Purpose

The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age.

Methods

Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6.

Results

CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin.

Conclusions

CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

Purpose

Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.

Methods

A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.

Results

An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.

Conclusions

A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0^ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0^ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0^ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0^ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0^ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

Purpose

To investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract.

Methods

The formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers.

Results

Postnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared.

Conclusions

CP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.

Purpose

To review, contrast, and compare current known tapetal mechanisms and review the implications for the evolution of the tapetum.

Methods

Ocular specimens of representative fish in key piscine families, including Acipenseridae, Cyprinidae, Chacidae; the reptilian family Crocodylidae; the mammalian family Felidae; and the Lepidopteran family Sphingidae were reviewed and compared histologically. All known varieties of tapeta were examined and classified and compared to the known cladogram representing the evolution of each specific family.

Results

Types of tapeta include tapetum cellulosum, tapetum fibrosum, retinal tapetum, invertebrate pigmented tapetum, and invertebrate thin-film tapetum. All but the invertebrate pigmented tapetum were examined histologically. Review of the evolutionary cladogram and comparison with known tapeta suggest that the tapetum evolved in the Devonian period 345 to 395 million years ago. Tapeta developed independently in at least three separate orders in invertebrates and vertebrates, and yet all have surprisingly similar mechanisms of light reflection, including thin-film interference, diffusely reflecting tapeta, Mie scattering, Rayleigh scattering, and perhaps orthogonal retroreflection.

Conclusion

Tapeta are found in invertebrates and vertebrates and display different physical mechanisms of reflection. Each tapetum reflects the wavelengths most relevant to the species' ecological niche. With this work, we have hypothesized that the tapetum evolved independently in both invertebrates and vertebrates as early as the Devonian period and coincided with an explosion of life forms.

Purpose

We employed in vivo, 1.0-µm axial resolution visible-light optical coherence tomography (OCT) and ex vivo electron microscopy (EM) to investigate three subcellular features in the mouse outer retina: reflectivity oscillations inner to band 1 (study 1); hyperreflective band 2, attributed to the ellipsoid zone or inner segment/outer segment (IS/OS) junction (study 2); and the hyperreflective retinal pigment epithelium (RPE) within band 4 (study 3).

Methods

Pigmented (C57BL/6J, n = 10) and albino (BALB/cJ, n = 3) mice were imaged in vivo. Enucleated eyes were processed for light and electron microscopy. Using well-accepted reference surfaces, we compared micrometer-scale axial reflectivity of visible-light OCT with subcellular organization, as revealed by 9449 annotated EM organelles and features across four pigmented eyes.

Results

In study 1, outer nuclear layer reflectivity peaks coincided with valleys in heterochromatin clump density (-0.34 ± 2.27 µm limits of agreement [LoA]). In study 2, band 2 depth on OCT and IS/OS junction depth on EM agreed (-0.57 ± 0.76 µm LoA), with both having similar distributions. In study 3, RPE electron dense organelle distribution did not agree with reflectivity in C57BL/6J mice, with OCT measures of RPE thickness exceeding those of EM (2.09 ± 0.89 µm LoA). Finally, RPE thickness increased with age in pigmented mice (slope = 0.056 µm/mo; P = 6.8 × 10-7).

Conclusions

Visible-light OCT bands arise from subcellular organization, enabling new measurements in mice. Quantitative OCT-EM comparisons may be confounded by hydration level, particularly in the OS and RPE. Caution is warranted in generalizing results to other species.

Purpose

To investigate diurnal variation in the length of mouse rod outer segments in vivo.

Methods

The lengths of rod inner and outer segments (RIS, ROS) of dark-adapted albino mice maintained on a 12-hour dark:12-hour light cycle with light onset 7 AM were measured at prescribed times (6:30 AM, 11 AM, 3:30 PM) during the diurnal cycle with optical coherence tomography (OCT), taking advantage of increased visibility, after a brief bleaching exposure, of the bands corresponding to RIS/ROS boundaries and ROS tips (ROST).

Results

Deconvolution of OCT depth profiles resolved two backscatter bands located 7.4 ± 0.1 and 10.8 ± 0.2 µm (mean ± SEM) proximal to Bruch's membrane (BrM). These bands were identified with histology as arising from the apical surface of RPE and ROST, respectively. The average length of dark-adapted ROS at 6:30 AM was 17.7 ± 0.8 µm. By 11 AM, the average ROS length had decreased by 10% to 15.9 ± 0.7 µm. After 11 AM, the ROS length increased steadily at an average rate of 0.12 µm/h, returning to baseline length by 23.5 hours in the cycle.

Conclusions

The diurnal variation in ROS length measured in these experiments is consistent with prior histological investigations showing that rodent rod discs are phagocytosed by the RPE maximally over several hours around the time of normal light onset. The rate of recovery of ROS to baseline length before normal light onset is consistent with the hypothesis that disc membrane synthesis is fairly constant over the diurnal cycle.

Determining which biological traits affect taxonomic durations is critical for explaining macroevolutionary patterns. Two approaches are commonly used to investigate the associations between traits and durations and/or extinction and origination rates: analyses of taxonomic occurrence patterns in the fossil record and comparative phylogenetic analyses, predominantly of extant taxa. By capitalizing upon the empirical record of past extinctions, paleontological data avoid some of the limitations of existing methods for inferring extinction and origination rates from molecular phylogenies. However, most paleontological studies of extinction selectivity have ignored phylogenetic relationships because there is a dearth of phylogenetic hypotheses for diverse non-vertebrate higher taxa in the fossil record. This omission inflates the degrees of freedom in statistical analyses and leaves open the possibility that observed associations are indirect, reflecting shared evolutionary history rather than the direct influence of particular traits on durations. Here we investigate global patterns of extinction selectivity in Devonian terebratulide brachiopods and compare the results of taxonomic vs. phylogenetic approaches. Regression models that assume independence among taxa provide support for a positive association between geographic range size and genus duration but do not indicate an association between body size and genus duration. Brownian motion models of trait evolution identify significant similarities in body size, range size, and duration among closely related terebratulide genera. We use phylogenetic regression to account for shared evolutionary history and find support for a significant positive association between range size and duration among terebratulides that is also phylogenetically structured. The estimated range size - duration relationship is moderately weaker in the phylogenetic analysis due to the down-weighting of closely related genera that were both broadly distributed and long lived; however, this change in slope is not statistically significant. These results provide evidence for the phylogenetic conservatism of organismal and emergent traits, yet also the general phylogenetic independence of the relationship between range size and duration.

Purpose

To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.

Methods

We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers.

Results

F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers.

Conclusions

These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

Aberrant connectivity between the dorsolateral prefrontal cortex (DLPFC) and the subgenual cingulate cortex (SGC) has been linked to the pathophysiology of depression. Indirect evidence also links hippocampal activation to the cognitive side effects of seizure treatments. Magnetic seizure therapy (MST) is a novel treatment for patients with treatment resistant depression (TRD). Here we combine transcranial magnetic stimulation with electroencephalography (TMS-EEG) to evaluate the effects of MST on connectivity and activation between the DLPFC, the SGC and hippocampus (Hipp) in patients with TRD. The TMS-EEG was collected from 31 TRD patients prior to and after an MST treatment trial. Through TMS-EEG methodology we evaluated significant current scattering (SCS) as an index of effective connectivity between the SGC and left DLPFC. Significant current density (SCD) was used to assess activity at the level of the Hipp. The SCS between the SGC and DLPFC was reduced after the course of MST (p < 0.036). The DLPFC-SGC effective connectivity reduction correlated with the changes in Hamilton depression score pre-to-post treatment (R = 0.46; p < 0.031). The SCD localized to the Hipp was reduced after the course of MST (p < 0.015), and the SCD change was correlated with montreal cognitive assessment (MOCA) scores pre-post the course of MST (R = -0.59; p < 0.026). Our findings suggest that MST treatment is associated with SGC-DLPFC connectivity reduction and that changes to cognition are associated with Hipp activation reduction. These findings demonstrate two distinct processes which drive efficacy and side effects separately, and might eventually aid in delineating physiological TRD targets in clinical settings.