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doi: 10.1242/10.1242/jcs.00325
Research Article |
A-crystallin-knockout mouse
1 Department of Ophthalmology and Visual Sciences, Washington University School
of Medicine, St Louis, MO 63110, USA
2 Department of Biochemistry and Molecular Biophysics, Washington University
School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: andley{at}vision.wustl.edu)
Accepted 17 December 2002
A-Crystallin (A) is a molecular chaperone expressed
preferentially in the lens. A transcripts are first detected during the
early stages of lens development and its synthesis continues as the lens grows
throughout life. A–/– mouse lenses are smaller
than controls, and lens epithelial cells derived from these mice have
diminished growth in culture. In the current work, we tested the hypothesis
thatA prevents cell death at a specific stage of the cell cycle in
vivo. Seven-day-old 129Sv (wild-type) and A–/–
mice were injected with 5-bromo-2'-deoxyuridine (BrdU) to label newly
synthesized DNA in proliferating cells. To follow the fate of the labeled
cells, wholemounts of the capsule epithelial explants were made at successive
times after the BrdU pulse, and the labeling index was determined.
Immunofluorescence and confocal microscopy showed that both wild-type and
A–/– cells had a 3-hour labeling index of 4.5%
in the central region of the wholemount, indicating that the number of cells
in S phase was the same. Twenty-four hours after the pulse, individual cells
labeled with BrdU had divided and BrdU-labeled cells were detected in pairs.
The 24-hour labeling index in the wild-type lens was 8.6%, but in the
A–/– lens it was significantly lower, suggesting
that some of the cells failed to divide and/or that the daughter cells died
during mitosis. TUNEL labeling was rarely detected in the wild-type lens, but
was significant and always detected in pairs in the
A–/– wholemounts. Dual labeling with TUNEL and
BrdU also suggested that the labeled cells were dying in pairs in the
A–/– lens epithelium. Immunolabeling of
wholemounts with ß-tubulin antibodies indicated that the anaphase spindle
in a significant proportion of A–/– cells was
not well organized. Examination of the cellular distribution of A in
cultured lens epithelial cells showed that it was concentrated in the
intercellular microtubules of cells undergoing cytokinesis. These data suggest
that A expression in vivo protects against cell death during mitosis in
the lens epithelium, and the smaller size of the
A–/– lens may be due to a decrease in the net
production of epithelial cells.
Key words: A-Crystallin, Lens, Chaperone, Cell death
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