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.

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy.

Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.

Arecoline suppresses epithelial cell viability by upregulating tropomyosin-1 through the transforming growth factor-ß/Smad pathway.

CONTEXT: Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. OBJECTIVES: This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. MATERIALS AND METHODS: HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. RESULTS: The IC50 of arecoline was approximately 50 µg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-ß receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. DISCUSSION AND CONCLUSIONS: Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-ß/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.

The molecular mechanisms of a high Ca2+-sensitivity and muscle weakness associated with the Ala155Thr substitution in Tpm3.12.

Substitution of Ala for Thr residue in 155th position in γ-tropomyosin (Tpm3.12) is associated with muscle weakness. To understand the mechanisms of this defect, we studied the Ca2+-sensitivity of thin filaments in solution and multistep changes in mobility and spatial arrangement of actin, Tpm, and myosin heads during the ATPase cycle in reconstituted muscle fibres, using the polarized fluorescence microscopy. It was shown that the Ala155Thr (A155T) mutation increased the Ca2+-sensitivity of the thin filaments in solution. In the absence of the myosin heads in the muscle fibres, the mutation did not alter the ability of troponin to switch the thin filaments on and off at high and low Ca2+, respectively. However, upon the binding of myosin heads to the thin filaments at low Ca2+, the mutant Tpm was found to be markedly closer to the open position, than the wild-type Tpm. In the presence of the mutant Tpm, switching on of actin monomers and formation of the strong-binding state of the myosin heads were observed at low Ca2+, which indicated a higher myofilament Ca2+-sensitivity. The mutation decreased the amount of myosin heads bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation. It is suggested that direct binding of myosin to Tpm may be one оf the reasons for muscle weakness associated with the A155T mutation. The use of reagents that decrease the Ca2+-sensitivity of the troponin complex may not be adequate to restore muscle function in patients with the A155T mutation.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

INTRODUCTION: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. METHODS: Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. RESULTS: We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. CONCLUSIONS: We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Anatomy of the red cell membrane skeleton: unanswered questions.

The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism.

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization.

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMß causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.

3-OST-7 regulates BMP-dependent cardiac contraction.

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

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.

Regulation of nonmuscle myosin II by tropomyosin.

The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.

Tropomyosin dynamics.

Tropomyosin is a two chained α-helical coiled coil protein that binds actin filaments and interacts with various actin binding proteins. Tropomyosin function depends on its ability to move to distinct locations on the surface of actin in response to the binding of different thin filament effectors. Tropomyosin dynamics plays an important role in these fluctuating interactions with actin and is thought to be fundamental to many of its biological activities. For example tropomyosin concerted movement on the surface of actin triggered by Ca(2+) binding to troponin or myosin head binding to actin has been argued to be key to the cooperative allosteric regulation of muscle contraction. These large-scale motions are affected by tropomyosin internal dynamics and mechanical properties. Tropomyosin internal dynamics corresponding to smaller and more localised structural fluctuations are increasingly recognised to play an important role in its function. A thorough understanding of the coupling between local and global structural fluctuations in tropomyosin is required to understand how time dependent structural fluctuations in tropomyosin contribute to the overall thin filament dynamics and dictate their various biological activities.

Nuclear tropomyosin and troponin in striated muscle: new roles in a new locale?

Tropomyosin and troponin have well known Ca(2+)-regulatory functions in the striated muscle sarcomere. In this review, we summarize experimental evidence that tropomyosin and troponin are localized, with as yet unidentified functional roles, in the striated muscle cell nucleus. We also apply bioinformatics approaches that predict localization of some tropomyosin and troponin to the nucleus, and that SUMOylation could be a covalent modification that modulates their nuclear localization and function. Further, we provide examples of cardiomyopathy mutations that alter the predicted likelihood of nuclear localization and SUMOylation of tropomyosin. These observations suggest novel mechanisms by which cardiomyopathy mutations in tropomyosin and troponin might alter not only cardiac contractility but also nuclear function.

Cytoskeletal tropomyosins: choreographers of actin filament functional diversity.

The actin cytoskeleton plays a central role in many essential cellular processes. Its involvement requires actin filaments to form multiple populations with different structural and therefore functional properties in specific subcellular locations. This diversity is facilitated through the interaction between actin and a number of actin binding proteins. One family of proteins, the tropomyosins, are absolutely essential in regulating actin's ability to form such diverse structures. In this review we integrate studies from different organisms and cell types in an attempt to provide a unifying view of tropomyosin dependent regulation of the actin cytoskeleton.

Vardenafil improves penile erection in type 2 diabetes mellitus patients with erectile dysfunction: role of tropomyosin.

INTRODUCTION: Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases. AIM: The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins. METHODS: Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics. MAIN OUTCOME MEASURES: International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs). RESULTS: The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and ß-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only ß-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-ß1 subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 µg/mL) did not modify sGC-ß1 subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin. CONCLUSION: Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma ß-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs.

TrkB (tropomyosin-related kinase B) controls the assembly and maintenance of GABAergic synapses in the cerebellar cortex.

Inhibitory interneurons play a critical role in coordinating the activity of neural circuits. To explore the mechanisms that direct the organization of inhibitory circuits, we analyzed the involvement of tropomyosin-related kinase B (TrkB) in the assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex. We show that TrkB acts directly within each cell-type to regulate synaptic differentiation. TrkB is required not only for assembly, but also maintenance of these synapses and acts, primarily, by regulating the localization of synaptic constituents. Postsynaptically, TrkB controls the localization of a scaffolding protein, gephyrin, but acts at a step subsequent to the localization of a cell adhesion molecule, Neuroligin-2. Importantly, TrkB is required for the localization of an Ig superfamily cell adhesion molecule, Contactin-1, in Golgi and granule cells and the absence of Contactin-1 also results in deficits in inhibitory synaptic development. Thus, our findings demonstrate that TrkB controls the assembly and maintenance of GABAergic synapses and suggest that TrkB functions, in part, through promoting synaptic adhesion.

MicroRNA-21 regulates vascular smooth muscle cell function via targeting tropomyosin 1 in arteriosclerosis obliterans of lower extremities.

OBJECTIVE: The goal of this study was to determine the expression signature and the potential role of microRNAs in human arteries with arteriosclerosis obliterans (ASO). METHODS AND RESULTS: The expression profiles of microRNAs in human arteries with ASO and in normal control arteries were determined by quantitative reverse transcription-polymerase chain reaction array. Among the 617 detected microRNAs, multiple microRNAs were aberrantly expressed in arteries with ASO. Some of these dysregulated microRNAs were further verified by quantitative reverse transcription-polymerase chain reaction. Among them, microRNA-21 (miR-21) was mainly located in arterial smooth muscle cells (ASMCs) and was increased by more than 7-fold in ASO that was related to hypoxia inducible factor 1-α. In cultured human ASMCs, cell proliferation and migration were significantly decreased by inhibition of miR-21. 3'-Untranslated region luciferase assay confirmed that tropomyosin 1 was a target of miR-21 that was involved in miR-21-mediated cellular effects, such as cell shape modulation. CONCLUSION: The results suggest that miR-21 is able to regulate ASMC function by targeting tropomyosin 1. The hypoxia inducible factor-1 α/miR-21/tropomyosin 1 pathway may play a critical role in the pathogenesis of ASO. These findings might provide a new therapeutic target for human ASO.

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments.

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.

Preservation of tropomyosin-related kinase B (TrkB) signaling by sodium orthovanadate attenuates early brain injury after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Recent studies reported that apoptosis was involved in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). The aim of this study was to examine whether sodium orthovanadate (SOV) prevents post-SAH apoptosis by modulating growth factors and its downstream receptor tyrosine kinases. Method- Rats were operated on with the endovascular perforation model. SAH animals were treated with vehicle, 3 mg/kg and 10 mg/kg SOV, and evaluated regarding neurofunction and brain edema. The expression of growth factors such as mature brain-derived neurotrophic factor, insulin-like growth factor-1, and vascular endothelial growth factor and phosphorylation of tropomyosin-related kinase B, which is a receptor tyrosine kinase for brain-derived neurotrophic factor and the downstream pathway in antiapoptosis, was examined by Western blot analysis. Neuronal cell death was measured with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. We also administered K252a, a tropomyosin-related kinase B antagonist, to examine the mechanisms for neuroprotective effects by SOV. RESULTS: SOV significantly improved neurofunction and reduced brain edema after SAH. SOV increased mature brain-derived neurotrophic factor and prevented post-SAH tropomyosin-related kinase B inactivation and caspase-3 activation, resulting in attenuation of neuronal cell death in the cortex and hippocampal CA1 region. Preinjection of K252a abolished the beneficial effects of SOV. CONCLUSIONS: The current study showed that brain-derived neurotrophic factor-induced tropomyosin-related kinase B activation by SOV was necessary for protection against early brain injury after SAH.

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast.

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.