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βA3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. βA3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, βA3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of βA3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the βA3/A1-crystallin knockout (KO) mouse and explored the function of βA3/A1-crystallin in lens development. Our results showed that βA3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of βA3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the [alpha]- and [beta]-crystallins. Here, we investigated the effects of the deamidation of [alpha]A- and [alpha]B-crystallins on their interaction with [beta]A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT [alpha]A- and WT [alpha]B-crystallins and their deamidated mutants with [beta]A3-crystallin. The deamidated mutants of [alpha]A-crystallin ([alpha]A N101D and [alpha]A N123D) displayed lower adherence propensity for [beta]A3-crystallin relative to the binding affinity shown by WT [alpha]A-crystallin. Among [alpha]B-crystallin mutants, [alpha]B N78D displayed higher adherence propensity whereas [alpha]B N146D mutant showed slightly lower binding affinity for [beta]A3-crystallin relative to that shown by WT [alpha]B-crystallin. Under the in vivo condition (FLIM-FRET), both [alpha]A-deamidated mutants ([alpha]A N101D and [alpha]A N123D) exhibited strong interaction with [beta]A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT [alpha]A-crystallin (18±4%). Similarly, the [alpha]B N78D and [alpha]B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with [beta]A3-crystallin compared to 18±4% FRET efficiency of WT [alpha]B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of [alpha]A- and [alpha]B-crystallins with [beta]A3-crystallin suggested that interaction sites most likely reside in the [alpha]A CTE and [alpha]B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT [alpha]A- and WT[alpha]B-crystallins, the deamidated mutants showed strong interactionfor [beta]A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of [alpha]-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

[beta]A3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. [beta]A3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, [beta]A3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of [beta]A3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the [beta]A3/A1-crystallin knockout (KO) mouse and explored the function of [beta]A3/A1-crystallin in lens development. Our results showed that [beta]A3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of [beta]A3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.

The icosahedral double-stranded DNA bacteriophages and herpes viruses package their genomes into preformed proheads by a powerful ATP driven motor. The packaging motor, an oligomer of gp17 (large terminase) in phage T4, is assembled at the special portal vertex of the empty prohead. The T4 motor is the fastest and most powerful motor reported to date. gp17 has an N-terminal ATPase that powers DNA translocation and a C-terminal translocase that causes DNA movement. The dynamic interactions between the motor (gp17) and the portal (gp20) are however poorly understood. Here, using biochemistry, bioinformatics, structure, and molecular genetics, the site in gp17 that interacts with the dodecameric portal protein is determined. Biochemical and structural studies suggest that the N-terminal domain of gp17 interacts with gp20, and that the stoichiometry of prohead-gp17 complex is five subunits of gp17 to twelve subunits of gp20. Sequence alignments predict that there are two potential portal binding sites in gp17. Mutational studies show that the portal binding site I in the N-terminal domain is critical for function whereas the site II in the C-terminal domain is not critical. Second site suppressors of site I D331Q mutant (temperature sensitive) show a single intragenic mutation in the helix-loop-helix (HLH) of N-terminal sub-domain II, suggesting the importance of this motif in portal interaction. Fitting the X-ray structure of gp17 into the cryo-EM density of portal-motor complex showed the same HLH (amino acids 333-352) in contact with gp20. A peptide corresponding to the HLH motif specifically binds to proheads as well as inhibits DNA packaging in vitro. Swapping of non-conserved residues of the helix, but not the conserved residues of the loop, from T4-family phages relieves the DNA packaging inhibition. Together these data for the first time identify a HLH motif in gp17 that interacts with gp20, leading to models for symmetry mismatch between the packaging motor and the portal as well as implications to the mechanism of viral DNA translocation.

The interaction of amyloid-β (Aβ) peptides with the plasma membrane (PM) is a potential trigger that initiates the formation of higher-order aggregates, membrane alterations/damage, and progressive neurotoxicity in Alzheimers disease (AD). In a previous study, we showed that oligomers of Aβ1-42 (oAβ1-42) induced PM damage, resulting in PM repair cascade via lysosomal exocytosis coupled with endocytosis, and facilitation of tunneling nanotubes (TNTs)-like membrane protrusions to promote direct cell-to-cell transfer of aggregates. In this study, we demonstrated that PM damage induced by oligomers of the aggregation-prone peptide Aβ1-42 significantly facilitates PM repair by enhancing phosphorylated p21-activated kinase 1 (pPAK1)-dependent endocytosis and Rab3a-dependent exocytosis in SH-SY5Y and SK-N-SH neuronal cells compared to control and oAβ1-40 treated cells. We studied the kinetics of pPAK1-dependent endocytosis and the fusion of EGFP-Rab3a vesicles near the PM using total internal reflection fluorescence (TIRF) microscopy. IPA-3, a selective non-ATP competitive inhibitor of PAK1, inhibits endocytosis of oAβ peptides and Rab3a-dependent PM repair. Further, shRNA-mediated knockdown of the Rab3a gene inhibits pPAK1 and disrupts PM repair. Repair of damaged PM is a vital protective mechanism for non-proliferative cells like neurons, as disruption in PM repair leads to gradual neuronal cell death. However, there was no explicit understanding of PM repair in response to Aβ oligomers. This study revealed the interconnected action of Rab3a and pPAK1 in PM repair in response to oAβ -mediated damage, and its potential correlation in AD pathogenesis.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Modified the special characters \"beta\" in the abstract.