Dataset Information

Dysregulation of Non-Muscle Myosin IIA Assembly and Phosphorylation in S100A4 Null Mouse Lens

ABSTRACT: Non-muscle myosin IIA (NMIIA), a motor protein plays a critical role in regulating cell morphology, adhesion, migration, contractility, and mechanotransduction across various tissues, including the ocular lens. S100A4, a known NMIIA-interacting protein, is abundantly expressed in lens fibers and shows discrete spatial distribution. Loss of S100A4 has previously been shown to associate with late-onset lens opacification in mice. However, its role in regulating NMIIA activity, assembly, and actin cytoskeletal organization in the lens remains unclear. Using S100A4-null mice, this study reveals that S100A4 co-immunoprecipitates with NMIIA and that its absence leads to decreased NMIIA (Ser1943) phosphorylation, impaired NMIIA filament assembly, and disruption in actin cytoskeletal organization and polymerization in lens. Quantitative proteomic analysis of cytoskeletal- and membrane-enriched fractions from S100A4-null versus wild-type lenses further identified decreased levels of CLIC5 and RNA binding protein-SERBP1. Moreover, treatment of wild-type mouse lenses in ex-vivo with trifluoperazine, a known S100A4 inhibitor, induced lens opacification in association with increased insolubilization of S100A4, NMIIA and actin. Collectively, these findings demonstrate that S100A4 plays a critical role in regulating NMIIA activity and assembly and maintaining actin cytoskeletal organization in the ocular lens, thereby contributing to lens transparency and homeostasis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Fibroblast

SUBMITTER: Nik Skiba

LAB HEAD: Ponugoti V. Rao

PROVIDER: PXD071603 | Pride | 2026-06-29

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	02_13_20_Rupa_S100A4_A5_IP.MSMS.mgf	Mgf
	02_13_20_Rupa_S100A4_A5_IP.mzid	Mzid
	03_12_21_Rupa_lens_cytosk_S100KO1_1.raw	Raw
	03_12_21_Rupa_lens_cytosk_S100KO1_2.raw	Raw
	03_12_21_Rupa_lens_cytosk_S100KO2_1.raw	Raw

Items per page:

1 - 5 of 37

Publications

Dysregulation of Non-Muscle Myosin IIA Assembly and Phosphorylation in S100A4 Null Mouse Lens.

Maddala Rupalatha R Lankford Levi K LK Skiba Nikolai P NP Rao Ponugoti V PV

Journal of cellular physiology 20260601 6

Non-muscle myosin IIA (NMIIA), a motor protein plays a critical role in regulating cell morphology, adhesion, migration, contractility, and mechanotransduction across various tissues, including the ocular lens. S100A4, a known NMIIA-interacting protein, is abundantly expressed and exhibits a discrete spatial distribution in lens fibers. Loss of S100A4 has previously been shown to associate with late-onset lens opacification in mice. However, its role in regulating NMIIA activity, assembly, and a ...[more]

PMID: 42312500

Similar Datasets

Project description:Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract in humans. Here, we applied an integrated approach to elucidate the molecular pathology of cataract in Tdrd7 targeted-knockout (Tdrd7-/-) mice. Tdrd7-/- animals precipitously develop lens fiber cell abnormalities early in life, suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing followed by iSyTE-integrated bioinformatics-based analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1 (HSP27), among the high-priority down-regulated candidates in Tdrd7-/- lens. Moreover, a protein 2-D fluorescence difference gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 to be reduced in Tdrd7-/- lens, offering independent support for focusing efforts on this factor to explain Tdrd7-/- cataract. Reduction of HSPB1 preceded lens morphological abnormalities, suggesting that cytoskeletal defects underlie the Tdrd7-/- cataract phenotype. In agreement, scanning electron microscopy revealed abnormal fiber cell membrane protrusions in Tdrd7-/- lenses. Significantly, abnormal F-actin staining was detected specifically in Tdrd7-/- fiber cells that exhibit nuclear degradation, thereby revealing that there are distinct mechanisms based on pre- or post-nuclear degradation differentiation stage for F-actin cytoskeletal maintenance in fiber cells. Further, RNA-immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA-imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in a specific pre-nuclear degradation area of differentiating fiber cells, indicating that TDRD7-ribonucleoprotein complexes are necessary for controlling optimal levels of key factors in lens development. Together, these data uncover a novel role for TDRD7 in regulating elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

Project description:Introduction: Cataracts are the world’s leading cause of reversible blindness. Although cataract formation is commonly initiated by lens fiber cell defects, cataractogenesis can be characterized by aberrant proliferation and migration of lens epithelial cells, particularly in posterior capsule opacification. Subsequent overproduction of extracellular matrix components such as fibronectin and collagen by epithelial cells is associated with fibrosis of the lens. Little is known about the role of platelet-derived growth factor receptor β (PDGFRβ) in lens fibrosis. Here, we investigated mice with a conditional knock-in driving mesenchymal-specific PDGFRβ hyperactivation (Fsp1-cre;Pdgfrb+/D849V), which consistently develop cataracts at a young age. Methods: Lenses from Fsp1-cre;Pdgfrb+/D849V mice and age-matched controls were dissected and visualized via microscopy from 9-15 weeks. Early transcriptional changes were investigated between 10-12 day old Fsp1-cre;Pdgfrb+/D849V and control mice via RNA sequencing followed by gene set enrichment analysis. Confirmation of RNA sequencing results and mechanistic investigation of PDGFRβ-induced cataractogenesis were determined in lenses isolated from 15-week-old Fsp1-cre;Pdgfrb+/D849V and control mice. Results: Gross examination of cataractous lenses from Fsp1-cre;Pdgfrb+/D849V mice revealed complete opacification by 15 weeks of age compared to no opacification in age-matched controls. Structural changes in the anterior, equatorial, and posterior lens were observed in histology. RNA sequencing revealed significant enrichment of gene sets related to extracellular matrix deposition and reorganization. Mechanistic investigation revealed major roles for TGFβ, SOCS2, and STAT5-IGF1 signaling axes in PDGFRβ-induced cataract formation. Conclusion: PDGFRβ promoted cataractogenesis by modulating pro-fibrotic extracellular matrix changes, likely through TGFβ, SOCS2, and the STAT5-IGF1 pathways. Future experiments will delineate the precise role of the STAT5-IGF1 signaling pathway in PDGFRβ-mediated fibrosis and the interplay between PDGFRβ and TGFβ in the lens and whether this signaling is targetable to modulate cataractogenesis.

Project description:We report the application of RNA-sequencing technology for the high-throughput profiling of mammalian lens gene expression at embryonic day 15.5. The lens has a particularly biased transcriptome, with the top 50 genes encoding approximately 90% of the protein content. Using RNA-Seq, we have shown that there are over 7,700 genes being expressed in the lens at embryonic day 15.5. As expected, the crystallins and many structural genes were amoung the most highly expressed; however, numerous genes expressed at lower transcript abundance were also identified. This study provides a framework for the application of RNA-Seq technology towards characterization of the mammalian lens transcriptome during development. Using high-throughput RNA-sequencing, gene expression in the mammalian lens was compared between an inbred C56Bl/6<har> strain and a mix background strain at E15.5. This analysis identifies almost 2,000 genes being differentially expressed between the inbred and mixed background lenses, ranging from 6.5 fold upregulated to 5.2 fold downregulated in the mixed background compared to the inbred strain. This list does not include unknown/predicted genes or pseudogenes which are known to change between strains. Further, it appears that approximately 98% of these genes are altered at levels less than 2.5 fold. This study therefore provides a fold change threshold cutoff (2.5 fold) to use in the analysis of differentially expressed lens genes at E15.5 using RNA-Seq technology as it takes into account genetic variation due to background strain differences. SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases where it functionally links TGFb signaling to the loss of epithelial preferred gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes involved in EMT/cancer. Furthermore, 34% of the upregulated genes in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes downregulated in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts. RNA-Seq of inbred background wild type lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and inbred wild type (C57Bl/6<har>) lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and Sip1 conditional knockout lenses at E15.5

Project description:Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity.