Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy

BACKGROUND: Compaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses. METHODS: Transparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 μm were compared. RESULTS: Diabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts. CONCLUSION: As in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant

Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy

BACKGROUND: Compaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses. METHODS: Transparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 μm were compared. RESULTS: Diabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts. CONCLUSION: As in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant

Simultaneous Stretching and Contraction of Stress Fibers In Vivo

To study the dynamics of stress fiber components in cultured fibroblasts, we expressed α-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and α-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and α-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider

Differential susceptibility of S and M phase cyclin/CDK complexes to inhibitory tyrosine phosphorylation in yeast

Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B-type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation

A Role for Cdc2- and PP2A-Mediated Regulation of Emi2 in the Maintenance of CSF Arrest

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell cycle arrest by inhibiting the anaphase promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood

Predicted Light Scattering from Particles Observed in Human Age-Related Nuclear Cataracts Using Mie Scattering Theory

PURPOSE. To employ Mie scattering theory to predict the lightscattering from micrometer-sized particles surrounded by lipid shells, called multilamellar bodies (MLBs), reported in human age-related nuclear cataracts. METHODS. Mie scattering theory is applicable to randomly distributed spherical and globular particles separated by distances much greater than the wavelength of incident light. With an assumed refractive index of 1.40 for nuclear cytoplasm, particle refractive indices from 1.33 to 1.58 were used to calculate scattering efficiencies for particle radii 0.05 to 3 m and incident light with wavelengths (in vacuo) of 400, 550, and 700 nm. RESULTS. Surface plots of scattering efficiency versus particle radius and refractive index were calculated for coated spherical particles. Pronounced peaks and valleys identified combinations of particle parameters that produce high and low scattering efficiencies. Small particles (Ͻ0.3 m radius) had low scattering efficiency over a wide range of particle refractive indices. Particles with radii 0.6 to 3 m and refractive indices 0.08 to 0.10 greater (or less) than the surrounding cytoplasm had very high scattering efficiencies. This size range corresponds well to MLBs in cataractous nuclei (average MLB radius, 1.4 m) and, at an estimated 4000 particles/mm 3 of tissue, up to 18% of the incident light was scattered primarily within a 20°forward cone. CONCLUSIONS. The calculated size of spherical particles that scatter efficiently was close to the observed dimensions of MLBs in cataractous nuclei. Particle refractive indices only 0.02 units different from the surrounding cytoplasm scatter a significant amount of light. These results suggest that the MLBs observed in human age-related nuclear cataracts may be major sources of forward light scattering that reduces contrast of fine details, particularly under dim light. (Invest Ophthalmol Vis Sci. 2007;48:303-312) DOI:10.1167/iovs.06-0480 A ssessments of light-scattering by the ocular lens depend critically on the measurement geometry. For example, slit lamp images obtained by ophthalmologists detect light scattered mainly by small particles that preferentially scatter at high angles. 1-3 Light scattered at low angles (forward scattering) is more difficult to measure in vivo, although indirect methods have been proposed, 11 Mie calculations are presented that explore parameters influencing particle-scattering efficiency and that compare predicted relative scattering from transparent and cataractous lens nuclei. It is important to note that Rayleigh and Mie scattering are not two different kinds of scattering, but are two different methods of evaluating light-scattering from particles of various sizes. Mie calculations are rigorous for spheres and correct for any size. Rayleigh calculations are correct only for very small particles, less than one tenth the wavelength of the incident light. METHODS Morphologic Analysis Data from a recent study of eight transparent and eight cataractous nuclei were used to estimate the properties of a typical MLB along the optic axis and determine the range of variables used to calculate Mie scattering surface plots. 11 Briefly, fresh lenses or nuclei were sectioned on a microtome (model 1000; Vibratome, St. Louis, MO) and the 200-m-thick sections were fixed by immersion (2% paraformaldehyde, 1% tannic acid in 0.1 M cacodylate buffer [pH 7.2]; 0.5% glutaraldehyde for LM, and 2.5% glutaraldehyde for TEM) for 12 to 18 hours at room temperature with rotation. TEM samples were postfixed in osmium tetroxide (1% for 1 hour at 4°C). Both were treated with uranyl acetate (2% for 1 hour at room temperature) followed by alcohol dehydration. For LM, microtome-cut thick sections were embedded in LR-White resin (EMS, Hatfield, PA), sectioned to 0.7-m thickness, and stained with toluidine blue oxide. These histologic sections were examined on a light microscope (DMR; Leica Bannockburn, IL) equipped with a 12-megapixel charge-coupled device (CCD) digital camera (DC-500; Leica). For TEM, microtome-cut thick sections were embedded in Epon epoxy resin (EMS), thin-sectioned to 70 nm with a diamond knife (Diatome; EMS), and grid stained with uranyl acetate and lead citrate. Thin sections were examined with a transmission electron microscope (FEI-Philips Tecnai 12; FEI Co., Hillsboro, OR) at 80 kV using a 1 k ϫ 1 k CCD digital camera (model 694l; Gatan, Pleasanton, CA). Protocols were reviewed by the Institutional Review Board and the research was conducted according to the tenets of the Declaration of Helsinki for the protection of human subjects. Theoretical Analysis The scattering cross section of a single MLB of radius r is equal to qr 2 , where q is the scattering efficiency of the MLB. Because the volume fraction of the MLBs in the lens is quite small (Ͻ0.005%) and they are randomly distributed, they can be considered as independent scatterers. If no MLB shadowed another, the relative scattering cross section of N MLBs would equal Nq r 2 /A where A is the cross-sectional area of the central square millimeter of the lens nucleus. In this region N is equal to tA, where is the density of MLBs (per cubic millimeter) and t is the thickness of the nucleus (along the path of incident light). Therefore, the relative scattering cross section of all the MLBs in the center of the lens nucleus would be 11 S/A ϭ tqr 2 However, the values must be corrected for the fact that each incident photon has a probability of being scattered by an MLB before it reaches another MLB deeper in the lens nucleus. This process is analogous to the exponential decrease in probability that a raindrop will hit a dry spot after previous raindrops have begun covering a dry surface. This issue has been addressed in a derivation of Beer's Law, The scattering efficiency, q, was calculated employing Mie theory implemented by using Mätzler's algorithms 16 in commercial software (MatLab; MathWorks, Natick, MA). Each point on the surface plots represents a single calculation for ranges of parameters relevant to the observed MLBs. The key input parameters were the ratios of particle diameters to wavelength and the ratios of internal refractive indices of the MLBs to cytoplasmic refractive index, measured directly to be 1.40. 14 This average scattering angle, , is given by where I() is the intensity of light scattered at the angle . For small particles, which scatter as the average scattering angle is 90°. For large particles, the average angle can be less than 10°. The relative scattering cross section, S/A, was calculated for three wavelengths of unpolarized light, given the observed MLB radii and their densities in transparent and cataractous nuclei. 11 RESULTS Morphologic Analysis Further characterization of MLBs along the optic axis are presented in Figures 1 to 3, which display new images of MLBs, emphasizing their unique features and the distribution of sizes not previously published. Light microscopy of toluidine blue oxide histologic sections reveals MLBs as circular profiles with white borders 10,11 These particles present circular profiles in sections, and, since two thirds of the particles examined here present circular profiles in sections, it is reasonable to assume that they are also spherical. The remaining one third are slightly oval or have several components, at least one of which appears to be spherical Particle dimensions for all 117 MLBs observed in the cataractous nuclei (including the embryonic, fetal and juvenile nuclei) have been presented as a frequency plot 11,20 The corrected diameter is 2.7 Ϯ 0.7 m. The distribution of particle diameters is almost normal, with the exception of a tail at larger diameters indicated by the observation of several particles more than 3.5 m in diameter. A larger number of measurements would be necessary to determine whether this minor deviation from normal distribution is significant. The shape of the frequency distribution and the Even when more than one MLB was visible in the same field of view, they were separated by distances much larger than the wavelength of incident light. The MLB at the upper right had a core visible at a different plane of focus. Edge processes (arrowheads) were numerous profiles also found with a similar density in transparent lenses. close correspondence of the mean and median (2.7 m vs. 2.6 m, respectively) suggest that the value of 2.7 m is a reasonable representation of a typical MLB diameter in the human nuclear cataracts examined. The range of particle sizes used in the Mie scattering calculations was adjusted to include the full range of particle sizes observed. From TEM images at high magnification, there appeared to be 3 to 10 thin bilayers forming the coat of the MLBs Differences between the refractive indices of the particles and the surrounding cytoplasm are essential to produce observable light scattering. TEM images clearly show that many MLBs have interiors that are very different from their surroundings 21 Although this appears to be a high refractive index for an ocular lens, it is noted that this refractive index is still somewhat less than the value of 1.55 reported for the nuclear core of fish lenses. Mie Theory Calculations of Particle Light Scattering The scattering efficiency, q, per MLB in unpolarized light of 550 nm wavelength was calculated for a range of particle sizes and refractive indices of the MLB interior, assuming that the lipid-rich coat has n ϭ 1.50 and the cytoplasm has n ϭ 1.40 This surface plot reveals the remarkable result that there is the potential for particles to have very large scattering efficiencies. Because they have such strong interactions with light at visible wavelengths, their scattering cross section is nearly three times larger than the actual particle cross section. This finding is important from another viewpoint. The surface plot suggests that relatively few super scattering particles can generate more scattered light than a much larger number of less efficient scattering particles. It should also be noted that valleys (blue regions) give valuable information. Thus, the valley at n ϭ 1.40 indicates that, regardless of particle size in the range displayed, if the interior refractive index is less than 1.42 (ratio 1.42/1.40 ϭ 1.0143), then these particles will generate very little scattered light. In addition, over the entire range of refractive indices displayed, particles with radii ranging from 0.05 to 0.3 m are extremely inefficient scatterers. Finally, particles with internal refractive indices lower than the surrounding cytoplasm scatter light with roughly the same efficiency. For the typical MLB, based on the actual particles observed and an assumed internal refractive index of 1.49, the calculated scattering efficiency was 2.6, which would place the particle near the top of the steep slope of the surface plot It has been shown in a polar plot that incident light is scattered by typical MLBs (1.36 m radius, internal n ϭ 1.49) FIGURE 3. Frequency plot of MLBs in cataractous nuclei. A total of 117 MLBs were observed in the juvenile, fetal, and embryonic nuclei of human age-related cataracts. 11 The diameter of MLBs presenting a circular profile (two thirds of all particles) was measured on light micrographs to the outer edge of the clear rim around the particle. For complex MLBs, the length and width were measured and averaged to give a diameter of an equivalent circular profile. The distribution was nearly normal, with a tail containing several particles larger than 3.5 m diameter. Mean Ϯ SD of the raw data is 2.13 Ϯ 0.64 m. with high intensity in the forward direction within a narrow cone of 20°scattering angle (i.e., Ϯ10°around the optic axis). 11 This angular dependence of scattered light is more completely displayed in the surface plot calculated using unpolarized 550-nm incident light 9,13,14 Of particular interest is the relative amount of light scatter predicted by Mie scattering theory for the measured density of MLBs reported. 11 The relative scattering cross section, S/A, is calculated for the observed MLBs (summarized in DISCUSSION Mie scattering theory is appropriate for the coated particles observed in nuclei of aged transparent lenses and age-related cataracts. The theory gives exact solutions for spherical particles of any size and refractive index, and gives fairly accurate solutions for globular particles in general. 9,12-14 The conditions for Mie scattering calculations, mainly the random distribution of globular particles separated by distances greater than the wavelength of incident light, are well met by the observed MLBs. 11 In contrast, the utility of Rayleigh scattering theory, even incorporating modifications or approximations such as Debye-Gans, 9,13,14 is limited for large particles. Wavelength dependence of Rayleigh scattering theory for independent scatterers and the predicted scattering roughly equal in all directions is suitable for small particles about one tenth of the wavelength of light, 9,13,14 but cannot predict correctly the low-angle scattering or the scattering efficiency of the MLBs. The algorithms for Mie scattering theory permit the calculation of three-dimensional surface plots describing potential scattering from coated spherical particles over a wide range of sizes, refractive index properties and wavelengths of incident light. 308 Costello et al. IOVS, January 2007, Vol. 48, No. 1 Downloaded from iovs.arvojournals.org on 06/28/2019 FIGURE 7. Surface plots of percentage light scatter (rainbow scales, 0%-70%) as a function of particle radius and refractive index of the particle interior. Predicted scattering from MLBs is based on observed density in transparent nuclei (A-C) and cataractous nuclei (D-F) Fiber cell membranes have also been implicated in cataract formation through direct scattering of membrane components, alterations of membranes and deposition of proteins on membranes. Recently, Michael et al., 11,29 An additional factor is that the scattering from the membranes is dependent mainly on the membrane thickness rather than the membrane topology. Minimal light is scattered by sheets that are thin compared with the wavelength of incident light, even if their topology is complex. The surface plots highlight the features of particles responsible for forward scattering and give direct visualization of the scattering performance of experimentally observed MLBs. Although the observed MLBs displayed a range of particle sizes, for simplicity, the average diameter measured in sections was used for the Mie calculations. The critical property of the ratio of internal to cytoplasmic refractive indices (1.49/1.40) was chosen to illustrate the effect of having particles that are significantly different as indicated from electron micrographs

A Role for PP1 in the Cdc2/Cyclin B–mediated Positive Feedback Activation of Cdc25

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry