S584 Biochemical Society Transactions (1997) 25
178
Lens cell organelle loss during differentiation
versus stress-induced apoptotic changes
RALF DAHh4, CHRIS GRIBBON, ROY A. QUINLAN, and ALAN R.
PRESCOTr
Dept of Biochemistry, University of Dundee, DD14HN, UK
The lens of the eye is composed of only one cell type and is
enclosed by the tissue's basement membrane, the lens capsule.
Undifferentiated epithelial cells cover its anterior surface. In the
equatorial region these cells differentiate into the highly elongated
fibre cells that make up the bulk of the lens as concentric layers. New
layers of differentiating lens fibres are continuously laid down on top
of existing layers thereby creating a gradient of differentiation stages
as one moves into the lens.
During differentiation, the fibres lose all their membranous
organelles, including their nuclei [l], and form the organelle-free
zone (OFZ)in the centre of the lens. This loss is a necessary
prerequisite to lens transparency as it greatly reduces light scattering
and absorption and facilitates the formation of a homogenous fibre
cell cytoplasm. Disruption of the denucleation of lens fibres has been
found to be a characteristic of certain congenital cataracts in humans
[2] and animal model systems [3]. As there is no turnover of lens
cells and new fibre cells are continuously formed throughout life, the
lens and therefore the OF2 expands with age.
The process of programmed organelle loss in the vertebrate lens
appears superficially similar to that seen during apoptosis: The ER
and Golgi apparatus vesiculate and vanish [4 and our own data], the
mitochondria lose their function [5] and are degraded [our own data].
In the nuclei, the lamina is broken down, accompanied by chromatin
aggregation and DNA cleaveage (DNA laddering) and ultimately the
release of nucleosome-sized fragments into the cytosol[6,7, and our
own data]. In contrast to earlier reports the breakdown of DNA in
lens fibre cells has been shown to be a rapid process probably
mediated by DNAse I-like enzyme [6]. Their findings seem to
question earlier studies that failed to detect DNAse I transcripts in
lens fibre cells [8]. It should however been mentioned that a range of
nuclease activities have been reported in the chicken lens by a number
of authors.
There are however significant differences between lens fibre cell
differentiation and apoptosis. For example, the timescales of the two
processes are vastly different: hours in the case of apoptosis, days
during fibre differentiationin the adult lens. Also, in contrast to cells
undergoing apoptosis, differentiating fibre cell plasma membranes do
not bleb and the cells do not shrink. Instead lens fibres elongate and
form elaborate plasma membrane interdigitations with their
neighbours that allow them to maintain their highly ordered tissue
organisation during accomodation. Moreover, the fibre cells are not
phagocytosed by adjacent cells but persist throughout life.
To determine the mechanisms underlying organelle loss in the
mammalian lens, we have stained bovine lens cryosections with a
range of antibodies against nuclear or mitochondrial marker proteins.
We have mapped the changes in the nuclear lamina as the nuclei are
broken down and have correlated this to changes in the chromatin
and DNA degradation using the DNA stain propidium iodide and
TUNEL-labelling by laser scanning confocal microscopy (LSCM).
Additionally, we have correlated mitochondrial and nuclear
breakdown during fibre cell differentiation.
The nuclear lamina is one of the fiist targets for degradation during
both apoptosis and lens fibre cell differentiation. Due to the
difference in timescales over which the two processes occur, we
were able to observe the onset of the degradation of the nuclear
lamina preceding DNA degradation in differentiatinglens fibre cells.
Accompanying the breakdown of the nuclear lamina in the bovine
lens is the entry of the intermediate fiiament protein CP49 into the
nuclear compartment [9] and a general change in shape of the nucleus
from oval to a smaller and more spherical appearance.
Shortly after the first changes in the nuclear lamina become
apparent, and before the nuclei start rounding up, the chromatin
aggregates into large clumps. At later stages of chromatin
aggregation, when the nuclear lamina has been almost entirely
degraded, DNA fragmentation sets in as shown by TUNEL labelling.
Despite the fact that the nuclear lamina is one of the earliest targets
during the denucleation of a differentiating fibre cell, the lamin
proteins A, B. and C are expressed at all stages of nuclear
breakdown including in the pyknotic nuclear fragments. These
results are in line with those obtained on embryonic chicken lenses
[a]. that shown lamin B2 to be present in the nuclear remnants of
late-stage differentiating fibre cells.
DNA degradation in differentiatingbovine fibre ells, as evidenced
by TUNEL labelling, occurs only after the nuclear lamina has almost
completely been broken down. This again reflects the situation found
in the embryonic chicken lens [a]. where the first structures to be
TUNEL-positive are the condensed nuclei of late stage-fibre cells. As
in the bovine lens, " E L labelling extends to the nuclear debris
deep into the organelle free zone.
In parallel with these changes in the nucleus, the mitochondria are
lost from differentiating fibre cells. Bovine lens cryosection triplelabelled with antibodies against the mitochondrial marker protein
BAP37, lamin B and the DNA stain DAPI show that the
mitochondria are degraded in parallel with the breakdown of the
nuclear lamina and the nuclei adopting a more spherical shape. At the
stage when the nuclei are reduced to pyknotic fragments. no
mitochondria are left. Equivalent results were obtained when bovine
lens cryosections were stained with antibodies against another
mitochondrial protein, prohibitin.
During the course of apoptosis in several cell types,mitochondria
appear to play a crucial role by releasing factors into the cytoplasm
that function as signals to activate the cellular apoptotic machinery.
Two of these factors have so far been identified - cytochrome c and
the Apoptosis Induction Factor (AIF) [101. AIF-independent
apoptosis, however, has been observed [ll], suggesting that there is
more than one apoptotic pathway. In this light lens cell differentiation
might be seen as yet another form of (parrial) cellular degradation.
DNA fragmentation appears to be a relatively late event during
fibre cell differentiation. We have shown that both the nuclear lamina
and the mitochondrial membranes in the bovine lens are degraded
before DNA fragmentation occurs. Also the data on the embryonic
chicken show that not only the nuclear and mitochondrial
membranes, but also the Golgi apparatus and the endoplasmatic
reticulum are lost 2-3 days before DNA degradation sets in. Taken
together these results seem to indicate a general difference between
lens cell differentiation and apoptosis which in many cell types is
chracterised by DNA damage preceding degradation of organelles.
Despite the obvious differences between lens fibre cell
differentiation and apoptosis, work on p53 and Rb nil mice [12]
indicates that the two processes might still share some fundamental
mechanisms. The fact that the distance between the outer edge of the
OFZ and the surface of the lens in the chicken stays constant during
development, gave rise to the hypothesis that the organelle
breakdown might be triggered by spatial signals, such as a drop in
nutrient or oxygen concentration. These primary signals might cause
the breakdown of the ER and later the mitochondria and subsequently
(potentially in concert with calcium released from these organelles)
cause the degradation of the other organelles [6]. The transparency of
the ocular lens (which greatly facilitates light microscopical studies),
the fact that the position of a fibre cell in the lens can be correlated to
its differentiation stage and the time-scale of events suggest that this
tissue might serve as a good model system for the study of
programmed organelle breakdown and mechanisms involved in the
apoptotic process.
References
1. Piatigorsky, J. (1981) Differentiation 19.134-153
2. Zimmermann, L.E., and Font, R.L. (1966) J.A.M.A. 196,684-692
3. Capetanaki, Y., Smith, S.,and Heath, J.P. (1989) J. Cell Biol. 109,
1653-64
4. Bassnett, S. (1995) Invest. Ophthalmol. Vis. Sci. 36.1783-1803
5. Bassnett, S. (1992) Cum. Eye Res. 11,1227-1232
6. Bassnett. S. and Mataic. D. (1997) J. Cell Biol. 137.37-49
7. Chaudun, E., h t i , C., Courtois, Y., Ferrag, F., Jeanny, J.C., Patel,
B.N., Skidmore, C., Toniglia, A., and Counis, M.F. (1994) J. Cell
Physiol. 158,354-364
8. Hess, J.F. and Fitzgerald, P. (1996) Mol. Vis.2,8
9. Sandilands, A., Rescott, A.R., Carter, J.M., Hutcheson, A.M.,
Quinlan,R.A., Richards, J., and Fitzgerald, P.G. (1995) J. Cell Sci.
108,1397-1406
10. Petit, P. X., Susin, S-A.. Zamzami, N., Mignotte, B.. and Kroemer,
G.. (1996) FEBS lett. 396.7-13
11. I(Luck. R. M.. Bossy-Wet&, E.,Green, D. R. and Newmyer, D. D. ,
(1997) Science 275,1132-1 136
12. Pan, H. and Griep, A.E. (1994) Genes & Dev. 8,1285-1299