Sondip K. Biswas

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC‐induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF‐α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.

Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens.

PURPOSE Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. METHODS The loss of AQP0 in AQP0(-/-) lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0(-/-) lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. RESULTS Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. CONCLUSIONS This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.

Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. In the present study, dynamics of the spindle pole body (SPB) during the cell cycle was examined using freeze-substitution and serial thin-sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar-shaped in G1 through G2 phases. At the beginning of prophase, globular elements of the SPB enlarged, associated with numerous cytoplasmic microtubules, and separated on the nuclear envelope. At prometaphase, the SPBs entered the nuclear region by breaking a part of the nuclear membrane, were located at the isthmus, and were associated with numerous nuclear microtubules. The nuclear division process was carried out in the daughter cell, though the nucleolus remained in the mother cell. At anaphase, one half of the nucleus returned to the mother cell. At telophase, the SPB element was extruded back to the cytoplasm from the nuclear region. By analyzing serial sections of 63 cells, duplication of the SPB was found to take place in the early G1 phase. Thus, the location, structure, and duplication cycle of the C. neoformans SPB are different from those of Saccharomyces cerevisiae, but have similarities to those of Schizosaccharomyces pombe.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

The spindle pole body of the pathogenic yeast Cryptococcus neoformans: variation in morphology and positional relationship with the nucleolus and the bud in interphase cells

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Cryptococcus neoformans was studied in detail by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar shaped. The dumbbell-shaped SPBs were 228-365 nm long with amorphous spheres on each end, each sphere being 78-157 nm in diameter. The bar-shaped SPBs were 103-260 nm long and 32-113 nm thick. They consisted of filamentous materials. The dumbbell-shaped SPBs were more frequent (61%) than the bar-shaped SPBs. The bar-shaped SPBs may be regarded as dumbbell-shaped SPBs whose spherical parts became sufficiently small. There seemed to be no relationship between the SPB shape and the cell cycle stage of G1-G2, since both types of SPB appeared not only in unbudded cells but also in budded cells and their appearance seems to be random. It is not clear at present whether morphological changes between dumbbell- and bar shapes have any physiological function. The SPB tended to be localized away from the nucleolus (141 degrees +/- 44 degrees), but localized randomly to the bud (97 degrees +/- 50 degrees). The present study highlights the necessity of observing a large number of micrographs in three dimensions to describe accurately the ultrastructure of the SPB in yeast.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

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.

Connexin 50 Functions as an Adhesive Molecule and Promotes Lens Cell Differentiation

Connexins play essential roles in lens homeostasis and development. Here, we identified a new role for Cx50 that mediates cell-cell adhesion function. Cx50 enhanced the adhesive capability of AQP0. Interestingly, the expression of Cx50 alone promoted cell adhesion at a comparable level to AQP0; however, this cell adhesive function was not observed with other lens connexins, Cx43 and Cx46. Moreover, the adhesive property occurred in both homotypic with Cx50 expressed in both pairing cells and heterotypic with Cx50 in only one pairing cell, and this function appears to be unrelated to its role in forming gap junction channels. Cx50 KO lenses exhibited increased intercellular spaces between lens fiber cells. The second extracellular loop domain (E2) is primarily responsible for this adhesive function. Treatment with a fusion protein containing E2 domain inhibited cell adhesion. Furthermore, disruption of cell adhesion by the E2 domains impaired primary lens cell differentiation. Five critical amino acid residues in the E2 domain primarily are involved in cell adhesive function as well as lens epithelial-fiber differentiation. Together, these results suggest that in addition to forming gap junction channels, Cx50 acts as an adhesive molecule that is critical in maintaining lens fiber integrity and epithelial-fiber differentiation.

Tropomyosin 3.5 protects F-actin networks required for tissue biomechanical properties

ABSTRACT Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. Mouse lenses contain Tpm3.5 (also known as TM5NM5), a previously unstudied isoform encoded by Tpm3, which is associated with F-actin on lens fiber cell membranes. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin–F-actin and α-actinin–F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience, leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that a Tpm protein is essential for cell biomechanical stability in a load-bearing non-muscle tissue, and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivo. This article has an associated First Person interview with the first author of the paper. Highlighted Article: Tropomyosin 3.5 stabilizes F-actin in eye lens fiber cells and promotes normal tissue biomechanical properties. Tpm3.5 deficiency leads to F-actin network rearrangements and decreased lens stiffness and resilience.

A Correlative Immunoconfocal and Electron Microscopic Study of Gap Junctions in Interlocking Domains of the Lens

Ball-and-sockets and protrusions are specialized interlocking membrane domains between fiber cells of the avascular lenses in all species studied [1, 2]. Although both domains are similar in their shape, surface morphology and are highly enriched with aquaporin-0 water channel protein, our previous studies reveal that ball-and-sockets and protrusions possess important structural and functional differences during fiber cell differentiation and maturation [2, 3]. Specifically, gap junctions are regularly associated with all ball-and-sockets examined in metabolically active young cortical fibers, but not with protrusions. Also, while many ball-and-sockets are distributed primarily on the broad and narrow surfaces of young hexagonal fiber cells, numerous protrusions are located along the angles of the cells throughout the entire lens. It was proposed that the unique ball-and-socket-associated gap junctions may significantly facilitate cell-to-cell communication between young cortical fiber cells since they often protrude deeply into neighboring cells to increase membrane surface areas [2]. In contrast, protrusions are shown to play important interlocking role in maintaining fiber-to-fiber stability during visual accommodation [3].