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Lens structure in MIP-deficient mice.

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Lens Structure in MIP-Deficient Mice
Department of Anatomy and Cell Biology, Rush-Presbyterian-St. Luke’s
Medical Center, Chicago, Illinois
Department of Ophthalmology, Rush-Presbyterian-St. Luke’s Medical Center,
Chicago, Illinois
Department of Pathology, Rush-Presbyterian-St. Luke’s Medical Center,
Chicago, Illinois
Department of Basic and Health Sciences, Illinois College of Optometry,
Chicago, Illinois
Department of Ophthalmology and Visual Science, Washington University School of
Medicine, St. Louis, Missouri
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri
In this study we used correlative light, scanning, and transmission (freeze-etch) electron microscopy to characterize
lens structure in normal mice and compare it with that in mice deficient in the major intrinsic protein (MIP) of fiber
cells. Grossly, wild-type lenses were transparent and had typical Y sutures at all of the ages examined. These lenses had
fibers of uniform shape (hexagonal in cross section) arranged in ordered concentric growth shells and radial cell
columns. In addition, these fibers had normal opposite end curvature and lateral interdigitations regularly arrayed
along their length. Ultrastructural evaluation of these fibers revealed anterior and posterior end segments characterized by square array membrane on low-amplitude wavy fiber membrane. Approximately 13% of the equatorial or mid
segments of these same fibers were specialized as gap junctions (GJs). In contrast, heterozygote lenses, while initially
transparent at birth, were translucent by 3 weeks of age, except for a peripheral transparent region that contained
fibers in the early stages of elongation. This degradation in clarity was correlated with abnormal fiber structure.
Specifically, although the mid segment of these fibers was essentially normal, their end segments lacked normal
opposite end curvature, were larger than normal, and had a distinct non-hexagonal shape. As a result, these fibers
failed to form typical Y sutures. Furthermore, the nuclear fibers of heterozygote lenses were even larger and lacked any
semblance of an ordered packing arrangement. Grossly, homozygote lenses were opaque at all ages examined, except
for a peripheral transparent region that contained fibers in the early stages of elongation. All fibers from homozygote
lenses lacked opposite end curvature, and thus failed to form any sutures. Also, these fibers were essentially devoid of
interlocking devices, and only 7% of their mid segment was specialized as GJs. The results of this study suggest that
MIP has essential roles in the establishment and maintenance of uniform fiber structure, and the organization of fibers,
and as such is essential for lens function. Anat Rec Part A 273A:714 –730, 2003. © 2003 Wiley-Liss, Inc.
Key words: MIP; crystalline lens; knockout mice; sutures; gap junctions; structure; electron microscopy; freeze-etch; freeze-fracture
The crystalline lens is a major optical component of the
dynamic focusing process. During this process it develops
and maintains an apparently simple, but in reality complex, tissue structure predicated on the formation and
precise organization of specialized cells, the lens fibers. All
vertebrate lenses develop and grow as inverted, stratified
epithelia. The fibers are long, crescent-like cells arranged
end to end in growth shells (GSs). As such, as more fibers
are formed throughout life, the outer, younger GSs constitute the lens cortex, while the inner, older GSs make up
the lens nucleus. However, while this basic lens architecture is characteristic of all vertebrate lenses, differences
in fiber anatomy significantly affect the optical quality of
the lens (see Fig. 1).
Grant sponsor: NIH NEI; Grant numbers: EY06642; EY11411;
Grant sponsor: Louise C. Norton Trust; Grant sponsor: Doctor
Bernard and Jennie M. Nelson Fund.
*Correspondence to: J.R. Kuszak, Ph.D., Departments of Ophthalmology and Pathology, Rush-Presbyterian-St. Luke’s Medical
Center, 1653 West Congress Parkway, Chicago, IL 60612. Fax:
(312) 942-2371. E-mail:
Received 11 February 2003; Accepted 28 March 2003
DOI 10.1002/ar.a.10080
Fig. 1. Whole fixed lenses as seen through a surgical dissecting
microscope (A–C) and 3D-CADs (D–L) of lenses with umbilical sutures
(left column), line sutures (center column), and “Y” sutures (right column).
A posterior view of an avian lens with branchless, umbilical sutures (A
and D) has no discernable surface features. All fibers are sequentially
overlaid in register onto existing GSs of fibers, resulting in RCCs that
extend from the center to the periphery of the lens (G). These fibers are
straight meridians that extend from pole to pole (J). Lenses with line
sutures (e.g., rabbit and frog lenses) feature two branches oriented 180°
apart to form a vertical line suture anteriorly and a horizontal line suture
posteriorly (B and E). As new fibers are overlaid in register onto existing
GSs, suture branches are also overlaid in register. Thus, two continuous,
triangular suture planes are formed that extend from the center of the
lens to its periphery (H). Within GSs, fibers are either straight or s-shaped
(K). The position of four straight fibers effectively defines the orientation
of the four suture branches. Between straight fibers, the ends of sshaped fibers are aligned as longitudinal arc lengths (suture branches).
The ends of fibers from two adjacent quadrants abut and overlap to form
suture branches (E, H, and K). An anterior view of a Y-suture lens shows
that there are three anterior suture branches oriented 120° apart that
reach confluence at the pole (C and F). The position of six straight fibers
effectively defines the orientation of the six suture branches. Between
straight fibers, the ends of s-shaped fibers are aligned as longitudinal arc
lengths (suture branches). The ends of fibers from two adjacent sextants
abut and overlap to form six suture branches. All of the fibers are
overlaid in register onto existing GSs, and hence the six suture branches
also become overlaid in register to form six triangular suture planes that
extend from the center to the lens periphery (F, I, and L).
For example, avian lens fibers are meridians, tapering
at the ends as they extend from pole to pole (Fig. 1, left
column). In contrast, fibers of all other vertebrate lenses
are not meridians. These fibers have ends that flare and
curve away from the poles in opposite directions. As a
result, the end-to-end arrangement of these fibers produces “lens suture branches.” In line sutures (e.g., rabbit
and frog lenses), two anterior branches are oriented at
180° to each other to form a vertical “line” suture, while
opposite end curvature results in two posterior branches
forming a horizontal line suture (Fig. 1, center column).
Most other vertebrate lenses (e.g., in mice, rats, pigs, cats,
dogs, bovines, and primates at birth) have “Y” sutures,
three anterior branches oriented at 120° to each other to
form a Y suture pattern, while opposite end curvature
results in three posterior branches forming an inverted Y
suture (Fig. 1, right column). In primate lenses, sutural
complexity increases as fibers are formed during specific
periods of life. A Y suture is formed during fetal development, a six-branch “simple star” evolves during infancy, a
nine-branch “star” is laid down throughout adolescence,
and a 12-branch “complex star” is produced during the
adult years. The precise variation in the amount of fiber
end flare and opposite end curvature necessary to create
the different suture types is well documented (Kuszak,
1995a). In addition, both the effect of different suture
types on lens optical quality, and the negative effects of
abnormal sutural anatomy as a consequence of both naturally occurring and experimentally induced pathology on
lens function is calculable (Kuszak et al., 1991, 1994,
1999, 2000, 2002; Sivak et al., 1994; Al-Ghoul et al., 1998,
1999). However, the factors that direct the exact arrangement of fibers into the form that follows function, and are
compromised such that malformation leads to malfunction, are not known.
A major consequence of fiber terminal differentiation is
the transformation of selected cuboidal lens epithelial
cells, ⬍5 ␮m high, into the ribbon-like fibers that range in
length from 150 ␮m to 2.5 mm in small lenses (e.g., in
mice), and from 150 ␮m to 15 mm in large lenses (e.g., in
rabbit and bovine). The creation of these exceedingly long
fibers is characterized by the elaboration of specific cytoplasmic and membrane proteins: the crystallins and major
intrinsic protein (MIP), respectively. MIP is the most
abundant fiber surface marker (Gorin et al., 1984), and is
generally considered to comprise at least 50% of fiber
plasma membrane proteins (Alcala et al., 1975; Alcala and
Maisel, 1985).
Thus, the significance of MIP in lens function has been
the target of numerous biochemical, morphological, immunocytochemical, and physiological investigations. It was
proposed that MIP is the fiber gap junction (GJ) protein
(Goodenough, 1979; Kuszak et al., 1981). However, subsequent studies revealed that MIP was found both in association with fiber communicating junctions and distributed throughout the nonjunctional membrane (Bok et al.,
1982; Fitzgerald et al., 1983; Paul and Goodenough, 1983).
Subsequently, MIP was also localized to other fiber membrane specializations, including lateral interdigitations
(balls and sockets, and flaps and imprints) and regions of
close membrane apposition (i.e., square array membrane)
(Fitzgerald et al., 1985; Sas et al., 1985; Dunia et al., 1987;
Costello et al., 1989; Gruijters, 1989; Zampighi et al.,
1989). This suggests that MIP may also have a role in fiber
adhesion and the regulation of extracellular space volume.
Significant sequence homology with several transmembrane channel proteins responsible for the transport of
small molecules indicated that MIP may function as a
water channel (Sandal and Marcker, 1988; Shiels et al.,
1988; Baker and Saier, 1990; Rao et al., 1990). When MIP
was reconstituted into unilamellar vesicles and bilayers, it
demonstrated properties consistent with both adhesion
and fluid transport (Ehring et al., 1990; Michea et al.,
1994). Subsequently, MIP was identified as a member of a
sequence-related family of proteins, including the aquaporins and the aquaglyceroporins (Reizer et al., 1993;
Froger et al., 1998); therefore, it is now often referred to as
aquaporin 0. MIP exhibited water transport in Xenopus
oocyte membranes, which was qualitatively similar to
aquaporin 1, but was quantitatively of significantly less
magnitude (Chandy et al., 1997). In isolated lens fiber
membranes, MIP displayed moderate but consistent water transport activity (Varadaraj et al., 1999).
Recent structural studies reiterated the idea that MIP
may have more than one functional role in the lens. MIP
was consistently found by freeze-fracture immunolabeling
to localize only to the periphery of fiber GJs, which indicates that it may be involved in the formation of these
cell-to-cell communicating plaques (Dunia et al., 1998). In
addition, purified, reconstituted MIP displayed surface
tongue-and-groove contours consistent with a role for adhesion of apposing protein-membrane lattices (Fotiadis et
al., 2000).
In order to further elucidate the role of MIP in vertebrate lenses, we characterized lens structure in MIP-deficient mice. We utilized correlative light microscopy (LM)
to examine lens histology, scanning electron microscopy
(SEM) to characterize fiber surface morphology, and
transmission electron microscopy (freeze-etch) to assess
the ultrastructure of fiber membrane in lenses from agematched wild-type, heterozygote, and homozygote mice.
Our data indicate that a lack of MIP results in dysplastic
fibers, which adversely affect the exact fiber architecture
necessary for lens function.
MIP-deficient mice with a hybrid 129S5/C57BL/6J-albino genetic background were generated from a library of
gene-trap embryo stem cells, as described previously
(Shiels et al., 2001). Sequence analysis showed that the
gene-trap vector had inserted into the first exon of the
MIP gene, resulting in an in-frame translation stop signal
at codon 57. MIP transcript and protein levels in heterozygous mutants were reduced relative to wild-type levels,
and no MIP transcripts or translation products were detected in homozygous mutants as determined by Northern
and Western blot analyses, respectively. At 3 weeks of age
(postnatal day [P] 21), lenses from homozygous mice displayed polymorphic opacities, whereas lenses from heterozygous mice were translucent and did not develop
frank opacities until ⬃24 weeks of age.
All animals were handled in compliance with institutional animal care and use guidelines, and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision
Lenses from heterozygote and homozygote MIP-deficient mice, as well as from wild-type mice, were examined
by LM and SEM at the following time points: birth (P1), 1
week old (P7), 2 weeks old (P14), and 3 weeks old (P21).
For freeze-etch it was necessary to utilize older mice in
order to accurately dissect the lenses. Only 10-, 18-, and
24-week-old mice were examined by the freeze-etch technique. For each of the above time points, n ⫽ 8 –12 mice.
Animals were killed by intraperitoneal injection of sodium
pentobarbital or by CO2 asphyxiation. The lenses were
dissected from the enucleated eyes and then processed for
LM, SEM, or freeze-etch. All lenses were fixed in 2.5%
gluteraldehyde in 0.07 M sodium cacodylate buffer at pH
7.2 at room temperature for 2–3 days, with fresh fixative
changes daily. After the lenses were washed overnight in
0.2 M sodium cacodylate buffer, axial lens dimensions
were taken and the lenses were photographed under a
surgical dissecting microscope (Zeiss, New York, NY).
For histology, the lenses were postfixed in 1% aqueous
osmium tetroxide at 4°C overnight, then washed in cacodylate buffer and dehydrated through a graded ethanol
series to propylene oxide. Tissue was infiltrated and flat
embedded in epoxy resin. The embedded lenses were sectioned along the polar axis. Tissue sections 1–2 ␮m thick
were cut with a glass knife, stained with a 1:1 mixture of
methylene blue and azure II, and photographed with an
Olympus Vanox AHBS3 microscope (Olympus America
Inc., Melville, NY) equipped with a 35-mm camera. Color
slides were digitized using a Polaroid Sprint Scan 35 (Polaroid Corp., Bedford, MA) and processed using Adobe
PhotoShop version 6 (Adobe Systems Inc., San Jose, CA)
on a Pentium III PC platform.
To expose suture patterns at the poles and/or fiber surface morphology along the fiber length, the lenses were
dissected to remove the elongating fiber layers, as previously described (Al-Ghoul et al., 1998). To enable a morphological examination of the cortical and nuclear fibers,
the lenses were split along the polar axis, thereby exposing radial cell columns (RCCs) and GSs. The tissue was
postfixed overnight in 1% aqueous osmium tetroxide at 4°,
washed in cacodylate buffer, and dehydrated through a
graded ethanol series. After the tissue was dehydrated
overnight in 100% ethanol, the ethanol was replaced
through a graded ethanol/freon 113 series to pure freon
113. Specimens were critical-point dried in Freon 23 (Dupont, Wilmington, DE) in a Balzers CPD 020 (Balzers,
Hudson, NH), secured on aluminum stubs with silver
paste, sputter-coated with gold, and examined in a JEOL
JSM 35c scanning electron microscope (JEOL USA, Peabody, MA) at 15 kV.
As described earlier, all vertebrate lenses are stratified
epithelia that develop and grow in an inverted vesicle
configuration. As a result, unlike other stratified epithelia,
lenses retain every cell formed throughout life. Thus, in
any lens there are terminally differentiating fibers with a
full complement of cytoplasmic organelles, mature fibers
that have eliminated most cytoplasmic organelles, and, in
long-lived species, fibers with an age range that can span
decades. In addition, a unique polar fiber morphology has
been proposed to subserve lens physiology. Mathias et al.
(1997) theorized that current circulates around and
through the lens, inwardly at the poles and outwardly at
the equator. The inward movement of fluid is described as
being along the polar intercellular clefts, or by structural
definition the sutures, convecting glucose to the innermost
fibers, where it is used for anaerobic metabolism. The
outward movement of fluid is described as being intracellular, convecting waste products of metabolism out of the
lens at the equator (presumably through GJs). Previous
ultrastructural studies confirmed that GJs are not uniformly distributed along the fiber length (Fitzgerald, 1986;
Kuszak, 1995b). Fiber ends, or those portions of fibers
involved in sutures and thus the inward movement of
fluid, have few GJs. Fiber mid-portions, or those segments
of fibers not involved in sutures, are thought to be responsible for outward movement of fluid, through their numerous GJs. In addition, the density of GJs conjoining the
mid-segment of fibers varies between species (Kuszak et
al., 1985; Fitzgerald, 1986; Lo and Harding, 1986; Kuszak,
1995b). The mid-segments of chick, rat, frog, and human
fibers are reported to have 65%, 33%, 12%, and 5%, respectively, of their membrane specialized as GJs. Note
that the lenses with the most GJs have the simplest suture system, while lenses with the fewest GJs have the
most complex suture system.
The above findings indicate that no one group of fibers
from any vertebrate lens, or one lens from any species, is
completely representative of all fibers or all lenses. When
analyzing fiber ultrastructure, it is imperative that both
the region of a fiber along its length and the age of the
fiber be qualified. Therefore, we developed (and routinely
employ) a dissection protocol that guarantees the unequivocal retrieval of anterior and posterior end segments of
fibers, as well as the mid-segment of the same fibers, at
progressive depths in any lens. The dissection protocol is
shown in Figure 2.
Briefly, under a surgical dissecting microscope equipped
with an ocular reticle, the axial dimensions and suture
patterns of a lens are recorded prior to dissection in buffer.
The Y sutures are apparent in a fixed, normal mouse lens
(Figs. 1C and 2A). With a pair of #5 electron microscopy
(EM) forceps, the fiber ends that abut and overlap at two
adjoining suture branches are separated. Then, by gently
sliding the tips of the forceps beneath the group of fibers
that had been part of the two adjoining suture branches,
the entire group of fibers can be peeled off of the lens (Fig.
2A). The resulting crescent-shaped group of fibers is
shown in Figure 2B at the time of dissection, and in Figure
2D by SEM. Because of fiber opposite end curvature, the
peel contains an intact suture branch, as confirmed by
SEM (Fig. 2D, arrow). Additional peels containing other
suture branches are then retrieved. At this point, the
equatorial diameter of the remaining lens mass is recorded. By subtracting this measure from that of the original, undissected lens, it can be ascertained whether the
fiber peels contain superficial cortical (peripheral 200 ␮ at
the equator) and/or cortical fibers (the next 500 – 800 ␮ at
the equator). The peels are then cut into three pieces
containing an intact anterior suture branch, mid-segment
of fibers, and posterior ends of fibers as seen at the time of
dissection (Fig. 2C) and confirmed by SEM (Fig. 2E and F).
Fig. 2. Dissection of mouse lenses for freeze-etch TEM. A–C: Photomicrographs taken through a surgical dissecting microscope. D–F:
SEM micrographs of comparable specimens. To obtain specimens containing either a fiber end or mid-segment, groups of fibers are carefully
peeled away from the lens, beginning at one of the poles (A). The
resulting crescent-shaped specimens (B and D) are then cut to isolate
specifically the end segments from the mid-segment of fibers (C and E).
Because one set of fiber ends (either anterior or posterior
ends of fibers) are grasped by the EM forceps, they may be
crushed and therefore are discarded. Progressive dissections to retrieve peels of deep cortical fibers and, successively, nuclear fibers can then be performed. If intact
anterior suture branches were retrieved by the above procedure, then the contralateral lens is used for retrieval of
posterior suture branches. Note that further dissection of
groups of fibers into distinct ⬍1 mm3 pieces at or near
sutures (anterior and posterior segments), as well as off
sutures (mid-segment of fibers) guarantees that the distinct ultrastructure along fiber length can be studied without any overlap between adjacent segments. The above
procedure has been performed successfully on mouse, rat,
guinea pig, chicken, cat, dog, monkey, and human lenses.
Anterior, posterior, and equatorial fiber segments of
only mature cortical fibers from 10-, 18-, and 24-week-old
wild-type, heterozygote, and homozygote lenses were dissected from fixed lenses and prepared for freeze-etch analysis by standard techniques (Kuszak et al., 1982). Briefly,
after primary fixation, the tissue was cryoprotected by
gradual infiltration of 25% glycerol prepared in cacodylate
buffer. The specimens were mounted with appropriate
orientation (broad faces mounted parallel to the frozen
knife) on standard Balzers gold specimen carriers, and
snap-frozen in supercooled liquid nitrogen. Fracture and
replication were carried out at –115 C in a Balzers 301
freeze-etch unit according to standard techniques (Moor
and Muhlethaler, 1963). A drop of 0.5% colloidon was
placed on the replica to enhance its structural integrity.
Replicas were retrieved onto formvar-coated, 100-mesh
copper grids and examined at 2,500 –100,000⫻ magnification in a JEOL JEM 1200EX transmission electron microscope at 80 kV.
Quantification of GJ and non-GJ areas, as well as GJ
size and percent distribution from the anterior and posterior ends and equatorial segments, was accomplished using PC Scion Image, Beta v. 4.0.2 (Scion Corp., Frederick,
MD). Approximately 2,500 ␮m2 of mature cortical fiber
membrane were replicated in each of the three lens genotypes (wild-type, heterozygote, and homozygote). Of these
amounts, 400 –500 ␮m2 of membrane of each fiber region
were randomly selected for statistical analysis. A KruskalWallis one-way analysis of variance (ANOVA) was used to
test for significant differences between groups.
Fiber Morphology (LM and SEM)
At the lens equator, nascent fibers rotate 90° about their
axis as they begin elongating bidirectionally toward the
poles (Duke-Elder and Wybar, 1961). This area, which is
called the “bow” region because of the configuration of the
nuclei from the differentiating cells, was examined by LM
of thick (1–2 ␮m) sections. In all wild-type lenses, the
transparent bow region was structurally consistent with
normal fiber differentiation (Fig. 3A). The bow region of
lenses from both heterozygote and homozygote MIP-deficient lenses were comparable to the wild-type lenses at all
ages examined (Fig. 3B and C). Further histological examination of MIP-deficient lenses (translucent heterozygote lenses and opaque homozygote lenses) revealed structural abnormalities, especially at the fiber ends. The fiber
ends were markedly larger than normal, and/or excessively disorganized as compared to fiber ends in wild-type
lenses (Fig. 3D–I). These abnormalities became progressively more severe with time (data not shown).
SEM was used to evaluate fiber surface morphology and
to expand on LM observations. In wild-type lenses split
along the anterior–posterior axis, fibers were arranged in
ordered RCCs and GSs throughout the cortical and nuclear regions (Fig. 4A). In addition, all cortical fibers were
of uniform shape (hexagonal) and size, and had typical
lateral interdigitations regularly arrayed along their
length (Figs. 4B and 5A). In contrast, the shape and arrangement of fibers in the heterozygote lenses were less
uniform (Fig. 4C). Specifically, some cortical fibers were
enlarged (Fig. 4C, black asterisks), thereby disrupting the
ordered arrangement of RCCs and GSs in adjacent fiber
layers. Furthermore, the cortical fibers of heterozygotes
had lateral interdigitations that were smaller and less
regularly arrayed along the fiber length compared to the
age-matched wild-type lenses (Figs. 4D and 5B). By 3
weeks after birth, anterior regions of the heterozygote
lenses often displayed acellular material, which likely resulted from the breakdown of cortical fiber ends (Fig. 4C,
white asterisk). SEM of homozygotes showed that although these lenses were grossly of comparable shape and
size to both heterozygote and wild-type lenses, they had
almost no semblance of normal fiber shape or arrangement (Fig. 4E). Rather, these lenses had amorphous fibers
that were only arranged in a vague configuration of RCCs
or GSs (Fig. 4E and F). Nevertheless, very irregular lateral-edge processes were still present along the lengths of
homozygote cortical fibers (Figs. 4F and 5C)
Because LM examination revealed early and marked
structural compromise at the fiber ends, SEM was utilized
to assess fiber-end morphology and sutural architecture.
Wild-type lenses split along the polar axis revealed normal anterior and posterior suture planes (Fig. 4A, arrows;
and Fig. 6A, asterisks) wherein all fiber ends exhibited the
proper curvature to abut and overlap with opposing ends.
Fiber peels exposed the suture branches within GSs in the
deep cortex. The ends of mature wild-type fibers were
slightly flared and irregularly shaped (Fig. 6B), as is typical of properly formed sutures (Kuszak et al., 1996). In
contrast, the posterior ends of superficial cortical fibers
from heterozygotes were curved away from the polar axis
and toward the vitreous (Fig. 6C, arrows). Suture planes
(Fig. 6C, asterisks) were excessively disorganized due to
the presence of abnormally larger fiber ends. These markedly larger ends were especially obvious in fiber peels
showing the suture branches formed by mature cortical
fibers in heterozygotes (Fig. 6D). The anterior ends of
fibers often appeared to be globular (data not shown),
leading to complete cellular breakdown in this region (as
mentioned above). Consequently, anterior suture planes
were not present in most heterozygotes by 3 weeks of age
(see Fig. 4C). In homozygote lenses split along the polar
axis, there were no apparent suture planes at any age
examined (Fig. 4E and 6E). Similarly, fiber peels failed to
reveal any suture branches in these lenses (Fig. 6F). The
complete lack of uniformity in fiber size, shape, opposite
end curvature, and arrangement precluded suture formation in homozygote lenses.
It is well established that fiber morphology in the nuclear region is less uniform than in the cortical region in
vertebrate lenses (Willekens and Vrensen, 1982; Kuszak
et al., 1983; Kuszak, 1995b; Taylor et al., 1996; Al-Ghoul
and Costello, 1997; Shestopalov and Bassnett, 2000). The
nuclear fiber morphology in wild-type lenses observed in
the present study was consistent with the above-cited
reports. Specifically, nuclear fibers were wider than cortical fibers, displayed variability in width along the fiber
length (Fig. 7A, arrows), and had lateral interdigitations
of variable size and shape (Fig. 7A, arrowheads). In comparison, nuclear fibers in heterozygotes were markedly
more irregular in shape and appeared to have roughened
surface morphology, possibly due to membrane degeneration (Fig. 7B). As noted in cortical fibers, nuclear fibers in
homozygotes were amorphous and had no regular arrangement (Fig. 7C). At higher magnification, it was revealed that these fibers displayed a complete lack of interdigitations (Fig. 7D).
Freeze-etch was used to examine the ultrastructure of
cortical (fully elongated and mature) fiber membrane from
wild-type, heterozygote, and homozygote mouse lenses at
10, 18, and 24 weeks of age. Thus, while we assessed the
contribution of MIP to fiber development and lens growth
by LM and SEM, freeze-etch was used only to examine
cortical fiber membrane ultrastructure in adult lenses. All
of the quantitative data (mean, median, and size range of
GJs) from wild-type, heterozygote, and homozygote lens
cortical fibers are summarized in Table 1.
Fig. 3. Light micrographs of thick (1–2 ␮m) sections taken through
the polar axis of mouse lenses. Left column, wild-type; center column,
heterozygote; right column, homozygote. In the equatorial region of
wild-type (A), and MIP-deficient (B and C) lenses, morphology appeared
normal at all ages examined. It was apparent that the initial differentiation
and elongation of lens fibers was not adversely affected by MIP defi-
ciency. However, by 1 week of age, MIP-deficient lenses had abnormally
larger fibers that were not organized into proper RCCs and/or GSs. This
was especially evident at the anterior (D–F) and posterior (G–I) ends.
Panels A–C are at equivalent magnification, and panels D–I are at equivalent magnification.
Fiber peels comprising approximately one-third of the
lens equatorial diameter were removed from the periphery
of lenses. This initial set of peels (approximately 250 ␮m
thick) contained superficial cortical fibers (both elongating
and fully elongated and maturing), wherein the elaboration of MIP is an ongoing process. The second set of peels,
containing only mature cortical fibers, wherein the elaboration of MIP is complete, was taken for freeze-etch anal-
ysis. These peels were further split into anterior, mid, and
posterior segments (see Fig. 2).
Freeze-etch replicas of all wild-type and heterozygote
lenses simultaneously revealed the membrane ultrastructure of defined BFs and narrow faces (NFs) on a number of
fibers aligned in defined GSs and RCCs (Fig. 8A). In addition, replicas from all segments of wild-type fibers confirmed the uniformity in fiber shape (hexagonal) and typ-
Fig. 4. SEM micrographs of wild-type and MIP-deficient mouse
lenses split along the polar axis. Wild-type lenses (A and B) had transparent fibers of uniform shape and size arranged in ordered RCCs and
GSs. Normal anterior and posterior suture planes were present (arrows).
By comparison, heterozygote lenses (C and D) had translucent fibers
that were disordered due to occasional abnormally large fibers (black
asterisks). Regions of apparent fiber breakdown were also noted (white
asterisk). Homozygote lenses (E and F) had opaque fibers that displayed
a complete lack of uniformity with respect to size, shape, and arrangement. Specifically, these fibers were not arranged in any discernible
RCCs and GSs, and had not formed any sutures. It also appeared that
cortical fiber interdigitations in MIP-deficient lenses (D and F) were less
regular in size and shape as compared to those from wild-type lenses
(B). Panels A, C, and E are at equivalent magnification; white boxes
indicate the enlarged regions shown at medium magnification in B, D,
and F. Panels B, D, and F are at equivalent magnification.
ical, lateral edge processes arrayed along the fiber length
(Fig. 8A, arrowheads). The ability to simultaneously replicate identifiable BFs and NFs correlated well with the
SEM results (Figs. 4 – 6). The translucent, heterozygote
fibers still had a relatively hexagonal cross-sectional
shape. In contrast, freeze-etch replicas of the opaque homozygote cortical fibers never simultaneously revealed
defined BFs and NFs in any fibers, and the fibers were
never aligned in either defined GSs and/or RCCs (Fig. 8B).
The inability to simultaneously replicate identifiable BFs
and NFs on cortical fibers from homozygote lenses also
correlated well with the SEM results (Figs. 4 – 6). The
homozygote fibers always had a non-hexagonal, abnormally large, polygonal, cross-sectional shape.
All segments of wild-type fibers had a very high non-GJ
particle density, reflective of a full complement of MIP
having been produced and inserted into the fiber membrane. However, square array membrane (orthogonal arrays of MIP) was only observed at high magnification on
low-amplitude membrane of anterior and posterior end
segments from wild-type lenses (Fig. 9A). In addition,
these fiber segments were essentially devoid of GJs (⬎1%).
In contrast, 13.18% of the mid-segment of wild-type fibers
were specialized as GJs. Quantification of individual GJ
plaque areas revealed that wild-type GJ size ranged from
0.004 to 1.574 ␮m2.
Freeze-etch replicas of all segments of fibers from heterozygote lenses confirmed an alteration in fiber uniformity (shape and size), and a less than normal array of
lateral-edge processes initially found by SEM examination
(data not shown). All regions of these fibers had reduced
intramembrane particle (IMP) density, indicative of the
decreased production and/or lack of insertion of MIP into
fiber membrane (Figs. 9B and 10). Square array membrane was not observed on any segment of fibers from
heterozygote lenses (Figs. 9B and 10). Examination of the
mid-segment of heterozygote cortical fiber membrane revealed that the percentage (14.5%) of this membrane specialized as GJs, was essentially identical to that quantified in the comparable region of wild-type lenses (13.15%).
However, the mean, median, and range of GJ size between
these two groups was significantly different (P ⱕ 0.001).
Freeze-etch replicas from all segments of homozygote
lens fibers confirmed the lack of any semblance of fiber
uniformity in shape and size. In addition, there were reduced numbers and more irregularly arrayed lateral-edge
processes, as shown previously (Fig. 8B). Furthermore, as
expected, a paucity of non-GJ particles was observed, confirming that MIP production had been “knocked out” (Fig.
11). It was also noted that, as in the heterozygote fibers,
square arrays were not observed on any segment of the
homozygote fibers. In addition, only 6.81% of the midsegment of these fibers was specialized as GJs, literally
half of that quantified in both wild-type and heterozygote
lenses. Furthermore, whereas the heterozygote and homozygote cortical fibers had the same mean GJ size, the
median and range of GJ size between these two groups
was significantly different (P ⱕ 0.001).
Fig. 5. Higher-magnification SEM micrographs of the mid-segment
of cortical fibers. In wild-type lenses, lateral interdigitations formed at the
angle of two narrow faces were of consistent shape, size, and spacing
(A). High magnification confirmed that alterations to the lateral interdigitations on cortical fibers were present in MIP-deficient lenses. Specifically, interdigitations on both heterozygote (B) and homozygote lens
fibers (C) were smaller than on fibers from wild-type fibers (A). Additionally, in homozygotes these membrane specializations were less numerous and lacked the orderly arrangement seen in both wild-type and
heterozygote lenses. Panels A–C are at equivalent magnification.
This study details the morphology of lenses from MIPdeficient mice. An examination of heterozygote lenses revealed specific structural changes as a result of reduced
synthesis of MIP in mature lens fibers that was considerably more amplified in homozygote lenses.
At birth, the heterozygote lenses appeared unaffected.
However, by P7, these lenses were translucent and both
the anterior and posterior ends of mature cortical fibers
were abnormally large, with a very nonuniform shape. In
addition, these fibers had atypical lateral interdigitations.
These lateral-edge processes were smaller and less regularly arrayed along the fiber length than in age-matched
wild-type lenses. By P21, it was apparent that the atypical
structure of heterozygote cortical fiber ends precluded the
formation of normal Y sutures. The posterior ends of these
fibers were consistently seen to be aberrantly curved away
from the polar axis. Nuclear fibers from these lenses
lacked lateral interdigitations by 4 weeks of age.
In a related study (Shiels et al., 2001), it was shown that
the structural abnormalities in heterozygote lenses were
sufficient to effect a significant compromise in lens function (sharpness of focus). In the same study it was also
found that the structural abnormalities in homozygote
lenses were so severe that the lenses were rendered completely opaque, and thus were completely nonfunctional.
It is clear from this report that a deficiency in MIP does
not prevent lens fiber differentiation and elongation.
Fig. 6. SEM micrographs of fiber-end and sutural architecture.
Lenses split along the polar axis (left column) revealed RCCs and GSs in
the cortex as well as the suture planes (white asterisks). Crescentshaped fiber “peels” (right column) exposed the BFs of cortical fibers
within one GS, as well as the corresponding suture branch. In wild-type
lenses (A and B), fibers always featured proper end curvature to form
sutures. Fiber ends were slightly flared and irregular, forming continuous
suture planes and branches. However, in heterozygotes (C and D), the
posterior fiber ends were aberrantly curved away from the polar axis and
toward the vitreous (C, arrows). These cortical fiber ends were abnormally large, resulting in excessive disorder at sutures. Examination of
homozygote lenses (E and F) failed to reveal any suture branches or
planes. The lack of suture architecture in homozygote lenses was due to
the complete absence of fiber uniformity in size, shape, opposite end
curvature, and/or arrangement in proper RCCs and GSs.
These findings are consistent with results in two MIP
mouse mutants, the Fraser cataract mice (CATFr) and
lense opacity (LOP) models (Novak et al., 1999; Shiels et
al., 2000). A transposon-induced splicing error in the
CATFr mouse results in the addition of a long terminal
repeat sequence, and MIP remains in intracellular membranes. In the LOP mouse model, a missense mutation
inhibits targeting of MIP to the fiber membrane, and it
remains in the cytoplasm. In both of the mutants, MIP
was shown to have been produced but not inserted into
fiber plasma membrane. Comparable structural alterations in mature fibers were seen in both of these animal
models. Mature fibers in both the cortex and nucleus of
MIP-deficient lenses were unable to maintain the long,
ribbon-like shape that was achieved during elongation. In
addition, the concentric arrangement of fibers into highly
Fig. 7. SEM micrographs of nuclear fiber morphology. Transparent
nuclear fibers in wild-type lenses (A) were wider than cortical fibers,
displayed variability in width along fiber length (arrows), and had variably-sized interdigitations between cells (arrowheads). By comparison,
translucent heterozygote nuclear fibers (B) were more irregular in shape
and had roughened surface morphology. Opaque nuclear fibers in ho-
mozygotes (C) were amorphous and had no regular arrangement. In
addition, higher magnification revealed that these fibers completely
lacked interdigitations between cells (D). Panels A–C are at equivalent
magnification. White box in panel C indicates the region shown at higher
magnification in panel D.
TABLE 1. Quantitative data derived from freeze-etch replication of wild type, heterozygote and homozygote
mip knockout mice cortical fibers showing the number, mean and median size and size range of gap junctions
Number of GJs analyzed
Mean area/GJ ⫾ s.e.m. (␮m2)
Median area of a GJ (␮m2)
Range of GJ size (min. -max.; ␮m2)
Total GJ area (␮m2)
Total membrane area (␮m2)
% membrane specialized as GJ
Wild type
0.087 ⫾ 0.006
0.102 ⫾ 0.006*
0.102 ⫾ 0.015**
Statistical analysis was only used to compare mean GJ size and range in GJ size. These are the variables that were derived
from measurements made on the micrographs. The other values presented in the table (median size of GJs and % of membrane
specialized as GJ) are total values calculated from average values. Since there is only one value per variable, statistical
analysis of these values is not possible (Jaeger, 1993). P ⬍ 0.001 for the following comparisons: *Wild type vs. heterozygote;
**wild type vs. homozygote; ***heterozygote vs. homozygote.
ordered GSs was progressively degraded as fibers aged.
Results from the present study were consistent with observations in both CATFr and LOP mouse lenses (Shiels et
al., 2000). The similarities among these three models indicate that while MIP is not essential for fiber formation,
it is important in the establishment and maintenance of
both individual fiber shape and overall fiber arrangement.
One explanation for the observed changes in fiber structure is that MIP may be involved in the maintenance of
fiber–fiber interactions that regulate morphology. A clue
to the mechanism by which MIP could regulate lens fiber
structure may be seen in the morphology of fiber interdigitations shown in this study. Qualitatively, MIP-deficient
cortical fibers appeared to have fewer lateral interdigitations with altered size and shapes as compared to normal
control lenses. MIP-deficient nuclear fibers were even
more compromised, with sparse or no lateral interdigitations, as a function of development and growth. Fiber
lateral interdigitations, such as balls and sockets, flaps,
and imprints (Kuszak et al., 1996), and both high- and
Fig. 8. TEM of the mid-segment of freeze-etch replicated cortical
fibers from wild-type (A) and homozygote (B) lenses. The characteristic
cross-sectional shape (hexagonal) of a number of wild-type fibers (F1–
F3) is readily apparent. Large expanses of both BFs and NFs of these
fibers aligned in GSs and RCCs are apparent. In contrast, while the
mid-segment of freeze-etch-replicated homozygote cortical fiber membrane revealed large expanses of membrane, the markedly irregular
(non-hexagonal) shape of these fibers precluded positive identification
of either defined BFs or NFs.
Fig. 9. TEM of freeze-etch-replicated, cortical fiber-end segments
from wild-type (A) and heterozygote (B) lenses. Both the anterior and
posterior fiber-end segments of fibers from all three groups were characterized by a paucity of GJs (⬍1% of these fiber regions were special-
ized as GJs). While square array membrane, or orthogonal arrays, comprised of MIP, were readily observed in wild-type lenses, they were never
observed in either heterozygote or homozygote lenses. Panels A and B
are at equivalent magnifcation.
low-amplitude wavy membranes (which are equivalent to
polygonal domains of furrowed membrane seen by SEM
(Taylor et al., 1996; Kuszak and Costello, 2003)), keep
fibers in close apposition, minimizing extracellular space
and thereby contributing to transparency by reducing
large-particle scatter. Lateral interdigitations may also
serve to maintain the relative positions of fibers in RCCs
and concentric GSs during accommodation or dynamic
Fig. 10. TEM of the mid-segment of freeze-etch-replicated cortical
fibers from a heterozygote lens. Although the total percentage (14.5%) of
this fiber region specialized as GJs was the same as that quantified in
the comparable region of wild-type lenses (13.18%), the median and
range in size of the heterozygote GJs were significantly different (P ⱕ
0.001) from that of the wild-type.
focusing. Therefore, the absence of properly formed and
arrayed fiber lateral interdigitations would likely lead to a
degradation in lens optical quality.
The initial structural alteration that occurred at mature
fiber ends in the MIP knockout, LOP, and CATFr (Novak
et al., 1999; Shiels et al., 2000) mouse lenses may have
been the result of swelling. The unique vulnerability of
fiber ends to cellular swelling is consistent with the fact
that square array membrane, composed of MIP (Costello
et al., 1989; Chandy et al., 1997), was shown in this study
to occur predominantly at the anterior and posterior end
segments of fibers, while GJs were found to predominantly
conjoin the mid-segment of fibers. The fact that the size
range of fiber GJs was significantly altered in both heterozygote and homozygote lenses supports the proposition
that MIP also has a role in establishing and maintaining
fiber–fiber communication in the lens (Dunia et al., 1998).
The polar distribution of fiber membrane specializations
shown in this study may also relate to the prevailing
opinion that an internal circulatory system is an important component of lens physiology (Mathias et al., 1997).
However, a quantitative comparison of the polar distribution and the number of fiber membrane specializations in
vertebrate lenses remains to be conducted. Indeed, while
the predominance of GJs conjoining the middle segment of
Fig. 11. TEM of the mid-segment of freeze-etch-replicated cortical
fibers from a homozygote lens. The total percentage (6.81%) of this fiber
region specialized as GJ was literally half of that quantified in either
wild-type or heterozygote lenses. However, whereas the mean GJ size of
homozygote GJs was the same as that quantified for heterozgote GJs,
the median and range in size of the GJs were significantly different
between these two groups (P ⱕ 0.001), as well as between either of
these two groups and the GJs quantified from wild-type lenses. Note the
paucity of nonjunctional IMPs in this replica as compared to that seen in
heterozygote lenses (Fig. 10), owing to the lack of MIP production and
insertion into fiber membrane in these KO lenses.
fibers has been shown to be a consistent lens structural
feature in all vertebrates (Fitzgerald, 1986; Kuszak et al.,
1996), the amount of this fiber region specialized as GJs
varies considerably between species (Kuszak et al., 1985;
Lo and Harding, 1986; Kuszak et al., 1996). Freeze-etch
studies have shown that GJs constitute ⬃60%, 30%, 15%,
and 5% of middle-segment fiber plasma membrane in
chick, rat, frog, and human lenses, respectively. Comparable quantitative analyses of the amount of fiber plasma
membrane specialized as square array membrane between species have not been performed to date. Such
freeze-etch studies will be inherently more difficult to
conduct. Aggregates of MIP as square array membrane
are generally smaller and less circumscribed than fiber
GJs, since they do not exist as raised oval and circular
plaques on P-faces of replicas, or as similarly shaped depressions on E-face exposures. However, in bovine lenses,
vast expanses of fiber plasma membrane are specialized
as square array membrane (Costello et al., 1989; Zampighi
et al., 1989). Qualitatively, the amount of fiber membrane
specialized as square array membrane appears to be at
least as variable between species as are the GJs. Thus, if
both the square array membrane and the GJs are elements of a lens internal circulatory system, then variations in their polarized distribution and relative amounts
in different species would influence the effectiveness of
fluid transport in each lens type as a function of development, growth, age, and pathology.
Finally, the results reported herein may appear to be
inconsistent with those from a recent study by Zampighi
et al. (2002), but actually they are not. In the study by
Zampighi et al. (2002), freeze-fracture labeling was used
to assess the spatial arrangement and interactions of MIP
with other proteins in the plasma membrane of the most
nascent fibers. It was concluded that 1) MIP was arranged
in microdomains that extended along the long axis of
nascent fibers, and 2) the density of MIP varied along the
long axis of nascent fibers, being least at the apical end
and greatest at the mid-segment. Furthermore, while the
MIP microdomains of nascent fibers contained densely
packed MIPs, they were not arranged in characteristic
orthogonal arrays or square array membrane (Costello et
al., 1989). As described above, crystalline lenses consist of
fibers in different states of terminal differentiation, maturation, and aging. The nascent fibers replicated by
Zampighi et al. (2002) were probably only elongated to
approximately 5% of their eventual mature length (2 mm).
As such, they contained only a small portion of their
future full complement of the major plasma membrane
proteins (MIP, membrane protein 19kd [MP19], and GJ
connexins 46 and 50) and underlying cytoskeletal network. Furthermore, such nascent fibers have not even
begun the process of developing the extensive array of
fiber–fiber lateral interdigitations and sutures necessary
to establish and maintain the exact arrangement of fibers
as RCCs and GSs required for lens function (i.e., dynamic
focusing) (Kuszak et al., 1996). Therefore, the nascent
fibers examined by Zampighi et al. (2002) were different
from the mature cortical fibers investigated in the current
study. It is also important to note that since both studies
involved young animals (maximum age 6 months), the
effects of aging on the contribution of MIP to fiber ultrastructure must still be addressed. This is an important
consideration in lens physiology because the lens retains
every fiber formed throughout life.
Fig. 12. Color-coded 3D-CADs summarizing diagrammatically the
contributions of MIP to lens structure. Epithelial membrane is depicted
as white, MIP is yellow, and GJs are blue. A: During normal differentiation of epithelial cells into fibers, MIP is inserted into fiber plasma
membrane. The mid-segment of fibers contains both GJs (blue) and MIP
(yellow), and is therefore depicted as green (blue ⫹ yellow ⫽ green).
Mature cortical fibers have a uniform shape and size, resulting in ordered
GSs, RCCs, and sutures. B: In heterozygote lenses, the end segments
are depicted as a lighter yellow, while the mid-segment are depicted as
more blue. Both color changes are indicative of the reduction in MIP
synthesis and/or insertion into the membrane. The cortical fibers of
heterozygotes are less uniform in shape and size, especially at fiber
ends, resulting in less ordered GSs, RCCs, and sutures. C: During fiber
differentiation in homozygote lenses, MIP is not produced, and therefore
the end segments of these fibers are depicted as white. Consistent with
the lack of MIP resulting in essentially a 50% reduction in the percentage
of the mid-segment of these fibers being specialized as GJs, this region
of homozygote fibers is depicted as light blue. These cortical fibers lack
any semblance of uniformity in shape and size along their entire length,
resulting in malformation of GSs, RCCs, and sutures.
In summary, the contributions of MIP to lens structure
are shown diagrammatically by color-coded, three-dimensional, computer-assisted drawings (3D CADs) presented
in Figure 12. As fiber terminal differentiation occurs, the
principal membrane protein produced and inserted is MIP
(represented as yellow). Lens epithelial membrane, which
lacks MIP, is depicted as white. The mid-segment of fibers
are depicted as green, consistent with the presence of both
GJs (represented as blue) and MIP (blue ⫹ yellow ⫽
green). Mature fibers are of a uniform shape and size,
resulting in ordered GSs, RCCs, and sutures. By comparison, as fiber terminal differentiation occurs in heterozygote lenses, elongating fiber membrane is depicted as light
yellow at the ends, and more blue at the mid-segment,
consistent with a reduction in MIP production and/or insertion. The mature fibers of heterozygote lenses are of
less uniform shape and size, particularly at the fiber ends,
resulting in less ordered GSs, RCCs, and sutures. Finally,
in homozygote lenses, fiber membrane is depicted as white
at the fiber ends, and light blue at the mid-segment,
consistent with essentially a complete lack of MIP. The
mature fibers of homozygote lenses are of nonuniform
shape and size along their entire length, resulting in malformation of GSs, RCCs, and sutures.
In conclusion, even a 50% loss of MIP is sufficient to
significantly compromise lens function (dynamic focusing)
and eventually lead to cataract, because MIP is necessary
to establish and maintain fiber shape and (by extrapolation) lens sutures, and fiber lateral interdigitations. It is
also essential in establishing and determining the size
and distribution of fiber GJs (predominantly at a fiber’s
mid-segment), and establishing square array membrane
(predominantly at fiber-end segments).
This work was supported in part by NIH NEI grants
EY06642 (to J.R.K.) and EY11411 (to A.S.). The technical
assistance of Mr. Layne Novak and Kurt L. Peterson is
gratefully acknowledged.
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