Biology subjects

Wilmarth, P. A.

Publications and source records attributed to Wilmarth, P. A..

3 recordsLinked to original sources

bioRxiv2024

Analysis of mouse lens morphological and proteomic abnormalities following depletion of βB3-crystallin

Crystallin proteins serve as both essential structural and as well as protective components of the ocular lens and are required for the transparency and light refraction properties of the organ. The mouse lens crystallin proteome is represented by A-, B-, {beta}A1-, {beta}A2-, {beta}A3-, {beta}A4-, {beta}B1-, {beta}B2-, {beta}B3-, {gamma}A-, {gamma}B-, {gamma}C-, {gamma}D-, {gamma}E, {gamma}F-, {gamma}N-, and {gamma}S-crystallin proteins encoded by 16 genes. Their mutations are responsible for lens opacification and early onset cataract formation. While many cataract-causing missense and nonsense mutations are known for these proteins, including the human CRYBB3 gene, the mammalian loss-of function model of the Crybb3 gene remains to be established. Herein, we generated the first mouse model via deletion of the Crybb3 promoter that abolished expression of the {beta}B3-crystallin. Histological analysis of lens morphology using newborn {beta}B3-crystallin-deficient lenses revealed disrupted lens morphology with early-onset phenotypic variability. In-depth lens proteomics at four time points (newborn, 3-weeks, 6-weeks, and 3-months) showed both down- and up-regulation of various proteins, with the highest divergence from control mice observed in 3-months lenses. Apart from the {beta}B3-crystallin, another protein Smarcc1/Baf155 was down-regulated in all four samples. In addition, downregulation of Hspe1, Pdlim1, Ast/Got, Lsm7, Ddx23, and Acad11 was found in three time points. Finally, we show that the {beta}B3-crystallin promoter region, which contains multiple binding sites for the transcription factors AP-2, c-Jun, c-Maf, Etv5, and Pax6 is activated by FGF2 in primary lens cell culture experiments. Together, these studies establish the mouse Crybb3 loss-of-function model and its disrupted crystallin and non-crystallin proteomes.

developmental biology↗

bioRxiv2019

Identification of PKC -dependent phosphoproteins in mouse retina

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC), the targets of PKC phosphorylation in the retina have not been identified. PKC activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC antibody and antibodies to phosphorylated PKC motifs. PKC activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC knockout (PKC-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKC-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.\n\nSignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKC, though the specific mechanism by which PKC modulates RBC physiology is unknown. This study examined PKC phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC-dependent modulation of RBC physiology.\n\nHighlightsO_LIPKC is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.\nC_LIO_LIProteins showing differential phosphorylation between phorbol ester-treated wild type and PKC-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).\nC_LIO_LIThe PKC-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKC-dependent modulation of RBC physiology.\nC_LI

neuroscience↗

bioRxiv2017

ELMOD1 stimulates ARF6-GTP hydrolysis to stabilize apical structures in developing vestibular hair cells

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ARF small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMOD1, a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we used a mouse strain lacking functional ELMOD1 (roundabout; rda). In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5 (P5); large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cells apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles as compared to controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.\n\nSignificance StatementAssembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

neuroscience↗