Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8-8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of alpha-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.     

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1–4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1–3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.     

Cytoplasmic gamma-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

The sarcoplasmic reticulum (SR) serves as the Ca(2+) reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γ(cyto)-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γ(cyto)-actin and Tmod3 defined an SR microdomain that was distinct from another Z line-flanking SR microdomain containing Tmod1 and Tmod4. The γ(cyto)-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γ(cyto)-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3-γ(cyto)-actin-sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca(2+) release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γ(cyto)-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction

Glycerinated rabbit psoas myofibrils, F-actin, and myofibril ghosts were irradiated with ultraviolet light (UV) to investigate how UV blocks myofibril contraction. Myofibril contraction is most sensitive to 270- and 290-nm wavelength light. We irradiated I and A bands separately with 270- and 290-nm wavelength light using a UV microbeam and constructed dose-response curves for blocking sarcomere contraction. For both wavelengths, irradiations of A bands required less energy per area to block contraction than did irradiations of I bands, suggesting that the primary effects of both 270- and 290-nm wavelength light in stopping myofibril contraction are on myosin. We investigated whether the primary effect of UV in blocking I-band contraction is the depolymerization of actin by comparing the relative sensitivities of I-band contraction, F-actin depolymerization, and thin filament depolymerization to 270- and 290-nm light. We also compared the dose of UV required to depolymerize F-actin in solution with the dose needed to block I-band contraction and the dose required to alter thin filament structure in myofibril ghosts. The results confirm that UV blocks I-band contraction by depolymerizing actin. We discuss how the results might be relevant to the hypothesis that an actomyosin-based system is involved in chromosome movement.     

Genetic dissection of novel myopathy models reveals a role of CapZα and Leiomodin 3 during myofibril elongation

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.     

Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle.

Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.     

Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens

Hexagonal packing geometry is a hallmark of close-packed epithelial cells in metazoans. Here, we used fiber cells of the vertebrate eye lens as a model system to determine how the membrane skeleton controls hexagonal packing of post-mitotic cells. The membrane skeleton consists of spectrin tetramers linked to actin filaments (F-actin), which are capped by tropomodulin1 (Tmod1) and stabilized by tropomyosin (TM). In mouse lenses lacking Tmod1, initial fiber cell morphogenesis is normal, but fiber cell hexagonal shapes and packing geometry are not maintained as fiber cells mature. Absence of Tmod1 leads to decreased γTM levels, loss of F-actin from membranes, and disrupted distribution of β2-spectrin along fiber cell membranes. Regular interlocking membrane protrusions on fiber cells are replaced by irregularly spaced and misshapen protrusions. We conclude that Tmod1 and γTM regulation of F-actin stability on fiber cell membranes is critical for the long-range connectivity of the spectrin–actin network, which functions to maintain regular fiber cell hexagonal morphology and packing geometry.     

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.     

Intermediate filaments connect z-discs in adult chicken muscle.

When adult chicken skeletal myofibrils are treated with a myosin-extracting solution, the Z-discs with attached actin filaments retain their linear connections with one another in the extracted myofibril. The sarcomere length increases in the extracted myofibrils from a control lenght of 2.5 micrometer up to 6 micrometer. In a sarcomere, eight to fifty 10 nm filaments can be seen in parallel array in the H-zone. The 10 nm-wide filaments do not bind heavy meromyosin and are two to four micrometers in length. These intermediate filaments are postulated to be an integral part of the sarcomere, connecting Z-bands along the length of the myofibril.     

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis. This paper is dedicated to the memory of Professor Pavel P. Rumyantsev (1927–1988), a pioneer in studies of cardiac muscle differentiation, who is a lasting inspiration to all who worked with him.     

Leiomodin and tropomodulin in smooth muscle

Evidence isaccumulating to suggest that actin filament remodeling is critical forsmooth muscle contraction, which implicates actin filament ends asimportant sites for regulation of contraction. Tropomodulin (Tmod) andsmooth muscle leiomodin (SM-Lmod) have been found in many tissuescontaining smooth muscle by protein immunoblot and immunofluorescencemicroscopy. Both proteins cofractionate with tropomyosin in theTriton-insoluble cytoskeleton of rabbit stomach smooth muscle and aresolubilized by high salt. SM-Lmod binds muscle tropomyosin, abiochemical activity characteristic of Tmod proteins. SM-Lmod stainingis present along the length of actin filaments in rat intestinal smoothmuscle, while Tmod stains in a punctate pattern distinct from that ofactin filaments or the dense body marker -actinin. After smoothmuscle is hypercontracted by treatment with 10 mM Ca²⁺,both SM-Lmod and Tmod are found near -actinin at the periphery ofactin-rich contraction bands. These data suggest that SM-Lmod is anovel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth musclemay be capped by Tmod in localized clusters.

     

Actin-titin interaction in cardiac myofibrils: probing a physiological role.

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.     

Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.     

Caenorhabditis elegans UNC-96 is a new component of M-lines that interacts with UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.     

Sarcomere structures in the rabbit psoas muscle as revealed by fluorescent analogs of phalloidin

Summary The fluorescent analogs of phalloidin (rhodamine-and fluorescein-phalloidin) bind tightly to the skinned fibres of rabbit psoas muscle at essentially the same sites as phalloidin and mainly stain the known regions of actin localization in the sarcomere: the thin filaments and Z bands. On both sides of the Z bands, unstained zones were observed, suggesting the presence of proteins tightly bound to the thin filaments. In myofibrils which are stretched to such an extent that the actin and myosin filaments do not overlap, stained bands could also be seen at the myosin-band border, which suggests the localization of actin at these sites.     

Polysome structure in sea urchin eggs and embryos: an electron microscopic analysis

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.     

Mechanical strength of sarcomere structures of skeletal myofibrils studied by submicromanipulation

The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.     

Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.     

Actin dynamics at pointed ends regulates thin filament length in striated muscle

Regulation of actin dynamics at filament ends determines the organization and turnover of actin cytoskeletal structures. In striated muscle, it is believed that tight capping of the fast-growing (barbed) ends by CapZ and of the slow-growing (pointed) ends by tropomodulin (Tmod) stabilizes the uniform lengths of actin (thin) filaments in myofibrils. Here we demonstrate for the first time that both CapZ and Tmod are dynamic on the basis of the rapid incorporation of microinjected rhodamine-labelled actin (rho-actin) at both barbed and pointed ends and from the photobleaching of green fluorescent protein (GFP)-labelled Tmod. Unexpectedly, the inhibition of actin dynamics at pointed ends by GFP-Tmod overexpression results in shorter thin filaments, whereas the inhibition of actin dynamics at barbed ends by cytochalasin D has no effect on length. These data demonstrate that the actin filaments in myofibrils are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at pointed ends determines the length of thin filaments.     

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.