|Molecular Vision 2003;
Received 22 January 2003 | Accepted 15 March 2003 | Published 16 April 2003
Morphology and organization of posterior fiber ends during migration
Al-Ghoul,1,2,3 Jer R. Kuszak,2,3
Jeffrey Y. Lu,1 Michael J. Owens1
Departments of 1Anatomy & Cell Biology, 2Ophthalmology, and 3Pathology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL
Correspondence to: Kristin J. Al-Ghoul, Ph.D., Department of Anatomy & Cell Biology, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, 600 South Paulina Street, Chicago, IL, 60612; Phone: (312) 563-2672; FAX: (312) 942-5744; email: email@example.com
Purpose: To characterize structural parameters of the basal membrane complex (BMC) and to determine the arrangement and organization of posterior fiber ends during elongation/migration in lenses with branched sutures.
Methods: Lenses from normal, juvenile (4-6 week old) Sprague-Dawley rats (n=16) were utilized. Posterior fiber ends were assessed on both whole mounts of lens capsules and on decapsulated lenses. The size, shape and organization of migrating fiber ends was assessed by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) along the entire posterior surface. The area of the BMC was measured using image analysis software and subjected to statistical analysis.
Results: Posterior fiber ends had a characteristic regional arrangement during elongation and migration along the capsule. These regions were termed the equatorial, the lateral-posterior (posterior from the equator to within 150 μm of the sutures), the peri-sutural (150 μm surrounding the sutures), and the sutural. The area of fiber ends (seen by SEM) was compared to the area of fluorescent F-actin profiles (seen by LSCM). There was no significant difference (p=0.324) between the average basal end area (40.21 μm2) and the average area of F-actin profiles (40.65 μm2). The average fiber end area in the lateral-posterior, peri-sutural, and sutural regions was 63.19 μm2, 71.95 μm2, and 25.75 μm2, respectively. In the equatorial region, footprints were aligned in rows oriented toward the posterior pole, consistent with the arrangement of straight, meridional rows. Initially, fiber ends within the lateral-posterior region were arranged in short irregular rows having variable orientation with respect to the posterior pole. The remainder of these ends were randomly arranged. In the peri-sutural region, fiber ends approaching suture branches were aligned in short rows oriented at angles to the posterior pole. At the sutures, fiber ends appeared to become rounded, presumably during detachment from the capsule.
Conclusions: The results confirm that F-actin profiles delineate the BMC of posterior fiber ends. Furthermore, the average area, shape and arrangement of fiber ends varies in a predictable pattern during migration. The data suggests that elongating fiber ends follow defined migration patterns along the posterior capsule to their sutural destinations. This controlled process is crucial to the formation of ordered suture patterns, thereby minimizing their adverse effects on lens optical quality.
Lens fiber differentiation is a complex process wherein the cuboidal lens epithelial cells elongate into exceptionally long, ribbon-like cells. Elongation of nascent fibers occurs bi-directionally as fiber ends migrate both anteriorly and posteriorly with respect to the lens equator. Anterior fiber ends migrate along the apical surface of the epithelium, while posterior fiber ends migrate along inner surface of the capsule. When elongation is completed, the ends of lens fibers detach from the epithelium or capsule, and then abut with opposing fibers to form the lens sutures. Because the lens grows throughout life, new fibers are continually added onto the periphery of the lens. Consequently, fiber migration is an ongoing process and is subject to disruption from both senescent and pathological processes.
In avian lenses, developing fibers all elongate toward the poles and interdigitate. As a result, all fibers are essentially meridians, and branchless or umbilical sutures are formed at both poles (see  for review). In all other lenses, fibers within each growth shell exhibit variable length and curvature, resulting in the formation of suture branches. Thus, immature fibers originating from all equatorial locations must reach a precise sutural destination in order to establish and maintain a particular suture pattern in subsequent growth shells. For example, in lenses with line sutures, fiber ends curve in one of four directions to form a two-branched pattern. Similarly, in lenses with Y sutures, fiber ends curve in one of six directions to form a three-branched pattern. In general, lenses with more suture branches have more variations in fiber curvature (and length) and presumably, fiber ends have more complex migration patterns.
As stated above, posterior fiber ends interact with the lens capsule during elongation. Their coordinated adhesion to, movement along, and detachment from the capsule are mediated by the basal membrane complex (BMC), which has been characterized in avian lenses . The BMC of lens fibers consists of the basal domain of the fiber cell membrane, its integral membrane proteins and their associated cytoskeletal elements. This dynamic area constitutes a distinct structural and biochemical region, and includes components such as integrins, cadherins, actin, myosin, and caldesmon .
At the termination of their migration, basal fiber ends detach from the capsule and interdigitate to form the suture branches. Because the suture branches formed from each growth shell of fibers are successively overlain onto previous suture branches, suture planes are formed as the lens grows and ages. Improper or disorganized fiber end migration leads to the formation of irregular and/or excess suture branches that become aligned along the visual axis. This excessive disorganization has been shown to adversely affect lens optical quality [3-5] and, in severe instances, also compromises lens transparency [6,7]. Additionally, it has been established that a complete failure to form posterior sutures results in posterior subcapsular cataract (PSC) in two animal models of retinal degenerative disease [8-11], in a model to control the accumulation of fos-related antigens  and in the RLC mouse . Furthermore, faulty migration wherein the posterior fiber ends migrate into, rather than across, the capsule is involved in the formation of posterior cortical cataracts in SPARC-null mice [14,15]. It is evident from this that proper migration and detachment of fiber ends, especially basal ends, is critical in maintaining optimal lens function and transparency.
The goal of this study was to determine the overall arrangement and organization of migrating basal fiber ends in normal rat lenses, which possess a Y suture pattern. We examined both intact fiber ends on decapsulated lenses and fiber ends adhered to whole mounts of lens capsules. Our results indicate that the average area, shape and arrangement of basal fiber ends vary in a predictable pattern during fiber migration.
All animals were handled in compliance with institutional animal care and use guidelines and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A total of 20 normal, juvenile Sprague-Dawley rats (75-99 g) were utilized in this study. Animals were sacrificed by an intraperitoneal injection of a euthanasia agent (containing 6 g/ml sodium pentobarbital, 40.0% isopropyl alcohol, 2.5% propylene glycol, 0.05% disodium edetate, and 2.0% benzyl alcohol [Butler Company, Columbus, OH]). Immediately following sacrifice, eyes were enucleated and lenses were dissected from the orbit using a Zeiss surgical dissecting microscope (Zeiss USA, New York, NY).
Whole-mount lens capsules
Following animal sacrifice and enucleation, the posterior sclera and retina was removed to expose the lens, and the remaining anterior portion of the eye containing the lens was pre-fixed for 2 min in 2% paraformaldehyde. Light pre-fixing of the lens enables the basal fiber ends to adhere to the lens capsule when the capsule is removed from the lens. After a brief rinse in 0.07 M PBS, the lens was situated on dental wax on its posterior surface. Beginning at the anterior pole, the capsule was carefully peeled away from the lens around its diameter and pinned onto the dental wax (Figure 1A). The decapsulated lens was rolled off the posterior capsule and discarded (Figure 1B). Whole-mount lens capsules (with the sheared-off fiber ends adhered, Figure 1C) were then processed for examination with either scanning electron microscopy (SEM) or laser scanning confocal microscopy (LSCM). Using this procedure, only the posterior lens capsule and attached basal fiber ends were preserved intact. Typically, the anterior capsule, including the lens epithelial cells and the initial transitional zone cells were not obtained.
To expose intact basal fiber ends; fresh, unfixed lenses were placed in phosphate buffer immediately after dissection from the eye. Beginning at the anterior pole, the lens capsule was removed from the lens with #5 EM forceps. (Because the epithelial cells adhere to the capsule, the anterior epithelium was also removed.) The capsule plus epithelia was discarded and the lens mass was immediately fixed, then processed for SEM. Comparison of intact fiber ends from decapsulated lenses with fiber ends on whole mount lens capsules was made to ensure that the prefixation/decapsulation technique did not alter the morphology of fiber ends with respect to shape and arrangement.
Scanning electron microscopy (SEM)
Lenses were fixed in 2.5% gluteraldehyde (in 0.07 M cacodylate buffer, pH 7.2) for 3-5 days with fresh fixative changes daily. Whole mount lens capsules were fixed in the same fixative for 2 h. All specimens were then washed in 0.15 M cacodylate buffer, post-fixed overnight in 1% aqueous osmium tetroxide at 4 °C, washed in buffer, and dehydrated through a graded ethanol series. Following overnight dehydration in 100% ethanol, the alcohol was replaced with a graded ethanol/Freon 113 series (Dupont, Wilmington, DE) to 100% Freon 113. Specimens were then dried in 100% Freon in a critical-point drying apparatus (CPD 020, Balzers, Hudson, NH), secured on aluminum stubs with silver paste, sputter coated with gold, and examined in a scanning electron microscope (JSM 35c; JEOL, Peabody, MA) at 15 kV.
Laser scanning confocal microscopy (LSCM)
Whole-mount lens capsules were fixed for an additional 30 min in 2% paraformaldehyde, then washed in 0.07 M PBS. To label F-actin, specimens were incubated in phalloidin-FITC at 1:50 dilution of a methanolic stock (200 U/ml) solution (Sigma Chemical Company, St. Louis, MO). This was followed by thorough washing in 0.07 M PBS. The labeled capsule was then mounted on a glass coverslip (Gold Seal Products, Portsmouth, NH) with Vectashield mounting medium (Vector Laboratories, Inc., Burlington, CA) to prevent photobleaching. Specimens were examined using a Zeiss LSM5 laser scanning confocal microscope (Zeiss USA, New York, NY).
Morphometry and statistical analysis
Morphometry and morphology of the posterior fiber ends was assessed within 4 regions of fiber end migration, illustrated in Figure 2. These were defined as the equatorial region, the lateral-posterior region (posterior from the equator to within 150 μm of sutures), the peri-sutural region (150 μm surrounding the sutures), and the sutural region (directly adjacent to and at the suture branches). The initial region of fiber end migration, the equatorial region, was located just posterior to the equator. Transitional cells in this region migrate posteriorly and become nascent fibers.
The direction of fiber end migration was determined by reference to either the straight, meridional rows of basal ends in the equatorial zone or to the suture branches. At low magnification, the region of interest was compared to the appropriate referent and the migration direction was noted. This information was then used to indicate the direction of migration on micrographs. In some cases, basal ends of fiber cells had microspikes extending in the direction of migration, which could be used to confirm the direction of migration on high magnification micrographs.
SEMs were digitized at a resolution of 300 dpi on an Epson Perfection 1200 photo scanner (Epson America Inc., Long Beach, CA) and saved as TIFF files using a Pentium pc platform. Digital confocal data was converted to TIFF files. The data was viewed and measured using Scion Image, version beta 4.0.2 (Scion Corporation, Frederick, MD). Statistical analysis was conducted using Microsoft Excel 2000 (Microsoft Corporation, Redmond, WA). For the equatorial region, the area of posterior ends was measured on SEMs while the area of fluorescent F-actin profiles was measured on confocal micrographs. A total of 175 measures were selected at random (from a population of approximately 400) and used to compile descriptive statistics for each of the two sample populations. The data was subjected to analysis (student's t-test) to determine whether there was a statistically significant difference in the average area of fiber ends versus the average area of F-actin profiles in the BMC. A p value of less than 0.05 was considered significant. Images were viewed and analyzed using an LSM510 image browser (Carl Zeiss, Jena, Germany) and Adobe Photoshop version 5.5 (Adobe Systems Inc., San Jose, CA). For the remaining regions of fiber end migration, area measurements were made using both SEMs and confocal micrographs. A total of 200 measures in each region (randomly selected from a population of approximately 400) were used to compile descriptive statistics. The data was analyzed using the student's t-test to determine if the average fiber end area was significantly different between regions. A p value of less than 0.05 was considered significant.
Posterior fiber end morphology was characterized using both SEM and confocal microscopic localization of F-actin. The region termed "equatorial" was actually located slightly below the lens equator. In this area, the basal ends were those of transitional cells that had commenced posterior migration. As expected, basal ends in the equatorial region (Figure 3) were aligned in straight rows. In whole mount lens capsules, basal ends visualized by both SEM and confocal microscopy appeared comparable in size and arrangement (Figure 3). F-actin fluorescence appeared to be localized to the periphery of the BMC. In order to confirm that the fluorescent F-actin profiles delineated the extent of the BMC in fiber ends, we compared the areas of fiber ends visualized by SEM with the area of F-actin profiles visualized by LSCM (Table 1). The average basal end area measured on scanning electron micrographs (40.20 μm2) was virtually identical to the average area of fluorescent actin profiles (40.64 μm2). Statistical analysis revealed that there was no significant difference between the average areas of the two sample groups (p=0.324). The overall average area of basal ends for the equatorial region (using measurements from both techniques) was 40.07 μm2 (Table 2).
In the initial portion (approximately the first 100 μm) of the lateral posterior region, fiber ends were arranged in short, irregular rows (Figure 4A,B). However, as fiber ends migrated posteriorly, they were less ordered with respect to their orientation to the posterior pole (Figure 4C,D). In addition, the size and shape of fiber ends in the distal portion of the lateral-posterior region appeared more variable. Overall, the basal area of elongating fibers in the lateral-posterior region was 63.19 μm2 (Table 2). Statistical analysis revealed that the average BMC area for the lateral-posterior region was significantly larger than that in the equatorial region (p<0.001).
Fiber ends in the peri-sutural region (Figure 5) appeared more organized than in adjacent portions of the lateral-posterior region. For example, SEMs of intact fiber ends often displayed distinct directionality toward sutures (Figure 5A,B). Additionally, numerous microspikes were noted at basal ends of fibers as they migrated across the capsule toward the sutures (Figure 5B, black arrows). Approaching suture branches, basal fiber ends were arranged in either curved or straight rows (Figure 5C, dotted lines and Figure 6C, arrowheads) oriented at angles to the posterior pole. Their average area was 71.95 μm2. Compared to the lateral-posterior region, peri-sutural fiber ends had a significantly larger area (p<0.001), and were slightly more variable in size (Table 2).
At the sutures, fiber ends appeared to become rounded and decrease in size (Figure 6A,B). F-actin profiles showed a comparable pattern of numerous, small profiles that were randomly arranged (Figure 6C). The average area of fiber ends at posterior sutures was 25.75 μm2 (Table 2). These ends were significantly smaller than the average area of basal fiber ends in the peri-sutural region (p<0.001).
The morphology of fiber ends attached to whole-mount lens capsules was compared to intact fiber ends on decapsulated lenses (no prefixation) to insure that the prefixation/decapsulation technique did not alter the morphology of fiber ends with respect to shape and arrangement. In all regions examined, fiber end morphology was comparable whether or not fiber ends were attached to the posterior capsule (compare Figure 4A,C).
This study documented the morphology and organization of posterior fiber ends during migration in a Y-suture lens. The results indicate that the average area, shape and arrangement of posterior fiber ends varies during migration along the lens capsule. These data also suggest that elongating fiber ends follow defined migration patterns along the posterior capsule as they form the posterior suture branches. As described previously  the lens is an asymmetrical oblate spheroid, as is the Earth. Therefore, the position of any fiber can be described by its latitudinal and longitudinal position. Using this terminology, we propose that in lenses with branched sutures, posterior fiber ends migrate along meridians until they reach the latitudinal ring defined by the proximal ends of posterior suture branches (Figure 7). Only the fiber ends of straight fibers that will eventually elongate to the distal end of suture branches at the posterior pole continue to migrate along meridians. By comparison, fiber ends of straight fibers that elongate to the proximal ends of suture branches have completed their migration. All other fiber ends (fibers with opposite end curvature) migrate along paths which diverge from meridians after passing within the latitudinal ring described by the proximal ends of sutures. Specifically, these basal ends begin to migrate along a curved path, eventually elongating to the lateral aspects of the suture branches. This suggests that fiber development follows a mathematical construct that becomes progressively more complex in the umbilical, line, Y and star sutures of vertebrate lenses.
In a previous study of embryonic chick lenses, which feature the simple umbilical suture, it was demonstrated that basal fiber ends were always aligned in straight rows oriented toward the posterior pole, that is, along meridians . In contrast, the present study of juvenile rat lenses, which feature more complex Y-sutures, showed that basal fiber ends are not aligned along meridians throughout the elongation process (compare Figure 3, Figure 4, Figure 5, and Figure 6). It is logical to presume that lenses with star suture patterns (primates) would demonstrate yet more complex migration patterns.
The basal fiber ends visualized by SEM appear to be comparable to the fluorescent profiles produced by F-actin staining. The results of this study demonstrated that there was no significant difference between the average areas of the two samples groups (p=0.15), indicating that the fluorescent actin profiles delineated the BMC at the posterior fiber ends. This interpretation is consistent with prior investigations, where F-actin was found to be particularly abundant at the periphery of the lens fiber cells, at the point of contact with the capsule [2,16]. This finding provides a basis for future studies to utilize F-actin fluorescence to demarcate the BMC during localization of other molecular BMC components. Additionally, the results of this investigation define structural parameters of the BMC, such as arrangement and area, which provide the necessary premise for interpreting actin profiles in vibratome-sectioned specimens of the lens posterior surface .
The average area of the BMC increased as differentiating fibers migrated from the equatorial region through the lateral-posterior region and into the peri-sutural region toward the sutures (see Table 2). The gradual increase in the average BMC area across the posterior capsule suggests a correlation between fiber length and BMC area. One possible explanation for this apparent correlation is that the increased area reflects increased synthetic capacity during elongation.
The variability in the BMC area was also increased in the lateral-posterior and peri-sutural regions as compared to the equatorial region (Table 2). This large variability in area between adjacent fiber ends is one factor that contributed to the disorganized appearance of migrating basal fiber ends in both the lateral-posterior and peri-sutural regions. One explanation for these large variations in area within a given region is that the basal area of individual fibers changes repeatedly as they migrate across the capsule. This is typical of migration in various cell types, especially when the processes of membrane extension, adhesion and traction are not smoothly coordinated [18-21].
The average area of the BMC was abruptly and significantly reduced in the sutural region (Table 2). F-actin localization revealed numerous, small profiles at the CFI (Figure 6C). In addition, SEM analysis showed that the basal fiber ends had a rounded morphology, unlike the flattened basal ends in the adjacent peri-sutural region (compare Figure 5A,B and Figure 6B). Together, these data indicate that fibers probably detach partially from the capsule as they approach the suture branch.
The morphology of apical fiber ends appears to be comparable to basal ends. In avian lenses, apical fiber ends interfacing with transitional zone cells are hexagonal and uniform in size . As elongating fibers interface with the germinative zone, pregerminative zone, and central zone cells of the epithelium, their apical ends display only slight variations in cell shape and size [22,23]. This compares favorably with basal ends of elongating avian fibers, which appear to have a relatively stabile shape and size during migration . In contrast, the apical ends of mammalian (rodent and primate) lens fibers become quite variable in size and shape during migration across the apical surface of the epithelium [24,25]. Specifically, the irregularly polygonal, elongated shape of apical fiber ends that interfaced with the central zone epithelium was similar to the morphology of basal fiber ends detailed in the present report.
Despite the structural similarities between anterior and posterior ends of elongating fibers, it is likely that anterior and posterior fiber end migrations are independently controlled processes. Indirect evidence for this idea is provided by PSCs from several etiologies. In these models, the structural evidence suggested that proper migration and detachment of basal fiber ends was disrupted, resulting in a growth malformation which formed the PSC [8,11,12]. The fact that the anterior segments of the same fibers continued to migrate to the proper destination, detach at the correct time, and interdigitate with opposing fiber ends to form suture branches, supports the idea of independent apical and basal fiber end migration.
At posterior fiber ends, the BMC coordinates the adhesion, migration and detachment of fibers. Several components of the BMC such as actin, β-1 integrin, and N-cadherin have been studied to show their role in cell attachment, migration, and proliferation during fiber differentiation. Studies have definitively shown that β-1 integrins are the primary integrin receptor for the basal lamina proteins in the lens capsule . These integrins act as bi-directional signaling receptors, mediating interactions between the cytoskeleton and extracellular matrix which regulate adhesion and polarization . N-cadherin, a cell adhesion molecule, is also implicated in lens fiber differentiation. Through its linkage to actin, N-cadherin regulates differentiation-dependent cytoskeletal reorganization . Predictably, N-cadherin is co-localized with actin both within the native BMC and in cell culture [2,17,28]. Finally, actin has been found to play an important role in coordinating structural changes and maintaining the integrity of lens development during fiber cell elongation [2,29-31].
Our recent investigations have determined that the distribution of some BMC components alters as fibers approach their sutural destinations . Although the regulation of accurate fiber end tracking to the sutures and timely detachment from the capsule is not fully understood, it is clear that some of the molecules involved in the initiation of fiber differentiation (see for review) play a role in these later processes. Specifically, signaling from growth factors such as IGF-1 and TGF-β may be required for appropriate migration of elongating fibers [33,34]. Cdk5, an important regulator of neuronal migration, has been localized to the ends of elongating fibers and is likely to affect adhesion in both cell-cell and cell-matrix interactions in the lens [35,36]. In addition, the membrane protein MP20 may also be involved in cell-cell adhesion during fiber migration via its interaction with galectin .
It is well established that mechanical changes in the cytoskeleton participate in the regulation of cellular differentiation processes such as growth and migration (for review see). In particular, actin is a key player in the dynamic cytoskeletal reorganizations which are necessary during cell migration along a substratum. Tropomodulin, which caps actin filaments (and binds to tropomyosin), is expressed in lens fibers only after the commencement of differentiation and appears to stabilize actin filaments during fiber elongation [16,39]. In addition, tropomodulin is enriched at apical and basal ends of elongating chick fibers, suggesting that it may also have a role in migration . An extensive network of zonulae adherens/actin bundles, in which N-cadherin was identified as the principle adhesion protein, has been documented at apical ends of both epithelial and fiber cells in the developing lens . This raises the intriguing possibility that if the adherens structures persist after embryonic development, then N-cadherin would be perfectly positioned to mediate differentiation-dependent cytoskeletal reorganizations at apical ends of nascent fibers.
In summary, this investigation has characterized important structural parameters of the basal membrane complex (BMC) and has determined the arrangement and organization of posterior fiber ends during migration in lenses with branched sutures. Accurate migration, followed by detachment and interdigitation at sutures must occur sequentially in each growth shell of fibers in order to maintain the orderly array of cells which is necessary for optimum lens function and transparency . Disruption of these processes would be likely to result in faulty migration leading to sutural malformations which affect lens focusing [6,7], and in extreme cases may result in posterior subcapsular cataract formation [8,11,12]. The results presented in the present study establish some parameters of migrating basal fiber ends in normal, juvenile rat lenses and provide the necessary premise for accurate interpretation and evaluation of basal fiber ends in pathological situations entailing aberrant migration. Further elucidation regarding the regulation of fiber migration is clearly necessary in order to reveal the underlying mechanisms of posterior sutural malformations.
Presented in part at the 2002 Annual Meeting of the Association for Research in Vision and Ophthalmology, Ft. Lauderdale, FL. The technical assistance of Mr. Layne Novak is gratefully acknowledged. The authors thank Dr. Mei-Ling Chen of the Research Resource Center (University of Illinois at Chicago) for valuable technical advice. This work was supported by a grant from the Midwest Eye Banks & Transplantation Center, Ann Arbor, MI (KJA) and by the Doctor Bernard and Jennie M. Nelson Fund, Chicago, IL.
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