Tropomyosin 3.1 Association With Actin Stress Fibers is Required for Lens Epithelial to Mesenchymal Transition

Abstract

Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Selective targeting of actin stress fiber-associated tropomyosin (Tpm) in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT. METHODS. We identified Tpm isoforms in mouse immortalized lens epithelial cells and epithelial and fiber cells from whole lenses by semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To induce EMT, we treated cells or native lenses with TGFβ2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by analysis of alpha-smooth muscle actin (αSMA) mRNA (real-time RT-PCR), and protein (Western immunoassay [WES]). RESULTS. Lens epithelial cells express eight Tpm isoforms. Cell culture studies showed that TGFβ2 treatment results in the upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αSMA in TGFβ2-treated cells. Using an ex vivo lens culture model, TGFβ2 treatment results in stress fiber formation at the basal regions of the epithelial cells. Genetic knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial αSMA levels. CONCLUSIONS. Targeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to repress lens EMT.

Immune responses to injury and their links to eye disease

“…While generally a highly successful procedure, a large number of post-cataract surgery patients develop a fibrotic vision-impairing condition called Posterior Capsule Opacification (PCO) along the fiber cell-denuded region of posterior lens capsule [173]. At the cellular and molecular level, PCO is linked to the appearance of Collagen I-producing, αSMA+ myofibroblasts, of which one well-characterized source is the epithelial-mesenchymal transition (EMT) of lens epithelial cells [174][175][176][177][178][179][180][181][182][183][184][185][186][187][188]. The resident immune cells of the lens also have been identified as a progenitor source of αSMA+ myofibroblasts post-cataract surgery [165,189].…”

Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration

“…To identify novel actin filament interactions, we performed a proximity-dependent biotin identification (BioID) analysis (Roux et al, 2012) on human osteosarcoma cells (U2OS) using a biotin ligase fused to Tropomyosin-3.1 (Tpm3.1), a central actin filaments component (Supplementary Figure 1A; Parreno et al, 2020). Among the high-confidence interactors, CAV-1, which has been used as a measure of cytoplasmic trafficking of both individual and clustered forms of caveolae (Mundy et al, 2002;Echarri et al, 2012), was chosen for further investigation (Figure 1A).…”

Whole mount staining of lenses for visualization of lens epithelial cell proteins

“…The lens epithelial cells continually proliferate and differentiate into fiber cells to support lens growth, a process that occurs throughout life. However, failure to maintain homeostasis in this anterior region of the lens could result in epithelial to mesenchymal transition (EMT) and pathologies, such as anterior subcapsular or posterior capsular opacification [1 , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] . Visualizing proteins in lens epithelial cells within intact lenses will help understand lens homeostasis as well as pathological processes.…”

“…We demonstrated how tissue-scale mechanical loads are transmitted onto lens epithelial cells [15 , 16] ; moreover, others have demonstrated that whole lens stretch results in lens epithelial cell proliferation [16] . Furthermore, the ex vivo culture system has allowed for the study of lens EMT [4 , 5 , 7 , [17] , [18] , [19] . By culturing lenses in the presence of TGFβ, the lens EMT process (i.e., lens fibrosis) can be initiated and comprehensively examined.…”