Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Tropomodulin 1 controls erythroblast enucleation via regulation of F-actin in the enucleosome.

Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained. Here, we employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34+ hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. This novel structure, the "enucleosome," may mediate common cytoskeletal mechanisms underlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species.

Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells.

The short F-actins in the red blood cell (RBC) membrane skeleton are coated along their lengths by an equimolar combination of two tropomyosin isoforms, Tpm1.9 and Tpm3.1. We hypothesized that tropomyosin's ability to stabilize F-actin regulates RBC morphology and mechanical properties. To test this, we examined mice with a targeted deletion in alternatively spliced exon 9d of Tpm3 (Tpm3/9d-/- ), which leads to absence of Tpm3.1 in RBCs along with a compensatory increase in Tpm1.9 of sufficient magnitude to maintain normal total tropomyosin content. The isoform switch from Tpm1.9/Tpm3.1 to exclusively Tpm1.9 does not affect membrane skeleton composition but causes RBC F-actins to become hyperstable, based on decreased vulnerability to latrunculin-A-induced depolymerization. Unexpectedly, this isoform switch also leads to decreased association of Band 3 and glycophorin A with the membrane skeleton, suggesting that tropomyosin isoforms regulate the strength of F-actin-to-membrane linkages. Tpm3/9d-/- mice display a mild compensated anemia, in which RBCs have spherocytic morphology with increased osmotic fragility, reduced membrane deformability, and increased membrane stability. We conclude that RBC tropomyosin isoforms directly influence RBC physiology by regulating 1) the stability of the short F-actins in the membrane skeleton and 2) the strength of linkages between the membrane skeleton and transmembrane glycoproteins.

Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images.

The periodically arranged thin filaments within the striated myofibrils of skeletal and cardiac muscle have precisely regulated lengths, which can change in response to developmental adaptations, pathophysiological states, and genetic perturbations. We have developed a user-friendly, open-source ImageJ plugin that provides a graphical user interface (GUI) for super-resolution measurement of thin filament lengths by applying Distributed Deconvolution (DDecon) analysis to periodic line scans collected from fluorescence images. In the workflow presented here, we demonstrate thin filament length measurement using a phalloidin-stained cryosection of mouse skeletal muscle. The DDecon plugin is also capable of measuring distances of any periodically localized fluorescent signal from the Z- or M-line, as well as distances between successive Z- or M-lines, providing a broadly applicable tool for quantitative analysis of muscle cytoarchitecture. These functionalities can also be used to analyse periodic fluorescence signals in nonmuscle cells.

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens' optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens' ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Feisty filaments: actin dynamics in the red blood cell membrane skeleton.

PURPOSE OF REVIEW: This article discusses recent advances and unsolved questions in our understanding of actin filament organization and dynamics in the red blood cell (RBC) membrane skeleton, a two-dimensional quasi-hexagonal network consisting of (α1ß1)2-spectrin tetramers interconnecting short actin filament-based junctional complexes. RECENT FINDINGS: In contrast to the long-held view that RBC actin filaments are static structures that do not exchange subunits with the cytosol, RBC actin filaments are dynamic structures that undergo subunit exchange and turnover, as evidenced by monomer incorporation experiments with rhodamine-actin and filament disruption experiments with actin-targeting drugs. The malaria-causing parasite, Plasmodium falciparum, co-opts RBC actin dynamics to construct aberrantly branched actin filament networks. Even though RBC actin filaments are dynamic, RBC actin filament lengths are highly uniform (∼37 nm). RBC actin filament lengths are thought to be stabilized by the capping proteins, tropomodulin-1 and αß-adducin, as well as the side-binding protein tropomyosin, present in an equimolar combination of two isoforms, TM5b (Tpm1.9) and TM5NM1 (Tpm3.1). SUMMARY: New evidence indicates that RBC actin filaments are not simply passive cytolinkers, but rather dynamic structures whose assembly and disassembly play important roles in RBC membrane function.

Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle.

The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific. We then generate Tmod4(-/-) mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4(-/-) muscle fibers leads to thin filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts.

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane.

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (~25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ~60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.

Alterations in thin filament length during postnatal skeletal muscle development and aging in mice.

The lengths of the sarcomeric thin filaments vary in a skeletal muscle-specific manner and help specify the physiological properties of skeletal muscle. Since the extent of overlap between the thin and thick filaments determines the amount of contractile force that a sarcomere can actively produce, thin filament lengths are accurate predictors of muscle-specific sarcomere length-tension relationships and sarcomere operating length ranges. However, the striking uniformity of thin filament lengths within sarcomeres, specified during myofibril assembly, has led to the widely held assumption that thin filament lengths remain constant throughout an organism's lifespan. Here, we rigorously tested this assumption by using computational super-resolution image analysis of confocal fluorescence images to explore the effects of postnatal development and aging on thin filament length in mice. We found that thin filaments shorten in postnatal tibialis anterior (TA) and gastrocnemius muscles between postnatal days 7 and 21, consistent with the developmental program of myosin heavy chain (MHC) gene expression in this interval. By contrast, thin filament lengths in TA and extensor digitorum longus (EDL) muscles remained constant between 2 mo and 2 yr of age, while thin filament lengths in soleus muscle became shorter, suggestive of a slow-muscle-specific mechanism of thin filament destabilization associated with aging. Collectively, these data are the first to show that thin filament lengths change as part of normal skeletal muscle development and aging, motivating future investigations into the cellular and molecular mechanisms underlying thin filament adaptation across the lifespan.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Functional effects of mutations in the tropomyosin-binding sites of tropomodulin1 and tropomodulin3.

Tropomodulins (Tmods) interact with tropomyosins (TMs) via two TM-binding sites and cap the pointed ends of TM-coated actin filaments. To study the functional interplay between TM binding and TM-actin filament capping by Tmods, we introduced disabling mutations into the first, second, or both TM-binding sites of full-length Tmod1 (Tmod1-L27G, Tmod1-I131D, and Tmod1-L27G/I131D, respectively) and full-length Tmod3 (Tmod3-L29G, Tmod3-L134D, and Tmod3-L29G/L134D, respectively). Tmod1 and Tmod3 showed somewhat different TM-binding site utilization, but nearly all TM binding was abolished in Tmod1-L27G/I131D and Tmod3-L29G/L134D. Disruption of Tmod-TM binding had a modest effect on Tmod1's ability and no effect on Tmod3's ability to stabilize TM-actin pointed ends against latrunculin A-induced depolymerization. However, disruption of Tmod-TM binding did significantly impair the ability of Tmod3 to reduce elongation rates at pointed ends with α/ßTM, albeit less so with TM5NM1, and not at all with TM5b. For Tmod1, disruption of Tmod-TM binding only slightly impaired its ability to reduce elongation rates with α/ßTM and TM5NM1, but not at all with TM5b. Thus, Tmod-TM binding has a greater influence on Tmods' ability to inhibit subunit association as compared to dissociation from TM-actin pointed ends, particularly for α/ßTM, with Tmod3's activity being more dependent on TM binding than Tmod1's activity. Nevertheless, disruption of Tmod1-TM binding precluded Tmod1 targeting to thin filament pointed ends in cardiac myocytes, suggesting that the functional effects of Tmod-TM binding on TM-coated actin filament capping can be significantly modulated by the in vivo conformation of the pointed end or other factors in the intracellular environment.

Differential actin-regulatory activities of Tropomodulin1 and Tropomodulin3 with diverse tropomyosin and actin isoforms.

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ~5-fold more weakly than Tmod1 to α/ßTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/ßTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet ß/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate ß/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic ß- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-ß/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester ß- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.

Tropomyosin is required for cardiac morphogenesis, myofibril assembly, and formation of adherens junctions in the developing mouse embryo.

BACKGROUND: We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform. RESULTS: Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non-beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1-deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non-striated F-actin fibrils with α-actinin puncta dispersed irregularly along their lengths. αTM1's binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin-containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1-deficient cardiomyocytes, α-actinin/F-actin adherens belts fail to assemble at apical cell-cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1-deficient cardiomyocytes exhibit failure of α-actinin/F-actin adherens belt assembly. CONCLUSIONS: Absence of αTM1 resulting in unstable F-actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1-based pathway stabilizing F-actin at cell-cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis.

Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle.

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity-dependent manner.

Pointed-end capping by tropomodulin modulates actomyosin crossbridge formation in skeletal muscle fibers.

In skeletal muscle, thick and thin filaments are arranged in a myofibrillar lattice. Tropomodulin 1 (Tmod1) is a pointed-end capping and tropomyosin-binding protein that controls thin-filament assembly, stability, and lengths. It remains unknown whether Tmods have other functional roles, such as regulating muscle contractility. To investigate this, we recorded and analyzed the mechanical properties and X-ray diffraction patterns of single membrane-permeabilized skeletal muscle fibers from mice lacking Tmod1. Results show that absence of Tmod1 and its replacement by Tmod3 and Tmod4 may impair initial tropomyosin movement over actin subunits during thin-filament activation, thus reducing both the fraction of actomyosin crossbridges in the strongly bound state (-29%) and fiber force-generating capacity (-31%). Therefore, Tmods are novel regulators of actomyosin crossbridge formation and muscle contractility, and future investigations and models of skeletal muscle force production must incorporate Tmods.

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice.

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse.

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

A two-segment model for thin filament architecture in skeletal muscle.

Correct specification of myofilament length is essential for efficient skeletal muscle contraction. The length of thin actin filaments can be explained by a novel 'two-segment' model, wherein the thin filaments consist of two concatenated segments, which are of either constant or variable length. This is in contrast to the classic 'nebulin ruler' model, which postulates that thin filaments are uniform structures, the lengths of which are dictated by nebulin. The two-segment model implicates position-specific microregulation of actin dynamics as a general principle underlying actin filament length and stability.

Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2ß2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.