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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.

Small heat shock protein HSPB7 is highly expressed in the heart. Several mutations within HSPB7 are associated with dilated cardiomyopathy and heart failure in human patients. However, the precise role of HSPB7 in the heart is still unclear. In this study, we generated global as well as cardiac-specific HSPB7 KO mouse models and found that loss of HSPB7 globally or specifically in cardiomyocytes resulted in embryonic lethality before embryonic day 12.5. Using biochemical and cell culture assays, we identified HSPB7 as an actin filament length regulator that repressed actin polymerization by binding to monomeric actin. Consistent with HSPB7’s inhibitory effects on actin polymerization, HSPB7 KO mice had longer actin/thin filaments and developed abnormal actin filament bundles within sarcomeres that interconnected Z lines and were cross-linked by α-actinin. In addition, loss of HSPB7 resulted in up-regulation of Lmod2 expression and mislocalization of Tmod1. Furthermore, crossing HSPB7 null mice into an Lmod2 null background rescued the elongated thin filament phenotype of HSPB7 KOs, but double KO mice still exhibited formation of abnormal actin bundles and early embryonic lethality. These in vivo findings indicated that abnormal actin bundles, not elongated thin filament length, were the cause of embryonic lethality in HSPB7 KOs. Our findings showed an unsuspected and critical role for a specific small heat shock protein in directly modulating actin thin filament length in cardiac muscle by binding monomeric actin and limiting its availability for polymerization.

TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 ( Filia ), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3. Inducible disruption of TET dioxygenases in mouse embryonic stem cells results in chromosome mis-segregation and aneuploidies, due to Khdc3 downregulation suggesting a role for TET enzymes and DNA methylation patterns in maintaining genome stability.

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 α₂β₂-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.

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.

Abstract Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes interconnected by long spectrin molecules at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modelling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization of F-actin nodes in the network in situ is not understood. Here, we examined F-actin distribution in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) 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. Live cell imaging reveals dynamic lateral movements, appearance and disappearance of F-actin foci. Single molecule STORM imaging and computational cluster analysis of experimental and synthetic data sets indicate that individual filaments are non-randomly distributed, with the majority as multiple filaments, and the remainder sparsely distributed as single filaments. These data indicate that F-actin nodes are non-uniformly distributed in the spectrin-F-actin network and necessitate reconsideration of current models of forces accounting for RBC shape and membrane deformability, predicated upon uniform distribution of F-actin nodes and associated proteins across the micron-scale RBC membrane. Competing Interest Statement The authors have declared no competing interest.

The understanding of multiscale load transfer within complex soft tissues is incomplete. The eye lens is ideal for multiscale tissue mechanics studies as its principal function is to fine focus light at different distances onto the retina via mechanical shape changes. The biomechanical function, resiliency, and intricate microstructure of the lens make it an excellent non-connective soft tissue model. We hypothesized that compressive strain applied onto whole lens tissue leads to deformation of specific microstructures and that this deformation is reversible following removal of load. For this examination, mouse lenses were compressed by sequential application of increasing load. Using confocal microscopy and quantitative image analysis, we determined that axial strain 10% reduces capsule thickness, expands epithelial cell area, and separates fiber cell tips at the anterior region of the lenses. At the equatorial region, strain 6% increases fiber cell widths. The effects of strain on lens epithelial cell area, capsule thickness, and equatorial fiber cell widths are reversible following the release of lenses from strain. However, although fiber cell tip separation following the removal of low loads is reversible, the separation becomes irreversible with application of higher loads. This irreversible separation between fiber cell tips leads to incomplete bulk lens resiliency. The lens is an accessible biomechanical model system that provides new insights on multiscale transfer of loads in soft tissues.

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to fine 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. The major mouse lens Tpm is Tpm3.5 (TM5NM5), a previously unstudied isoform. 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 -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 Tpm 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 vivo. Footnotes * Supplemental figures now included in PDF

The biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon 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 in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylated in vivo in RBCs, indicating 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 disc cell shape. As structures similar to the RBC membrane skeleton are conserved 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.