achtstätter
T.
(
1987
).
Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution.
Exp. Cell Res
173
,
17
37
Fuchs
E.
,
Weber
K.
(
1994
).
Intermediate filaments: structure, dynamics, function and disease.
Annu. Rev. Biochem
63
,
345
382
Fuchs
E.
,
Cleveland
D. W.
(
1998
).
A structural scaffolding of intermediate filaments in health and disease.
Science
279
,
514
519
Gard
D. L.
,
Cha
B. J.
,
King
E.
(
1997
).
The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon f-actin and microtubules.
Dev. Biol
184
,
95
114
Gerdes
H.-H.
,
Kaether
C.
(
1996
).
Green fluorescent protein: applications in cell biology.
FEBS Lett
389
,
44
47
Green
K. J.
,
Geiger
B.
,
Jones
J. C. R.
,
Talian
J. C.
,
Goldman
R. D.
(
1987
).
The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.
J. Cell Biol
104
,
1389
1402
Heim
R.
,
Prasher
D. C.
,
Tsien
R. Y.
(
1994
).
Wavelength mutations and posttranslational autoxidation of green fluorescent protein.
Proc. Nat. Acad. Sci. USA
91
,
12501
12504
Hirokawa
N.
,
Tilney
L. G.
,
Fujiwara
K.
,
Heuser
J. E.
(
1982
).
Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells.
J. Cell Biol
94
,
425
443
Ho
C.-L.
,
Martys
J. L.
,
Mikhailov
A.
,
Gundersen
G. G.
,
Liem
R. K. H.
(
1998
).
Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras.
J. Cell Sci
111
,
1767
1778
Hofmann
I.
,
Franke
W. W.
(
1997
).
Heterotypic interactions and filament assembly of type I and type II cytokeratins in vitro: viscometry and determinations of relative affinities.
Eur. J. Cell Biol
72
,
122
132
Horwitz
B.
,
Kupfer
H.
,
Eshhar
Z.
,
Geiger
B.
(
1981
).
Reorganization of arrays of prekeratin filaments during mitosis.
Exp. Cell Res
134
,
281
290
Jones
J. C. R.
,
Goldman
A.
,
Yang
H.-Y.
,
Goldman
R. D.
(
1985
).
The organizational fate of intermediate filament networks in two epithelial cell types during mitosis.
J. Cell Biol
100
,
93
102
Kitajima
Y.
,
Inoue
S.
,
Yoneda
K.
,
Mori
S.
,
Yaoita
H.
(
1985
).
Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes.
Eur. J. Cell Biol
38
,
219
225
Kitajima
Y.
,
Inoue
S.
,
Yaoita
H.
(
1986
).
Reorganization of keratin intermediate filaments by the drug-induced disruption of microfilaments in cultured human keratinocytes.
J. InveSt Dermatol
87
,
565
569
Klymkowsky
M. W.
,
Maynell
L. A.
,
Nislow
C.
(
1991
).
Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1.
J. Cell Biol
114
,
787
797
Kreis
T. E.
,
Geiger
B.
,
Schmid
E.
,
Jorcano
J. L.
,
Franke
W. W.
(
1983
).
De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin.
Cell
32
,
1125
1137
Ku
N. O.
,
Liao
J.
,
Omary
M. B.
(
1998
).
Phosphorylation of human keratin 18 serine 33 regulates binding to 14–3-3 proteins.
EMBO J
17
,
1892
1906
Kuruc
N.
,
Leube
R. E.
,
Moll
I.
,
Bader
B. L.
,
Franke
W. W.
(
1989
).
Synthesis of cytokeratin 13, a component characteristic of internal stratified epithelia, is not induced in human epidermal tumors.
Differentiation
42
,
111
123
Lai
Y.-K.
,
Lee
W.-C.
,
Chen
K.-D.
(
1993
).
Vimentin serves as a phosphate sink during the apparent activation of protein kinases by okadaic acid in mammalian cells.
J. Cell. Biochem
53
,
161
168
Lane
E. B.
,
Goodman
S. L.
,
Trejdosiewicz
L. K.
(
1982
).
Disruption of the keratin filament network during epithelial cell division.
EMBO J
1
,
1365
1372
Lawrence
J. B.
,
Singer
R. H.
(
1986
).
Intracellular localization of messenger RNAs for cytoskeletal proteins.
Cell
45
,
407
415
Leube
R. E.
,
Bader
B. L.
,
Bosch
F. X.
,
Zimbelmann
R.
,
Achtstaetter
T.
,
Franke
W. W.
(
1988
).
Molecular characterization and expression of the stratification-related cytokeratins 4 and 15.
J. Cell Biol
106
,
1249
1261
Leube
R. E.
,
Wiedenmann
B.
,
Franke
W. W.
(
1989
).
Topogenesis and sorting of synaptophysin: synthesis of a synaptic vesicle protein from a gene transfected into nonneuroendocrine cells.
Cell
59
,
433
446
Liao
J.
,
Lowthert
L. A.
,
Ku
N.-O.
,
Fernandez
R.
,
Omary
M. B.
(
1995
).
Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues.
J. Cell Biol
131
,
1291
1301
Liao
J.
,
Omary
M. B.
(
1996
).
14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor.
J. Cell Biol
133
,
345
357
Ludin
B.
,
Doll
T.
,
Meili
R.
,
Kaech
S.
,
Matus
A.
(
1996
).
Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins.
Gene
173
,
107
111
McLean
W. H. I.
,
Lane
E. B.
(
1995
).
Intermediate filaments in disease.
Curr. Opin. Cell Biol
7
,
118
125
Martys
J. L.
,
Ho
C.-L.
,
Liem
R. K. H.
,
Gundersen
G. G.
(
1999
).
Intermediate filaments in motion: Observations of intermediate filaments in cells using green fluorescent protein-vimentin.
Mol. Biol. Cell
10
,
1289
1295
Miller
R. K.
,
Vikstrom
K.
,
Goldman
R. D.
(
1991
).
Keratin incorporation into intermediate filament networks is a rapid process.
J. Cell Biol
113
,
843
855
Miller
R. K.
,
Khuon
S.
,
Goldman
R. D.
(
1993
).
Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo.
J. Cell Biol
122
,
123
135
Moll
R.
,
Franke
W. W.
,
Schiller
D. L.
,
Geiger
B.
,
Krepler
R.
(
1982
).
The catalog of human cytokeratin polypeptides: patterns of expression of specific cytokeratins in normal epithelia, tumors and cultured cells.
Cell
31
,
11
24
Olson
K. R.
,
McIntosh
J. R.
,
Olmsted
J. B.
(
1995
).
Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.
J. Cell Biol
130
,
639
650
Osborn
M.
,
Franke
W. W.
,
Weber
K.
(
1977
).
Visualization of a system of filaments 7–10 nm thick in cultured cells of an epitheloid line (PtK2) by immunofluorescence microscopy.
Proc. Nat. Acad. Sci. USA
74
,
2490
2494
Paramio
J. M.
,
Jorcano
J. L.
(
1994
).
Assembly dynamics of epidermal keratins K1 and K10 in transfected cells.
Exp. Cell Res
215
,
319
331
Paramio
J. M.
(
1999
).
A role for phosphorylation in the dynamics of keratin intermediate filaments.
Eur. J. Cell Biol
78
,
33
43
Prahlad
V.
,
Yoon
M.
,
Moir
R. D.
,
Vale
R. D.
,
Goldman
R. D.
(
1998
).
Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks.
J. Cell Biol
143
,
159
170
Rizzuto
R.
,
Brini
M.
,
Pizzo
P.
,
Murgia
M.
,
Pozzan
T.
(
1995
).
Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.
Curr. Biol
5
,
635
642
Schmidt
A.
,
Heid
H. W.
,
Schäfer
S.
,
Nuber
U. A.
,
Zimbelmann
R.
,
Franke
W. W.
(
1994
).
Desmosomes and cytoskeletal architecture in epithelial differentiation. Cell type-specific plaque components and intermediate filament anchorage. Eur.
J. Cell Biol
65
,
229
245
Sun
T.-T.
,
Green
H.
(
1978
).
Immunofluorescent staining of keratin fibers in cultured cells.
Cell
14
,
469
476
Toivala
D. M.
,
Goldman
R. D.
,
Garrod
D. R.
,
Eriksson
J. E.
(
1997
).
Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments.
J. Cell Sci
110
,
23
33
Tölle
H.-G.
,
Weber
K.
,
Osborn
M.
(
1987
).
Keratin filament disruption in interphase and mitotic cells—how is it induced?.
Eur. J. Cell Biol
43
,
35
47
Turner
B. M.
,
Ruane
M.
(
1985
).
Immunofluorescent localisation of cytokeratin antigens in mitotic HeLa cells using monoclonal antibodies.
Eur. J. Cell Biol
36
,
48
57
Wiche
G.
,
Krepler
R.
,
Artlieb
U.
,
Pytela
R.
,
Aberer
W.
(
1984
).
Identification of plectin in different human cell types and immunolocalization at epithelial basal cell surface membranes.
Exp. Cell Res
155
,
43
49
Wolf
K. M.
,
Mullins
J. M.
(
1987
).
Cytochalasin B-induced redistribution of cytokeratin filaments in PtK1cells.
Cell Motil. Cytoskel
7
,
347
360
Yang
Y.
,
Dowling
J.
,
Yu
Q.-C.
,
Kouklis
P.
,
Cleveland
D. W.
,
Fuchs
E.
(
1996
).
An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments.
Cell
86
,
655
665
Yoon
M.
,
Moir
R. D.
,
Prahlad
V.
,
Goldman
R. D.
(
1998
).
Motile properties of vimentin intermediate filament networks in living cells.
J. Cell Biol
143
,
147
157
This content is only available via PDF.

Supplementary information