Disease-related non-muscle myosin IIA D1424N rod domain mutation, but not R702C motor domain mutation, disrupts mouse ocular lens fiber cell alignment and hexagonal packing.

The mouse ocular lens is an excellent vertebrate model system for studying hexagonal cell packing and shape changes during tissue morphogenesis and differentiation. The lens is composed of two types of cells, epithelial and fiber cells. During the initiation of fiber cell differentiation, lens epithelial cells transform from randomly packed cells to hexagonally shaped and packed cells to form meridional row cells. The meridional row cells further differentiate and elongate into newly formed fiber cells that maintain hexagonal cell shape and ordered packing. In other tissues, actomyosin contractility regulates cell hexagonal packing geometry during epithelial tissue morphogenesis. Here, we use the mouse lens as a model to study the effect of two human disease-related non-muscle myosin IIA (NMIIA) mutations on lens cellular organization during fiber cell morphogenesis and differentiation. We studied genetic knock-in heterozygous mice with NMIIA-R702C motor domain or NMIIA-D1424N rod domain mutations. We observed that while one allele of NMIIA-R702C has no impact on lens meridional row epithelial cell shape and packing, one allele of the NMIIA-D1424N mutation can cause localized defects in cell hexagonal packing. Similarly, one allele of NMIIA-R702C motor domain mutation does not affect lens fiber cell organization while the NMIIA-D1424N mutant proteins disrupt fiber cell organization and packing. Our work demonstrates that disease-related NMIIA rod domain mutations (D1424N or E1841K) disrupt mouse lens fiber cell morphogenesis and differentiation.

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization.

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Nonmuscle Myosin IIA Regulates the Precise Alignment of Hexagonal Eye Lens Epithelial Cells During Fiber Cell Formation and Differentiation.

Purpose: Epithelial cells in the equatorial region of the ocular lens undergo a remarkable transition from randomly packed cells into precisely aligned and hexagon-shaped cells organized into meridional rows. We investigated the function of nonmuscle myosin IIA (encoded by Myh9) in regulating equatorial epithelial cell alignment to form meridional rows during secondary fiber cell morphogenesis. Methods: We used genetic knock-in mice to study a common human Myh9 mutation, E1841K, in the rod domain. The E1841K mutation disrupts bipolar filament assembly. Lens shape, clarity, and stiffness were evaluated, and Western blots were used to determine the level of normal and mutant myosins. Cryosections and lens whole mounts were stained and imaged by confocal microscopy to investigate cell shape and organization. Results: We observed no obvious changes in lens size, shape, and biomechanical properties (stiffness and resilience) between the control and nonmuscle myosin IIA-E1841K mutant mice at 2 months of age. Surprisingly, we found misalignment and disorder of fiber cells in heterozygous and homozygous mutant lenses. Further analysis revealed misshapen equatorial epithelial cells that cause disorientation of the meridional rows before fiber cell differentiation in homozygous mutant lenses. Conclusions: Our data indicate that nonmuscle myosin IIA bipolar filament assembly is required for the precise alignment of the meridional rows at the lens equator and that the organization of lens fiber cells depends on the proper patterning of meridional row epithelial cells. These data also suggest that lens fiber cell organization and a hexagonal shape are not required for normal lens size, shape transparency, or biomechanical properties.

Proteomic and functional analyses of the periodic membrane skeleton in neurons.

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories. We examined representative proteins in several of these categories using super-resolution imaging, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, and observed periodic distributions characteristic of the MPS along the neurites for ~20 proteins. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide insights into the interactome of the MPS and suggest previously unknown functions of the MPS in neurons.

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,ß1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton.

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.

Tropomodulins.

Arit Ghosh and Velia Fowler introduce the structural features and functions of tropomodulins - actin-binding proteins that cap the slow-growing (pointed) ends of actin filaments.

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

Purpose: 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 transcription-polymerase 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 TGFß2. 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 TGFß2 treatment results in the upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αSMA in TGFß2-treated cells. Using an ex vivo lens culture model, TGFß2 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.

The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology.

Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7-/- mice. Early postnatal Tdrd7-/- animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7-/- lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7-/- cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7-/- lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7-/- cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7-/- lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7-/- fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, 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 differentiating fiber cells, suggesting that TDRD7-ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7's novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

Non-uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation.

The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease.

Megakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9-related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/-GFP+/-, NMIIAD1424N+/-, and NMIIAE1841K+/-) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.

Age-related changes in eye lens biomechanics, morphology, refractive index and transparency.

Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.

MYH9-related disease mutations cause abnormal red blood cell morphology through increased myosin-actin binding at the membrane.

MYH9-related disease (MYH9-RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin-activated motor protein non-muscle myosin IIA (NMIIA). MYH9-RD patients suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA-expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F-actin) in the spectrin-F-actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled-coil rod, or non-helical tail) on RBC NMIIA function. We found that MYH9-RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron-scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9-RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F-actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub-clinical phenotype of MYH9-RD.

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and ßB2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.

Multifunctional roles of tropomodulin-3 in regulating actin dynamics.

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.

Tropomodulin Isoform-Specific Regulation of Dendrite Development and Synapse Formation.

Neurons of the CNS elaborate highly branched dendritic arbors that host numerous dendritic spines, which serve as the postsynaptic platform for most excitatory synapses. The actin cytoskeleton plays an important role in dendrite development and spine formation, but the underlying mechanisms remain incompletely understood. Tropomodulins (Tmods) are a family of actin-binding proteins that cap the slow-growing (pointed) end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown. In this study, we present evidence that Tmod1 and Tmod2 exhibit distinct roles in regulating spine development and dendritic arborization, respectively. Using rat hippocampal tissues from both sexes, we find that Tmod1 and Tmod2 are expressed with distinct developmental profiles: Tmod2 is expressed early during hippocampal development, whereas Tmod1 expression coincides with synaptogenesis. We then show that knockdown of Tmod2, but not Tmod1, severely impairs dendritic branching. Both Tmod1 and Tmod2 are localized to a distinct subspine region where they regulate local F-actin stability. However, the knockdown of Tmod1, but not Tmod2, disrupts spine morphogenesis and impairs synapse formation. Collectively, these findings demonstrate that regulation of the actin cytoskeleton by different members of the Tmod family plays an important role in distinct aspects of dendrite and spine development.SIGNIFICANCE STATEMENT The Tropomodulin family of molecules is best known for controlling the length and stability of actin myofilaments in skeletal muscles. While several Tropomodulin members are expressed in the brain, fundamental knowledge about their role in neuronal function is limited. In this study, we show the unique expression profile and subcellular distribution of Tmod1 and Tmod2 in hippocampal neurons. While both Tmod1 and Tmod2 regulate F-actin stability, we find that they exhibit isoform-specific roles in dendrite development and synapse formation: Tmod2 regulates dendritic arborization, whereas Tmod1 is required for spine development and synapse formation. These findings provide novel insight into the actin regulatory mechanisms underlying neuronal development, thereby shedding light on potential pathways disrupted in a number of neurological disorders.

Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties.

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. Mouse lenses contain Tpm3.5 (also known as TM5NM5), a previously unstudied isoform encoded by Tpm3, which is associated with F-actin on lens fiber cell membranes. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and α-actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience, leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that a Tpm protein is essential for cell biomechanical stability in a load-bearing non-muscle tissue, and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivoThis article has an associated First Person interview with the first author of the paper.

The effects of mechanical strain on mouse eye lens capsule and cellular microstructure.

The mouse eye lens was used as a model for multiscale transfer of loads. In the lens, compressive strain is distributed across specific lens tissue microstructures, including the extracellular capsule, as well as the epithelial and fiber cells. The removal of high loads resulted in complete recovery of most, but not all, microstructures.