Christine A. Lucas

Expression of extraocular myosin heavy chain in rabbit laryngeal muscle

SummaryThe intrinsic laryngeal muscles of mammals are functionally heterogeneous, some of these muscles (e.g. the thyroarytenoid) contract extremely rapidly, like extraocular muscle, whilst others (e.g. the cricothyroid) contract as fast as limb fast muscle. The extraordinarily rapid contraction speed of extraocular muscles is associated with a fast myosin not found in limb muscles. In this work we explored the possibility that the thyroarytenoid muscle may also express this extraocular-specific fast myosin by raising a monoclonal antibody (mab 4A6) against its heavy chain. Electrophoretic separation of native isomyosins revealed that both the extraocular and the thyroarytenoid have two similar bands migrating ahead of bands found in limb fast or cricothyroid myosins. These two bands bound mab 4A6. The thyroarytenoid muscle can be divided into two divisions, a vocalis division which is important in phonation and an external division which functions in closing the glottis. Fibres in the vocalis are heterogeneous, some stain with mab 4A6, whilst others stain with mabs against limb myosin heavy chains. Fibres in the external division stain almost homogeneous with mab 4A6. The immunohistochemical staining pattern in the cricothyroid muscle resembled that of fast limb muscle: no fibres stained with mab 4A6. Thus, the high speed of contraction of the thyroarytenoid is associated with the same myosin heavy chain found in extraocular muscles, this characteristic is presumably an evolutionary adaptation for rapid closure of the glottis to enhance airway defense mechanisms.

Fiber Types in Rat Laryngeal Muscles and Their Transformations After Denervation and Reinnervation

The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allo-types and that laryngeal muscle fibers are subject to neural regulation.

Cytoskeletal tropomyosin Tm5NM1 is required for normal Excitation- contraction coupling in skeletal muscle.

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation-contraction coupling in skeletal muscle.

An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake.

Actin has an ill‐defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1‐overexpressing mice, insulin‐stimulated glucose uptake was increased; while Tpm3.1‐null mice they were more sensitive to the impact of high‐fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3‐L1 adipocytes abrogates insulin‐stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.

Electrophoretic and immunochemical evidence showing that marsupial limb muscles express the same fast and slow myosin heavy chains as eutherians.

Limb muscles of eutherian (placental) mammals express a slow and three fast isoforms of myosin heavy chain (MyHC), but little is known about marsupial MyHCs. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of limb MyHCs from seven marsupial species, spanning two orders, revealed four components, each of which specifically cross‐reacted in Western blots with a monoclonal antibody (mAb) against a corresponding eutherian MyHC. For all seven species, the relative mobility of the band identified by each mAb matched that in the rat, suggesting that the four are homologous to eutherian slow, 2B, 2X and 2A MyHCs, respectively, in the order of decreasing mobility. Immunohistochemical analysis of fast marsupial limb muscles identitied four different fiber populations whose relative fiber size spectra (IIA

Jaw-closing muscles of kangaroos express α-cardiac myosin heavy chain

The masseter muscle of eutherian grazing mammals typically express β or slow myosin heavy chain (MyHC). Myosins in the masseter of 4 species of kangaroos and a slow limb muscle of one of them were compared with their cardiac myosin by pyrophosphate and sodium dodecyl sulphate (SDS) gel electrophoresis, immunoblotting and immunohistochemistry. It was found that ventricular muscle contains three isoforms homologous to V1 (α-MyHC homodimer), V2 (heterodimer) and V3 (β-MyHC homodimer) of eutherian cardiac muscle, and that the masseter contained V1, with traces of V2 and V3, in great contrast to eutherian ruminants, which express only V3. A polyclonal antibody (anti-KJM) was raised in rabbits against red kangaroo masseter myosin. After cross-absorption against limb muscle myofibrils, anti-KJM specifically reacted in Westerns with MyHCs from masseter but not limb muscles, and immunohistochemically with masseter, but not limb muscle fibers. In pyrophosphate Western blots, anti-KJM reacted with V1 but not with V3. However, a monoclonal antibody specific for eutherian slow myosin stained all kangaroo slow muscle fibers but only weakly stained scattered fibers in the masseter. The SDS-PAGE revealed that light chain composition of masseter and ventricular myosins is identical, but isoforms of both light chains of kangaroo limb slow myosin were observed. These results confirm that kangaroo jaw muscle express α-MyHC rather than β-MyHC. The difference in MyHC gene expression between marsupial and eutherian grazers may be related to the fact that kangaroos are not ruminants, and have only a single chance to comminute food into fine particles, hence the need for the greater speed and power of muscle contraction associated with V1 containing muscle fibers.

Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

Tropomyosin Tm5NM1 regulates cell proliferation and organ size. It mediates this effect by regulating the interaction of pERK and Imp7, leading to the regulation of pERK nuclear translocation. This demonstrates a role for a specific population of actin filaments in regulating a critical step in the MAPK/ERK signaling pathway.

Myosin isoforms and fibre types in limb muscles of Australian marsupials: adaptations to hopping and non-hopping locomotion

Using immunohistochemistry and SDS-PAGE, we studied the myosin heavy chain (MyHC) composition and fibre type distribution of hindlimb muscles of hopping and non-hopping Australian marsupials. We showed that hindlimb muscles of a bandicoot (Isoodon obesulus, order Peramelomorphia) and a small macropodoid, the brushtail bettong (Bettongia penicillata) expressed four MyHCs, slow, 2a, 2x and 2b, and had the corresponding fibre types as other macropods reported earlier. The fastest and most powerful 2b fibres predominated in most bettong hindlimb muscles, but were absent in the gastrocnemius and the flexor digitorum profundus, which are involved in elastic strain energy saving during hopping. The gastrocnemius of four large macropodids also showed little or no 2b MyHC, whereas this isoform was abundant in their tibialis anterior, which is not involved in elastic energy saving. In contrast, 2b MyHC predominated in the gastrocnemius of four non-hopping marsupials. These results suggest that absence of 2b fibres may be a general feature of macropodoid muscles involved in elastic energy saving. Large eutherians except llamas and pigs also have no 2b fibres. We hypothesize that 2x and 2a fibres perform better than 2b fibres in the storage and recovery of kinetic energy during locomotion in both marsupials and eutherians.

Tropomyosin isoforms support actomyosin biogenesis to generate contractile tension at the epithelial zonula adherens

Epithelial cells generate contractile forces at their cell–cell contacts. These are concentrated at the specialized apical junction of the zonula adherens (ZA), where a ring of stabilized E‐cadherin lies adjacent to prominent actomyosin bundles. Coupling of adhesion and actomyosin contractility yields tension in the junction. The biogenesis of junctional contractility requires actin assembly at the ZA as well as the recruitment of nonmuscle myosin II, but the molecular regulators of these processes are not yet fully understood. We now report a role for tropomyosins 5NM1 (Tm5NM1) and 5NM2 (Tm5NM2) in their generation. Both these tropomyosin isoforms were found at the ZA and their depletion by RNAi or pharmacological inhibition reduced both F‐actin and myosin II content at the junction. Photoactivation analysis revealed that the loss of F‐actin was attributable to a decrease in filament stability. These changes were accompanied by a decrease in E‐cadherin content at junctions. Ultimately, both long‐term depletion of Tm5NM1/2 and acute inhibition with drugs caused junctional tension to be reduced. Thus these tropomyosin isoforms are novel contributors to junctional contractility and integrity.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.