From the Department of Cell Biology, The Scripps
Research Institute, La Jolla, California 92037 and the
§ Department of Pathology, Yale University School of
Medicine, New Haven, Connecticut 06520
Received for publication, October 24, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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Terminal differentiation of lens fiber
cells resembles the apoptotic process in that organelles are lost, DNA
is fragmented, and changes in membrane morphology occur. However,
unlike classically apoptotic cells, which are disintegrated by membrane
blebbing and vesiculation, aging lens fiber cells are compressed into
the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein The spectrin-actin membrane skeleton underlies the plasma
membranes of all cells and is important for cellular shape, membrane stability and deformability, as well as the formation of membrane subdomains (1). The major component of the membrane skeleton, spectrin,
is composed of an Proteolysis of The terminal differentiation and aging of lens fiber cells are marked
by dramatic membrane morphological changes. As new cells arise on the
outside of the lens, older cells are pushed inward, where organelles
are lost, and cells fuse to form a syncytium (15, 16). Because these
cells are never lost from the lens, the most central cells are as old
as the organism. In addition, as lens fiber cells mature, specialized
membrane interdigitations develop, which are distributed regularly on
the lateral membranes (17). These age-related changes in membrane
morphology have been compared with the formation of apoptotic bodies
(18). In addition, they are believed to be important for lens
transparency by reducing light scattering at cell boundaries and by
allowing for protein turnover and ion homeostasis (16).
We have shown previously that spectrin and other components of the
membrane skeleton are associated with the plasma membranes of young and
old lens fiber cells (19). Here, we report that Antibodies--
Affinity-purified rabbit polyclonal antibodies
to bovine brain Isolation and Subcellular Fractionation of Lens Cells--
Whole
lenses were harvested from chickens or rats, and contaminating ciliary
epithelium was removed by careful dissection. For Fig. 3, cortical
fiber cells were isolated from nuclear fiber cells, which were
compacted more tightly, by dissection of adult chicken lenses (6-8
weeks old). Using fine forceps on the nuclear fiber cells, the outer
nuclear fiber cells were peeled away in layers from the inner nuclear
fiber cells. Whole lenses or isolated fiber cells were rinsed in
phosphate-buffered saline with 10 mM EGTA and homogenized
in lens buffer (100 mM NaCl, 25 mM Hepes, pH
7.4, 4 mM MgCl2, 10 mM EGTA, 1 mM dithiothreitol) at 30 mg/ml, using a Dounce homogenizer
(8-10 strokes with the tight pestle). Subcellular fractionation was
performed as described in Ref. 19. Briefly, lens fiber cell homogenates
were centrifuged at 30,000 × g for 20 min at 4 °C
to separate the cytosol supernatant from the membrane pellet. The
pellet was washed by two more rounds of resuspension and centrifugation
to prepare washed membranes, which were either resuspended in lens
buffer or extracted in lens buffer with 1% Triton for 1 h on ice.
Triton extracts were subsequently centrifuged at 30,000 × g for 20 min at 4 °C (supernatant 2 and pellet 2) (see
Fig. 4).
All procedures were performed on ice in the presence of the following
protease inhibitors: 100 µg/ml phenylmethylsulfonyl fluoride (Sigma),
1 µg/ml aprotinin, 15 µg/ml leupeptin, 5 µg/ml pepstatin A (Roche
Molecular Biochemicals, Indianapolis) and 100 µg/ml
tosyl-L-lysyl chloromethyl ketone (Calbiochem).
Electrophoresis and Western Blotting--
SDS-polyacrylamide gel
electrophoresis was performed on large pore 10% polyacrylamide gels
according to Dreyfuss et al. (22), and gels were transferred
to nitrocellulose in Tris-glycine transfer buffer (23) with the
addition of 0.01% SDS and the omission of methanol (this was optimal
for efficient transfer of spectrin). Broad range molecular mass
standards were purchased from Bio-Rad. The relative mobility of each
fragment was estimated based on standard RF
analysis. Unless otherwise indicated, Western blotting was performed as
described (24) except that antibodies were detected with protein
A-horseradish peroxidase (Sigma) followed by standard chemiluminescence
detection methods. To reprobe blots using a different antibody, blots
were first stripped in 62.5 mM Tris, pH 6.5, 2% SDS, and
100 mM 2-mercaptoethanol at 65 °C for 30 min. Stripped
blots were washed extensively with phosphate-buffered saline with 1%
Triton and then blocked and probed as usual. For the quantitation of
the ratio of full-length Purification of Fragments and N-terminal Sequencing--
Adult
chicken lenses (200) were homogenized using a Dounce homogenizer at 500 mg/ml in 10 mM NaHPO4, pH 7.4, 100 mM KCl, 5 mM EDTA, 5 mM EGTA, 0.5 mM dithiothreitol (lens buffer). Homogenates were
centrifuged at 30,000 × g to obtain a membrane pellet
(water-insoluble fraction), which was washed twice in lens buffer and
subsequently extracted in 10 mM NaHPO4, pH 7.4, 1.5 M KCl, 5 mM EDTA, 5 mM EGTA,
0.5% Triton, 0.5 mM dithiothreitol. Extracts were then
centrifuged at 30,000 × g, and the supernatant,
containing fragments and full-length spectrin, was retained (high salt
extract). The high salt extract was dialyzed into 10 mM
Tris, pH 8.0, 20 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol and then loaded
onto a 5-ml Resource Q anion exchange column (Amersham Pharmacia
Biotech). Bound proteins were eluted using a 94-ml linear gradient from
20 to 500 mM NaCl; the elution position of fragments was
determined by SDS-polyacrylamide gel electrophoresis and Coomassie
staining. Fractions enriched in Spectrin Is Cleaved to Discrete Fragments during Lens Development
and Aging--
We have shown previously that
To determine whether
Fragments of spectrin were also found to be present in the rat lens
(Fig. 2). As for the chicken lens, the
amount of fragmentation increased progressively with the postnatal age
of the rat lens. The ratio of intensities of Spectrin Is Completely Cleaved to Discrete Fragments in the Oldest
Fiber Cells of the Adult Lens--
The progressive increase in
spectrin fragmentation in older chicken and rat lenses suggested that
spectrin might be fragmented progressively with the age of the lens
fiber cell. To investigate this possibility, we compared the amount of
spectrin fragmentation in fiber cells of different ages in the
6-8-week-old adult chicken lens (Fig.
3). The youngest (~0-4 weeks old),
newly differentiated cortical fiber cells constitute the outermost
shell of the lens fiber cell mass, and are easily peeled away from the
remaining, older (nuclear) fiber cells. The outer nuclear fiber cells
(~4-6 weeks old) were then separated from the inner nuclear fiber
cells, the oldest fiber cells of the lens. Thus, for this experiment, the inner nuclear cells were ~6-8 weeks old. We estimate that our
inner nuclear preparation (~ 1-mm diameter core) contained ~8,000
cells out of 1.5 million total lens cells (30) based on the average
cross-sectional area of the human embryonic nuclear fiber cell
(31).
As expected, in newly differentiated fiber cells, spectrin fragments
were barely detectable relative to full-length by Western blotting
(Fig. 3). In older fiber cells (outer nuclear) the ratio of fragments
to full-length spectrin was ~1:1. In contrast, full-length
To investigate whether the spectrin-binding and membrane-binding
protein, ankyrin, is also proteolyzed during lens fiber cell aging,
Western blotting was performed on cortical and nuclear fiber cells of
the adult chicken lens (Fig. 3, right panel). In both
cortical and nuclear fiber cells, several ankyrin antibodies recognized
bands corresponding to the full-length ankyrin (220 kDa). However, in
nuclear fiber cells, an additional doublet at ~190 kDa was recognized
by antibodies raised against brain ankyrin, human erythrocyte ankyrin
(data not shown), or an N-terminal peptide of human erythrocyte ankyrin
(Fig. 3, right panel). These results suggest that ankyrin is
cleaved during lens fiber cell aging. Furthermore, because the
antibodies raised against an N-terminal peptide of ankyrin recognize
the 190-kDa cleavage product, the cleavage site is likely to be located
near the C terminus.
These results indicate that spectrin and ankyrin are progressively
cleaved during fiber cell aging. Moreover, the oldest fiber cells of
the adult lens are likely to contain only spectrin fragments and no
full-length spectrin.
A Caspase Is Responsible for Lens Spectrin Cleavage--
Both
calpains and caspases have been shown to cleave
To determine directly whether a caspase is responsible for lens
spectrin proteolysis, spectrin fragments were purified from adult
chicken lens membranes (see
"Experimental Procedures") and subjected to N-terminal sequencing
(Fig. 4B). No sequence could be obtained from the
The N-terminal sequence of the
In conclusion, the cleavage sites of lens spectrin, as well as the
sizes of the Spectrin Fragments Are Partially Dissociated from Lens Plasma
Membranes--
To investigate the biochemical consequences of caspase
fragmentation of lens spectrin, subcellular fractionation of adult lens
fiber cells was performed (Fig. 5).
Immunoblotting of cytosol and membrane fractions indicated that
However, the small proportion of
Interestingly, all three of the This is the first report of caspase cleavage of membrane skeleton
proteins to discrete and stable fragments during cellular maturation
and aging. We have shown that The cleavage of membrane skeleton components in the lens is specific.
In contrast to spectrin and ankyrin, other components of the membrane
skeleton, tropomodulin, tropomyosin, and actin, do not appear to be
proteolyzed during lens fiber cell aging (19). However, there are
reports of other membrane-associated lens proteins being proteolyzed to
discrete and stable fragments. Partial proteolysis of the major lens
membrane protein, MP-26 from a ~26- kDa to a ~20-kDa protein, has
been reported to occur during fiber cell maturation (37). The gap
junction protein connexin 50 ( The timing of spectrin fragmentation also implies roles in mediating
membrane morphological changes during lens fiber cell aging. Spectrin
fragments are first detected late during embryonic development,
coincident with formation of the organelle-free zone (28), but continue
to accumulate during development and after hatching. In the adult lens,
spectrin fragmentation appears to be restricted to older fiber cells,
and the amount of spectrin fragmentation increases with the age of the
lens fiber cell. Unfortunately, it is difficult to compare the timing
of spectrin fragmentation with that of other membrane-associated
proteins because a similar developmental analysis has not been
performed with other proteins. However, a functional syncytium of cells
within the organelle-free zone is first detected at day 12 of embryonic
development and expands with age (16). In addition, membrane
protrusions are not present in the day 7 embryonic lens but are first
observed later in the day 10 embryonic lens; these membrane protrusions become more elaborate with age (42). Moreover, in the adult chicken
lens, newly differentiated cortical fiber cells exhibit smooth
profiles, whereas older fiber cells display numerous membrane protrusions (43). In support of a role for spectrin fragmentation in
the development of membrane protrusions, antibodies, which recognize both full-length and fragments of This is the first report of caspase-mediated cleavage of
membrane-associated components in the lens. Our data suggests that caspase-3 may be involved in proteolysis of spectrin during lens fiber
cell maturation and aging. The sequence of the cleavage site for
chicken lens Caspase activity has previously been shown to be necessary for loss of
nuclei during lens fiber cell differentiation (46-48). In addition,
members of the caspase family (1-4, 6) and the caspase substrates, DNA
fragmentation factor and poly(ADP-ribose) polymerase, have been
identified in the lens (46). Like spectrin, poly(ADP-ribose polymerase
has also been reported to be cleaved late in lens development, after
organelle loss (46).
Although our data suggest that caspase cleavage of the membrane
skeleton occurs after organelle loss, others have reported that caspase
activation is required for organelle loss. This apparent discrepancy
may be explained by the idea that different caspases may be active at
different times during lens fiber cell maturation. According to Wride
et al. (46), particular caspases (i.e. caspase-3) are present early during lens fiber cell differentiation, whereas others (i.e. caspase-1) are predominant in older lens fiber
cells. Moreover, although inhibitors of caspases-1, -2, -6, and -9 appeared to inhibit nuclear loss in lens cell cultures, inhibitors of
caspase-3 and -8 were ineffective. Both of these observations suggest
that although the caspase pathway has not been delineated
precisely for lens cells, particular caspases are important at
different times during lens fiber cell differentiation. Thus, it is
possible that specific caspases (particularly caspase-3) are important for selective proteolysis of the membrane skeleton after organelle loss, whereas other caspases are important for initiating organelle loss.
What are the molecular consequences of caspase cleavage of spectrin and
ankyrin? We have shown that spectrin fragments are partially
dissociated from lens membranes. This may be due to cleavage of
ankyrin, which binds to both Cleavage of lens spectrin by caspase may lead to lowered membrane
affinity as well as calmodulin down-regulation of spectrin activities.
The caspase cleavage site on Interestingly, We have developed a speculative model (Fig.
6) to describe how specific and partial
proteolysis of membrane skeleton proteins could lead to membrane
morphological changes in the lens. In young (cortical) fiber cells
(Fig. 6A), as in other non-erythroid cells, -spectrin to ~150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave
-spectrin to
~150-kDa fragments and
-spectrin to ~120- and ~80-kDa
fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of
the adult lens, most, if not all, of the spectrin is cleaved to
discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the
organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the
membrane skeleton. Partial and specific proteolysis of membrane
skeleton components by caspases may be important for age-related
membrane changes in the lens.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
heterodimer that self-associates head-to-head
to form a 200-nm extended tetramer filament. Spectrin cross-links actin
filaments into an isotropic meshwork. This spectrin-actin meshwork is
attached to the membrane by direct interactions of
-spectrin with
membrane proteins and indirect interactions of
-spectrin with
membrane attachment proteins such as ankyrin (2).
-spectrin (
II-spectrin, non-erythroid spectrin, or
fodrin) to discrete fragments is implicated in changes in cell shape
and membrane morphology which occur in many cell types. During platelet
activation, which includes a cell shape transformation from discs into
irregular spheres, spectrin is cleaved to ~150-kDa fragments by the
calcium-dependent protease, calpain (3).
-Spectrin
cleavage by calpain has also been implicated in cellular hypoxia (4),
neuronal injury and degeneration (5), and neuronal growth cone
formation (6). However, in apoptotic cells,
-spectrin proteolysis to
~150-kDa fragments is mediated by caspases; in these cells, spectrin
proteolysis is thought to be important for the disintegration of the
plasma membranes via formation of vesicular "apoptotic bodies"
(7-12). Although calpain cleavage of spectrin is known to affect its
ability to bind membranes or actin filaments (13, 14), the detailed
consequences of caspase cleavage of spectrin have not been studied.
-spectrin is cleaved
to ~150-kDa fragments during terminal differentiation and aging of
lens fiber cells. In addition,
-spectrin, which dimerizes with
-spectrin, is also proteolyzed to ~120- and ~80-kDa fragments.
Fragmentation of spectrin progresses with lens and fiber cell age so
that in the oldest fiber cells of the adult lens, most, if not all, of
the spectrin is fragmented. The spectrin-binding and membrane-binding
protein, ankyrin, is also partially proteolyzed with lens fiber cell
age. N-terminal amino acid sequencing of the spectrin fragments reveals
that caspase cleavage is responsible for lens spectrin proteolysis.
Moreover, subcellular fractionation of lens fiber cells indicates that
caspase-cleaved spectrin fragments display reduced association with
lens membranes. The specific proteolysis of membrane skeleton
components by caspases may be important for age-related membrane
changes in the lens.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-spectrin (fodrin) were prepared as described
(R6017) (20). Rabbit polyclonal antibodies (pAb10D) were raised against
a recombinant peptide representing residues 1676-2204 of human
II-spectrin, spanning from repeat unit 13 to the COOH terminus (7,
21). Monoclonal antibodies to actin (C4) were a generous gift from J. Lessard (Children's Hospital Research Foundation, Cincinnati, OH).
Rabbit polyclonal antibodies to repeats 13-24 in the N-terminal ankyrin repeats domain of human red blood cell ankyrin were a generous
gift from P. S. Low (Purdue University, West Lafayette, IN).
-spectrin to fragments, autoradiographic
films were scanned into NIH Image. The total number of pixels in each
band was quantified in arbitrary units. To correct for nonspecific
binding, pixels from an unlabeled part of the film were subtracted.
Similar results were obtained from direct labeling of the blots with
125I-protein A followed by
-counting (data not shown).
-spectrin fragments were selected,
and the proteins were trichloroacetic acid precipitated, and run on
two-dimensional gels (25), followed by transfer to polyvinylidene
difluoride membranes in 10 mM
CAPS,1 10% methanol, 0.01%
SDS. Anion exchange fractions containing the
-spectrin 120-kDa
fragment were pooled separately, trichloroacetic acid precipitated,
solubilized in 0.1% SDS (with 20 mM Tris, pH 8.0, 2.5 mM EDTA, 2.5 mM EGTA), and loaded onto a
Sepharose CL-4B gel filtration column. Bound proteins were loaded and
eluted in the presence of 0.1% SDS as described in Ref. 26. Fractions enriched in the
-spectrin 120-kDa fragment were loaded on a
one-dimensional SDS-polyacrylamide gel and transferred to
polyvinylidene difluoride membranes. Proteins were eluted from
polyvinylidene difluoride and subjected to standard N-terminal
sequencing by J. Leszyk (University of Massachusetts Medical School,
Shrewsbury, MA). Molecular masses of fragments were estimated based on
amino acid composition using the ProtParam tool of the ExPASY
proteomics server (59).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-spectrin is partially
proteolyzed to three ~150-160-kDa fragments in the nuclear (oldest) fiber cells of the adult chicken lens (19). These results suggested either that spectrin fragmentation occurs only during terminal differentiation (i.e. organelle loss) of fiber cells in the
embryonic lens or that spectrin fragmentation progresses with aging of
fiber cells in post-hatched lenses. To determine when
-spectrin
proteolysis occurs, Western blotting was performed on proteins from
chicken lenses at different stages of embryonic and post-hatched
development (Fig. 1A). Of
particular interest were the day 6 embryo, when the lens first appears
transparent (27), the day 8-10 embryo, when organelle breakdown begins
(28), and the day 12 embryo, when an organelle-free zone is first
observed. When non-crystallin proteins were loaded equivalently (Fig.
1A, actin),
-spectrin fragments were barely detected at
day 17 of embryonic lens development; moreover, no fragments smaller
than ~150 kDa were detected. Strikingly, scanning quantitation of
blots indicated that the ratio of
-spectrin fragments to full-length
-spectrin increased dramatically with age after hatching (Fig.
1B). In the representative experiment shown, fragments were
first detected at day 17 of embryonic development (Fig. 1A).
However, when proteins from twice as much tissue (wet weight) were
loaded from day 12 embryonic lens proteins,
-spectrin fragments were
barely detected (data not shown). Nonetheless, fragments of
-spectrin were not detected from samples of day 6, 9, or 10 embryonic lens proteins, even when 3 × wet weight of tissue was
loaded (data not shown). These results suggest that
-spectrin
fragmentation may be initiated simultaneously with the formation of an
organelle-free zone. However, the extent of fragmentation of
-spectrin continues to increase in post-hatched lenses relative to
late embryonic lenses, suggesting that proteolytic processing of
-spectrin progresses with lens age.
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Fig. 1.
- and
-spectrin are cleaved to discrete fragments during
development and aging of the chicken lens. A, Western
blotting of lens proteins harvested from chickens at different days of
embryonic development (day 6, 9, 10, 12, 15, or 17) or ages after
hatching (1 day, 6 weeks, 5.5 months, 18 months).
-Spectrin is
partially proteolyzed to ~150-160-kDa fragments in the older
embryonic and post-hatched chicken lens. Simultaneously,
-spectrin
fragments at ~120 and ~80 kDa are also detected during late
embryonic development. Western blotting for actin indicates that lanes
were loaded relatively evenly for non-crystallin proteins (the
concentration of non-crystallin proteins decreases with lens age (56)).
Wet weight of tissue loaded is as follows: 0.7 mg (6E, 9E, 10E, 12E),
1.2 mg (15E, 17E, 1D, 6 weeks, 5.5 months), and 0.92 mg (18 months).
B, NIH Image quantitation of the ratio of intensities of the
full-length
-spectrin band relative to the intensities of the
-spectrin fragments. The average and standard error of two
experiments are shown. Comparison of Coomassie staining (data not
shown) and Western blotting indicates that our transfer and Western
blotting procedures slightly overestimate the amount of fragment to
full-length by 1.1-fold.
-spectrin, which forms a heterodimer with
-spectrin in most mammalian cells, is also proteolyzed, blots were
stripped and reprobed with a
-spectrin antibody (7, 21) (Fig.
1A). Two fragments were detected with the
-spectrin
antibody, which was raised against a peptide from the C-terminal half
of the protein (see "Experimental Procedures"). A ~120-kDa
fragment first appeared in day 17 embryonic lenses, when
-spectrin
fragments were first detected. In contrast, a smaller ~80-kDa
fragment was not detectable in day 17 embryonic lenses and was first
detected in older lenses. These results suggest either that the
~80-kDa fragment arises from cleavage at a site that is digested less readily than the site for the ~120-kDa fragment or that the ~80-kDa fragment arises from cleavage of the ~120-kDa fragment. An additional band at ~70 kDa was also barely detectable in the 18-month lens. We
also observed that, similar to
-spectrin, more
-spectrin fragments relative to full-length were observed with increasing age of
the lens.
-spectrin fragments to
full-length
-spectrin at postnatal day 3 in the rat lens was similar
to that of the day 17 embryonic chicken lens (data not shown; see Fig. 1A); this is consistent with the later development of an
organelle-free zone in rodent lenses as compared with chicken lenses
(28, 29). However, in addition to 150-160-kDa
-spectrin fragments,
an additional
-spectrin fragment at ~110 kDa was found in the
adult rat lens. Moreover, in addition to ~120- and ~80-kDa
-spectrin fragments, another fragment at ~50-kDa was also
detected. Similar-sized
-spectrin fragments were also observed in
the newborn bovine lens (data not shown). No
-spectrin fragments
smaller than ~110 kDa or
-spectrin fragments smaller than
~50 kDa were detected in bovine or rat lenses (data not shown). These
results indicate that both
- and
-spectrin are cleaved to
discrete fragments during development and aging of the chicken, rat,
and bovine lens.
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Fig. 2.
Spectrin fragmentation occurs during rat lens
development and aging. Western blotting of lens proteins from
postnatal day 3, day 10, and adult (6 weeks) rats using antibodies
raised against - and
-spectrin is shown. Note the presence of an
-spectrin fragment at 110 kDa.
-Spectrin is partially proteolyzed
to one ~120-kDa and three ~150-160-kDa fragments. Simultaneously,
-spectrin fragments at ~120, ~80, and ~50 kDa are also
detected during late embryonic development.
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Fig. 3.
Spectrin is cleaved to discrete fragments
during lens fiber cell aging. Left and center
panels, Western blotting for spectrin of lens proteins from
cortical (C), outer nuclear (oN), and inner
nuclear (iN) fiber cells of the adult chicken lens (6-8
weeks). We were unable to use actin as a loading control because the
high amounts of -crystallin in inner nuclear fiber cells interfered
with the detection of actin. However, proteins corresponding to 0.6 mg
of cortex (1×), 1.2 or 1.7 mg of outer nucleus (2×, 3×), and 1.2 mg
or 4.7 mg of inner nucleus (2×, 7×) were loaded. These are roughly
equivalent estimates of non-crystallin protein because
-crystallin
protein concentration increases (and thus, non-crystallin concentration
decreases) by ~3-fold from cortex to outer nucleus and an additional
~1.5-fold from outer nucleus to inner nucleus (57). Moreover, the
ratio of intensities of the full-length to fragment bands for each
sample did not change when different amounts of sample were loaded.
This experiment was repeated three times with similar results; a
representative experiment is shown. Right panel, Western
blotting for ankyrin of lens proteins from cortical and nuclear fiber
cells of the adult chicken lens (6-8 weeks).
- or
-spectrin was barely detectable in the oldest, inner nuclear fiber
cells (Fig. 3, left and middle panels).
Strikingly, ~150-kDa
-spectrin and ~120-kDa
-spectrin
fragments were easily detectable in the inner nuclear cells, suggesting
that spectrin fragments were not further degraded and lost from the
lens. In some inner nuclear preparations, which presumably contained a
smaller core of central fiber cells, only spectrin fragments and no
full-length spectrin at all could be detected by Western blotting or
silver staining (data not shown). In contrast, in cortical fiber cells, Western blotting revealed that the majority of the
- and
-spectrin was full-length (Fig. 3; also see Ref. 19). Although it is
technically difficult to isolate the undifferentiated epithelial cell
layer from adult chicken lenses, in the undifferentiated epithelial cells of day 15 embryonic lenses, most of the
-spectrin was
full-length (data not shown).
-spectrin to
~150-kDa fragments in vitro (7, 32). However, the lens
-spectrin fragment sizes are similar to that reported for in
vitro caspase cleavage of bovine brain
-spectrin (~110 and ~85 kDa) (7). In contrast, calpain cleavage of
-spectrin results in 165-, 125-, and 120-kDa fragments (32). The sequences of caspase-3
cleavage sites of
II- and
II-spectrin have been identified (7)
and are distinct from the sites of calpain cleavage of
II- and
II-spectrin (32).
-spectrin 160-kDa fragment, suggesting that this fragment contained
the blocked N terminus (7, 32). The N-terminal sequence of both of the
smaller
-spectrin fragments (
155 and
150) mapped to the
sequence DETD1185*SKTASP (with * representing the site of
cleavage and the beginning of the amino acid sequence obtained). This
sequence is the previously reported site of cleavage of bovine brain
II-spectrin by caspase-3 in vitro (7). However, this
amino acid sequence differs slightly from the GenBank sequence for
chicken
-spectrin, which reports the sequence
S1186KTVSP (33). Reverse
transcription-polymerase chain reaction amplification of chicken
stomach and lens spectrin cDNA followed by DNA sequencing confirmed
that the correct DNA and amino acid sequences, respectively, for that
region are TCTAAGACAGCCSTCTCCT3489 and
KTASP1190. Interestingly, the caspase cleavage
site in
-spectrin is located in repeat 11, just nine residues
upstream of the calmodulin binding site (32) (Fig. 4B).
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Fig. 4.
N-terminal sequencing indicates that a
caspase is responsible for lens spectrin cleavage. A,
- and
-spectrin fragments were purified from adult chicken lens
membranes and subjected to N-terminal sequencing. For
-spectrin, the
sequence across the predicted cleavage site was determined from reverse
transcription-polymerase chain reaction followed by DNA sequencing of
chicken lens and stomach
-spectrin RNA. For
-spectrin, the human
(34) and bovine (7) sequences were used. B, caspase cleavage
sites for
- and
-spectrin are indicated. Depicted is the domain
structure of spectrin, with boxes identifying each
~106-residue repeat. The placement of unknown cleavage sites
(?) was estimated based on the fragment size and known sites
for caspase-3 digestion of spectrin in vitro (7). The
location of binding sites for calmodulin (CaM), ankyrin,
actin, and ankyrin-independent membrane binding sites 1-3
(MAD1, 2, 3) are also shown (1). The
-spectrin antibody used (pAb10D) was raised against a peptide
spanning repeat 13 to the COOH terminus (7).
-spectrin 120-kDa fragment was
XKRLTVEKKFLE (Fig. 4A). Although the DNA or amino acid
sequence of chicken
II-spectrin is not known, this N- terminal
sequence of the 120-kDa fragment is homologous to the human
II-spectrin sequence DEVD1457*SKRLTVQTKFME in
repeat 11 (34). Importantly, this sequence is also the same site
as that reported for caspase-3 cleavage of bovine brain
II-spectrin
in vitro (7). Interestingly, this site was not located near
regions important for ankyrin, ankyrin-independent membrane (MAD), or
actin filament binding (1) (Fig. 4B). Unfortunately, we were unable to
obtain sufficient amounts of the
-spectrin 80-kDa fragment for
N-terminal sequencing.
- and
-spectrin fragments, indicate that a caspase
is responsible for spectrin cleavage in the lens. In particular, the
sequences of the cleavage sites strongly implicate caspase-3.
-spectrin fragments were considerably more abundant in the
30,000 × g supernatant fractions (66.3 ± 5.97%
in S1) than in the membrane pellet. In contrast, full-length
-spectrin was more abundant in the membrane pellet (only 15.6 ± 10.0% in S1), as expected (19, 35). Similarly, the majority of
-spectrin fragments remained in the 30,000 × g
supernatant (58.1 ± 3.82% in S1), whereas full-length
-spectrin was more abundant in the membrane pellet (only 16.8 ± 2.56% in S1), as shown previously (19, 35). However, although all
of the ~80-kDa
-spectrin fragment remained in the supernatant
(99.5 ± 1.53% in S1), a significant proportion (~40%)
of the ~120-kDa
-spectrin fragment was pelleted (58.1 ± 3.82% in S1). This difference in membrane association of the two
fragments may be a result of the 80-kDa fragment missing the C-terminal
membrane association domain (MAD2), as suggested in Fig.
4B.
View larger version (36K):
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Fig. 5.
Spectrin fragmentation results in partial
dissociation from lens membranes. A, adult chicken
lenses (6-8 weeks) were homogenized in a physiological buffer followed
by centrifugation at 30,000 × g to obtain
cytosol-enriched supernatants (S1) and membrane pellets (P1). Membranes
were then extracted to obtain 1% Triton-soluble (S2) extracts and
Triton-insoluble pellets (P2). Proteins were subjected to Western
blotting with anti- - and anti-
-spectrin antibodies. B,
histogram indicates fraction of total full-length spectrin or spectrin
fragments in S1. The percentage of soluble spectrin or spectrin
fragments was calculated by dividing the amounts in S1 by the sum of S1
and P1. For
-spectrin and
-spectrin, respectively, the average
percentage and standard deviation of six or three experiments is
shown.
- and
-spectrin fragments that
did pellet with the membranes appeared to be tightly associated with
the membranes. These fragments were not extracted in 1% Triton (S2)
and were only partially extracted by 1 M NaCl or by 1%
Triton with 1 M NaCl (data not shown). Thus, spectrin
fragments that are membrane-associated might constitute a different
population from the cytosolic fragments.
-spectrin fragments in the
supernatant appeared to fractionate together on gel filtration (data
not shown), suggesting that they were tightly associated in a complex.
On the other hand, the
-spectrin fragments did not fractionate
together, suggesting that they had dissociated from one another and
from the remaining full-length
-spectrin. Thus, our results
collectively indicate that caspase fragmentation of spectrin leads to
partial dissociation of both spectrin subunits from lens membranes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-spectrin is cleaved to ~150-kDa
fragments and
-spectrin to ~120- and ~80-kDa fragments during
lens fiber cell maturation and aging. The spectrin-binding protein,
ankyrin, is also cleaved to ~190-kDa fragments. These fragments
appear to be extremely stable and indeed, accumulate with age. In
contrast, in other cell types, caspase cleavage of spectrin precedes
cell death, thus ensuring a short half-life for the spectrin fragments
(7-12). Moreover, when cleavage of spectrin does not lead to cell
death, (i.e. neuronal remodeling), a brief period of
accumulation of fragments is followed by a decrease in the proportion
of fragments to full-length spectrin polypeptides, suggesting
replacement of spectrin fragments with newly synthesized full-length
spectrin (36). Our results indicate that in lens fiber cells, which do
not undergo cell death, the accumulation of caspase cleavage products
of spectrin and ankyrin may lead to a permanent remodeling of the
membrane skeleton.
8) has been shown to be cleaved by
calpain, leading to removal of the C-terminal tail of the protein from
the plasma membranes (38). Connexin 46 (
3) may also be cleaved in
mature fiber cells (39). The lens-specific intermediate filament
protein filensin is also proteolyzed to a discrete ~53-kDa band in
maturing fiber cells in the bovine lens (40) and multiple bands in the
chicken lens (19). Finally, ~150-kDa
-spectrin fragments have been
observed in the rabbit and guinea pig lens (41) in addition to our
observations in the chicken, rat, and cow lens. Partial proteolysis of
particular membrane-associated proteins (and not others) to discrete
and stable fragments may be important for membrane remodeling during lens fiber cell aging.
-spectrin stain
protrusions of nuclear fiber cells (19) as well as blebs decorating
differentiated lens cells in
culture.2
-spectrin is identical to that obtained by in
vitro cleavage of bovine brain
II-spectrin by caspase-3. In
addition, the sequences of cleavage for both chicken lens
- and
-spectrins (DETD*S, DEVD*S) match well with the consensus DXXD*S cleavage site identified for other caspase-3
substrates (44). In rat and cow lenses, the presence of the
~110-120-kDa
-spectrin fragment generated by caspase-3 cleavage
in vitro also suggests cleavage of endogenous lens
-spectrin by caspase-3 (7, 45). The lack of a ~120-kDa fragment of
-spectrin in the chicken lens may be due to conformational
differences between chicken and mammalian spectrins.
-spectrin and integral membrane
proteins. Calpain-cleaved ankyrin, which, like lens ankyrin, is also a
~190-kDa fragment derived from cleavage near the C terminus, exhibits
an 8-fold weaker affinity for erythrocyte membranes (49). Alternatively, cleavage of
-spectrin could lead to reduced membrane association. However, the initial site of cleavage near the middle of
the protein is not within its ankyrin binding site, actin binding site,
or within any of the ankyrin-independent MADs (1). Thus,
-spectrin
cleavage is unlikely to interfere with membrane association by direct
interference with its membrane binding sites, but perhaps indirectly by
affecting the conformation of these sites.
-spectrin is within 9 amino acids of
the calpain cleavage site and is proximal to the calmodulin binding
site (Fig. 4B). When calmodulin is bound to
-spectrin
during the action of calpain, both
- and
-spectrin subunits are
cleaved, and the heterotetramer dissociates irreversibly into its
component fragments (14). This results in complete loss of tetramer
formation, F-actin binding, or membrane binding. Such a dissociation of
spectrin fragments is reminiscent of the dissociation reported here
which results from caspase-mediated cleavage of both subunits in the
intact heterodimer.
- and
-spectrin are both cleaved at repeat 11, at
sites that interact in the
-
-spectrin heterodimer (50), suggesting that spectrin may be fully assembled before cleavage. In
contrast, in other types of cells (1), calpain may either cleave the
- or
-spectrin subunits separately or together in the intact
heterotetramer, thus leading to targeted loss of tetramer assembly,
F-actin binding, or membrane binding (13, 14). Thus, although other
cell types may use a multistep process involving calpain and calmodulin
to accomplish an incrementally regulated disassembly of the spectrin
cortical skeleton, lens cells may use predominately caspase and
calmodulin to disassemble their membrane skeleton in a single step process.
-
-spectrin
tetramers are likely cross-linked to short actin filaments. This
two-dimensional meshwork is then anchored to the membranes via spectrin
interactions with ankyrin and other membrane proteins (1, 51). In older
fiber cells (Fig. 6B), localized caspase activity could
result in proteolytic cleavage of specific regions of the membrane
skeleton, loosening constraints on the membranes and thus allowing for
membrane blebbing in specific sites. The extent and placement of
membrane blebbing could be regulated by whether ankyrin,
-spectrin,
and/or
-spectrin are cleaved, or by calmodulin binding. With age,
more caspase activity, and thus, more proteolytic processing, would
occur. This could result in the increased density of membrane
protrusions with fiber cell age (17). Membrane blebbing could lead to
repositioning of integral membrane proteins, such as ion channels,
which might be necessary for age-related changes in ion and water flow
(52). Moreover, membrane blebbing might lead to cell-cell fusion events that have been observed in maturing lenses (16, 17).
View larger version (24K):
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Fig. 6.
Caspase cleavage of spectrin and ankyrin may
lead to cell-cell fusion and the formation of membrane protrusions
during lens fiber cell aging (a model). A, in the
cortex, the spectrin-actin membrane skeleton is intact, contributing to
the stability and shape of the plasma membrane. Spectrin tetramers are
linked to short actin filaments, which are capped by tropomodulin (58).
Integral membrane proteins are positioned in the membrane skeleton via
interactions with ankyrin and also through direct binding to
-spectrin at MADs. Sites for caspase cleavage of
- and
-spectrin are located in regions of contact in the heterodimer (50).
B, in the nucleus, localized caspase activity leads to
cleavage of
- and
-spectrins and ankyrin. When both
- and
-spectrins are cleaved, complete loss of actin and membrane binding
abilities is likely, and the membranes are no longer constrained
(A). Fragmentation of ankyrin reduces its affinity for
membranes (B). In the presence of calmodulin and when only
-spectrin is cleaved, the ability to form tetramers and bind actin
filaments and membranes is lowered (C). The spatially
restricted reduction of membrane affinity allows for membrane blebbing
and repositioning of integral membrane proteins (D).
Membrane blebbing may lead to cell-cell fusion and the formation of
membrane protrusions.
Interestingly, cleavage of -spectrin by calpain, not by caspase, has
been associated with a number of cataract models (41, 53). Inhibition
of calpain inhibits cataract formation and spectrin cleavage (54), and
human lenses with age-related nuclear cataracts display a higher
density of finger-like membrane projections than transparent lenses of
the same age (55). Thus, it is possible that aberrant cleavage of
spectrin by calpain may lead to uncontrolled or incomplete membrane
furrowing and opacification of the lens, whereas specific cleavage of
spectrin by caspase may be important for normal physiological
functioning of the lens.
In conclusion, partial and specific proteolysis of spectrin and ankyrin
by caspases appear to effect an apoptosis-like program of membrane
changes during lens aging. Further characterization of lens membrane
morphology and membrane skeleton proteolysis in caspase knockouts, as
well as additional biochemical analysis of the lens membrane skeleton,
will lead to greater insight in the importance of caspase cleavage of
membrane skeleton components during lens development and function. It
is likely that the transparency of the aging lens nucleus may depend
not only on organelle loss but also on membrane skeleton remodeling. We
anticipate that these changes in the membrane skeleton, induced by
caspase-mediated but limited proteolysis, will modulate ion
homeostasis, the positioning of ion channels, the frequency of
intercellular fusion events, and lens deformability.
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ACKNOWLEDGEMENTS |
---|
We thank Jeannette Moyer for technical assistance in high salt extraction and gel filtration chromatography of lens membranes, Angels Almenar-Queralt and Robert Fischer for advice and for editorial comments, and Susan Glantz for help with interpretation of spectrin cleavage profiles.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants EY10814 (to V. M. F.) and NS32578 and DK43812 (to J. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF354639.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, MB24, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8277; Fax: 858-784-8753; E-mail: velia@scripps.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M009723200
2 B. Fischer and V. M. Fowler, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: CAPS, 3-(cyclohexylamino)propanesulfonic acid; MAD, membrane association domain; S1 and S2, supernatant 1 and supernatant 2, respectively.
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