The intracellular distribution of filamentous actin (F-actin), all actin isoforms and beta-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the beta-actin mRNA were used, and visualized with avidin-FITC. The intracellular localization of the beta-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The beta-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.

REFERENCES

Adams
A.
,
Fey
E. G.
,
Pike
S. F.
,
Taylorson
C. J.
,
White
H. A.
,
Rabin
B. R.
(
1983
).
Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.
Biochem. J
216
,
215
226
Batten
B. E.
,
Aalberg
J. J.
,
Anderson
E.
(
1980
).
The cytoplasmic filamentous network in cultured ovarian granulosa cells.
Cell
44
,
885
895
Ben-Ze'ev
A.
,
Duerr
A.
,
Solomon
F.
,
Penman
S.
(
1979
).
The outer boundary of the cytoskeleton: A lamina derived from plasma membrane proteins.
Cell
17
,
859
865
Cheng
H.
,
Bjerknes
M.
(
1989
).
Asymmetric distribution of actin mRNAand cytoskeletal pattern generation in polarized epithelial cells.
J. Mol. Biol
210
,
541
549
Hayashi
M.
,
Ninomiya
Y.
,
Hayashi
K.
,
Linsenmayer
T. F.
,
Olsen
B. R.
,
Trelstad
R. L.
(
1988
).
Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry.
Development
103
,
27
36
Hoock
T. C.
,
Newcomb
P. M.
,
Herman
I. M.
(
1991
).
Beta actin and its mRNA are localized at the plasma membrane and the regions of the moving cytoplasm during cellular response to injury.
J. Cell Biol
112
,
653
664
Jeffery
W. R.
,
Meier
S.
(
1983
).
A yellow crescent cytoskeletal domain in Ascidian eggs and its role in early development.
Dev. Biol
96
,
125
143
Lawrence
J. B.
,
Singer
R. H.
(
1986
).
Intracellular localization of messenger RNA for cytoskeletal proteins.
Cell
45
,
407
415
Lenk
R.
,
Ransom
L.
,
Kaufmann
Y.
,
Penman
S.
(
1977
).
A cytoskeletal structure with associated polyribosomes obtained from HeLa cells.
Cell
10
,
67
78
Lenk
R.
,
Penman
S.
(
1979
).
The cytoskeletal framework and poliovirus metabolism.
Cell
16
,
289
301
Lessard
J. L.
(
1988
).
Two monoclonal antibodies to actin, One muscle selective and one generally reactive.
Cell Motil. Cytoskel
10
,
349
362
Meier
S.
,
Hay
E. D.
(
1973
).
Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium.
Dev. Biol
35
,
318
331
Mowry
K. L.
,
Melton
D. A.
(
1992
).
Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.
Science
255
,
991
994
Nakajima-Iijima
S.
,
Hamada
H.
,
Reddy
P.
,
Kakunaga
T.
(
1985
).
Molecular structure of the human cytoplasmic-actin gene: Interspecies homology of sequences in the introns.
Proc. Nat. Acad. Sci. USA
82
,
6133
6137
Ponte
P.
,
Gunning
P.
,
Blau
H.
,
Kedes
L.
(
1983
).
Human actin genes are single copy for-skeletal and -cardiac actin but multicopy for-and -cytoskeletal genes: 3untranslated regions are isotype specific but are conserved in evolution.
Mol. Cell. Biol
3
,
1783
1791
Ramaekers
F. C. S.
,
Bennedetti
E. L.
,
Dunia
I.
,
Vorstenbosch
P.
,
Bloemendal
H.
(
1983
).
Polyribosomes associated with microfilaments in cultured lens cells.
Biochim. Biophys
740
,
441
448
Schliwa
M.
(
1981
).
Proteins associated with cytoplasmic actin.
Cell
25
,
587
590
Schliwa
M.
(
1982
).
Action of cytochalasin D on cytoskeletal networks.
J. Cell Biol
92
,
79
91
Singer
R. H.
,
Langevin
G. L.
,
Lawrence
J. B.
(
1989
).
Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.
J. Cell Biol
108
,
6
–.
Stepp
M. A.
,
Spurr-Michaud
S.
,
Gipson
I. K.
(
1993
).
Integrins in the wounded and unwounded stratified squamous epithelium of the cornea. Invest. Ophthalmol.
Vis. Sci
34
,
1829
1844
Sugrue
S. P.
(
1987
).
Isolation of collagen binding proteins from embryonic chicken corneal epithelial cells.
J. Biol. Chem
262
,
3338
3343
Sugrue
S. P.
(
1988
).
Identification and immunolocalization of the laminin binding protein from embryonic avian corneal epithelial cells.
Differentiation
38
,
169
176
Sugrue
S. P.
,
Hay
E. D.
(
1981
).
Response of basal epithelial cell surface and cytoskeleton to solubilized extracellular matrix molecules.
J. Cell Biol
91
,
45
54
Sugrue
S. P.
,
Hay
E. D.
(
1982
).
Interaction of embryonic corneal epithelium with exogenous collagen, laminin, and fibronectin: role of endogenous protein synthesis.
Dev. Biol
92
,
97
106
Sugrue
S. P.
,
Hay
E. D.
(
1986
).
The identification of extracellular matrix (ECM) binding sites on the basal surface of embryonic corneal epithelium and the effect of ECM binding on epithelial collagen production.
J. Cell Biol
102
,
1907
1916
Sundell
C. L.
,
Singer
R. H.
(
1990
).
Actin mRNA localizes in the absence of protein synthesis.
J. Cell Biol
111
,
2397
2403
Sundell
C. L.
,
Singer
R. H.
(
1991
).
Requirement of microfilaments in sorting of actin messenger RNA.
Science
253
,
1275
1277
Svoboda
K. K. H.
,
Hay
E. D.
(
1987
).
Embryonic corneal epithelial interaction with exogenous laminin and basal lamina is F-actin dependent.
Dev. Biol
123
,
455
469
Svoboda
K. K. H.
(
1991
).
Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia.
J. Cell Sci
100
,
23
33
Svoboda
K. K. H.
(
1992
).
Embryonic corneal epithelial actin alters distribution in response to laminin. Invest. Ophthalmol.
Vis. Sci
33
,
324
333
Taneja
K. L.
,
Lifshitz
L. M.
,
Fay
F. S.
,
Singer
R. H.
(
1992
).
Poly(A) RNA codistribution with microfilaments: Evaluation by in situ hybridizationand quantitative digital imaging microscopy.
J. Cell Biol
119
,
1245
1260
Wang
Y. L.
(
1984
).
Reorganization of actin filament bundles in living fibroblasts.
J. Cell Biol
99
,
1478
1485
Wolosewick
J. J.
,
Porter
K. R.
(
1976
).
Stereo high-voltage electron microscopy of whole cells of the human diploid line, WI-38.
Amer. J. Anat
147
,
303
324
Wolosewick
J. J.
,
Porter
K. R.
(
1979
).
Microtrabecular lattice of the cytoplasmic ground substance, artifact or reality?.
J. Cell Biol
82
,
114
139
Yisraeli
J. K.
,
Sokol
S.
,
Melton
D. A.
(
1990
).
A two-step model for the localization of maternal mRNA in Xenopus oocytes: Involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA.
Development
108
,
289
298
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