Tropomodulin 1 Constrains Fiber Cell Geometry during Elongation and Maturation in the Lens Cortex

Abstract

SummaryLens fiber cells exhibit a high degree of hexagonal packing geometry, determined partly by tropomodulin 1 (Tmod1), which stabilizes the spectrin-actin network on lens fiber cell membranes. To ascertain whether Tmod1 is required during epithelial cell differentiation to fiber cells or during fiber cell elongation and maturation, the authors quantified the extent of fiber cell disorder in the Tmod1-null lens and determined locations of disorder by confocal microscopy and computational image analysis. First, nearest neighbor analysis of fiber cell geometry in Tmod1-null lenses showed that disorder is confined to focal patches. Second, differentiating epithelial cells at the equator aligned into ordered meridional rows in Tmod1-null lenses, with disordered patches first observed in elongating fiber cells. Third, as fiber cells were displaced inward in Tmod1-null lenses, total disordered area increased due to increased sizes (but not numbers) of individual disordered patches. The authors conclude that Tmod1 is required first to coordinate fiber cell shapes and interactions during tip migration and elongation and second to stabilize ordered fiber cell geometry during maturation in the lens cortex. An unstable spectrinactin network without Tmod1 may result in imbalanced forces along membranes, leading to fiber cell rearrangements during elongation, followed by propagation of disorder as fiber cells mature. (J Histochem Cytochem 60:414-427, 2012)

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

“…77), it is reasonable to propose that, in newly differentiated wildtype lens fiber cells, connexons are inserted into small discontinuities in the actin-spectrin network on membranes of differentiating fiber cells. Indeed, in newly formed fiber cells, the actin-spectrin network is observed to be discontinuous and irregular, and as the fiber cells mature and elongate, the actin-spectrin network becomes smooth and continuous along fiber cell membranes, likely due to Tmod1 stabilization of F-actin filaments (72). Reinforcement of the actin-spectrin network coincides with the appearance of large gap junction plaques on the broad sides of differentiating fiber cells.…”

“…The generation of mixed-background FvBN/129SvEv/ C57BL/6 Tmod1 Ϫ/Ϫ mice is described elsewhere (35,36,66,68,71,72). Embryonic lethality in Tmod1 Ϫ/Ϫ mice (26) is rescued by a Tmod1-overexpressing transgene under the control of the cardiacspecific ␣-myosin heavy chain (␣MHC) promoter (66).…”

“…Eyeballs were fixed overnight in 1% paraformaldehyde at 4°C, washed in PBS, cryoprotected in sucrose, frozen in OCT medium (Sakura Finetek, Torrance, CA), and stored at Ϫ80°C until they were sectioned. Frozen 12-m-thick sections were collected with a Leica CM1950 cryostat and then permeabilized, blocked, and stained as previously described (71,72) using rabbit anti-Cx46 (1:200 dilution) or rabbit anti-Cx50 (1:100 dilution) with mouse monoclonal anti-␤2-spectrin (42/B, 1:100 dilution; BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were Alexa 488-conjugated goat anti-rabbit (1:100 dilution; Life Technologies) and Alexa 647-conjugated goat anti-mouse IgG (1:100 dilution; Life Technologies).…”

“…The actin filament linkages in this network are stabilized by ␥-tropomyosin and capped at their barbed and pointed ends by adducin and tropomodulin 1 (Tmod1), respectively (23,52,71,89,90). We previously showed that loss of Tmod1 destabilizes the actin-spectrin network and leads to local disorganization of lens fiber cells (36,71,72). Tmod1 is associated in a macromolecular complex with CP49 and filensin, as well as with actin and spectrin, suggesting that the actin-spectrin network and the beaded intermediate filaments are linked within lens fiber cells (36).…”

The lens actin filament cytoskeleton: Diverse structures for complex functions

“…Beaded intermediate filaments comprised of specialized intermediate filament proteins, CP49 (phakinin, Bfsp2 ) and filensin (CP115, Bfsp1 ) (Alizadeh et al, 2003; FitzGerald, 2009), are needed for mechanical integrity (Fudge et al, 2011; Gokhin et al, 2012) and maintaining life-long lens transparency (Gokhin et al, 2012; Oka et al, 2008; Sandilands et al, 1995). Of the F-actin networks, the best understood is the spectrin-actin membrane skeleton, composed of actin filaments cross-linked by α 2 β 2 -spectrin, which is integral for fiber cell packing (Gokhin et al, 2012; More et al, 2001; Nowak et al, 2009; Nowak and Fowler, 2012) and mechanical stiffness (Gokhin et al, 2012). In this review, we focus on the organization, regulation and functions of F-actin networks and their associated actin-binding proteins in the lens (summarized in Tables 1 and 2).…”