A Disfunção temporomandibular (DTM) é um termo coletivo que abrange um largo espectro de problemas clínicos da articulação e dos músculos na área orofacial; estas disfunções são caracterizadas principalmente por dor, sons na articulação, e função irregular ou limitada da mandíbula. Os músculos da mastigação podem estar envolvidos na DTM de origem muscular, e por definição são os músculos que promovem o toque dental, portanto os elevadores da mandíbula: masseter, temporal, pterigóideos medial e lateral. A origem muscular da DTM é a mais prevalente, sendo portanto,o entendimento funcional e estrutural da composição dos músculos da mastigação essencial para a compreensão desta DTM. Este estudo tem como objetivo analisar a estrutura dos músculos da mastigação quanto a variação do calibre das fibras lentas e rápidas, densidade capilar e da expressão da miosina neonatal com a variação da idade. Foram estudadas 37 amostras dos músculos temporal e masseter (20 amostras do sexo masculino e 17 do sexo feminino) de autópsias do Serviço de Verificação de Óbitos de São Paulo com intervalo pós-mortem de até 18 horas, de ambos os gêneros e com idades divididas por décadas (1a a 9a décadas). Foram realizadas reações imunoistoquímicas com os anticorpos Ulex europaeus biotinilada aglutinina, anti-miosina neonatal, anti-miosina rápida, anti-miosina lenta para análise da expressão das proteínas. A avaliação foi feita por dois observadores, após calibração intra observador, (duas contagens no mesmo campo pelo mesmo observador em tempos diferentes) e inter observador (contagem do mesmo campo por dois observadores), até se atingir uma margem de erro menor que 10%. Em relação ao número de capilares/fibra, nos músculos masseter e temporal, da primeira a nona década, em média respectivamente foi de 1 e 0,7 respectivamente. O número de capilares por mm², nos músculos masseter e temporal, não variou ao longo das nove décadas estudadas, e o número de capilares por mm², foi significantemente maior no músculo masseter quando comparado ao temporal, (p=0,025). A miosina neonatal manteve-se presente embora com decréscimo em todas as décadas dos músculos masseter e temporal. Observou-se o diâmetro das fibras do tipo II menores que as fibras do tipo I / Temporomandibular disfunction (TMD) is a colletive term that refers to different clinical problems of the temporomandibular joint and the jaw muscles. These disfunctions are characterizied meanly by pain, joint sounds and irregular or limited mandibular function. The jaw muscles can be involved in the TMD of muscle etiology, and by definition they are the ones which provide the teeth touch, then the jaw elevators: masseter, temporalis, medial pterygoid and lateral pterygoid. As the muscular etiology is the most prevalent cause of TMD, the detailed understanding of structural and functional composition of the masticatory muscles is paramount to better comprehend TMD due to muscle disorder. This study has the aim to analyze the jaw muscle structure concerning capillarie density, neonatal myosin expression, and the cross sectional area of the fast and slow fibers in temporalis and masseter muscles in autopsy samples from 1st to 9th decades of age. Thirty seven temporalis and masseter muscles samples were studied (20 from male and 17 from female) from Serviço de Verificação de Óbitos of São Paulo. The specimens were divided by gender and ages. The samples were collected up to 18 hours post-mortem. Imunohistochemistry stainning were made with antibodies to analize the protein expression. The evaluations were made by two observers, after intra observer calibration (two evaluations on the same field by the same observer in different times) and inter observer (evaluation of the same field by two observers), unti getting less than 10% of error. The number of capillaries per fiber in the masseter and temporalis muscle was in average 1 and 0,7 respectively. The number of capillaries per mm² was significantly higher in the masseter when compared to temporalis muscle (p=0.025). The neonatal myosin was present in all decades in both muscles, and it was observed that the cross sectional area of the type II fibers was smaller than the type I fibers

Os gânglios da raiz dorsal (GRD) armazenam neurônios TrkA-positivos. A percepção e transmissão de estímulos por estes neurônios dependem de uma neuritogênese adequada. Miosina (MioVa) é expressa no tecido nervoso e está presente em neuritos, corpo celular e cone de crescimento. Caracterizamos o padrão de expressão de MioVa na neuritogênese de células TrkA-positivas do GRD de galinha in vivo e in vitro. In vivo, MioVa é expressa em células que não começaram a emitir neuritos em HH25, e sua expressão persiste por toda neuritogênese. In vitro, é recrutada para o processo de re-emissão de neuritos de neurônios TrkA positivos na presença de NGF, sendo expressa em neuritos em diferentes estádios de neo-neuritogênese. Nos ensaios funcionais, observamos que a superexpressão do domínio globular de MioVa em culturas de GRD com 10 ou 100ng/ml de NGF reduz a população de células com neuritos longos e aumenta a população de células com neuritos curtos ou sem neuritos. Em conjunto, estes dados sugerem que a MioVa é importante para o estabelecimento de neuritos nociceptores. / The dorsal root ganglia (DRG) harbor the TrkA-positive neurons. The stimuli perception and transmission by these neurons depend on a proper neuritogenesis. Myosin (MyoVa) is widely expressed in nervous tissue and is present in neurites, cell body and growth cone. Here, we characterized the MyoVa expression pattern in chicken DRG TrkA-positive cells neuritogenesis, in vivo and in vitro. In vivo, at stage HH25, MyoVa was present both in cells with and without neurites and its expression persists throughout neuritogenesis. In vitro, it is recruited for the regeneration process and TrkA-positive neurons neurites re-emission in the presence of NGF, being expressed in neurites at different stages of neo-neuritogenesis. In functional assays, we observed that MyoVa globular tail overexpression in GRD cultures maintained with 10 or 100ng/ml NGF reduces the number of neurons with long neurites and increased the number of neurons with short neurites or no neurites. Taken together, these results suggest that Myosin Va is important for the establishment of nociceptor neurites.

A miosina Va é uma proteína motora envolvida no transporte e posicionamento de vesículas, organelas e mRNA. Além disso, postulou-se que a miosina-Va atua no seqüestro do fator pró-apoptótico, Bmf, no citoesqueleto de actina. Pesquisas realizadas em nosso laboratório demonstraram que um fragmento da miosina Va (MVaf), que corresponde ao sítio ligante de DLC2-Bmf, é capaz de induzir intensa apoptose em células de melanoma e de carcinoma in vitro. O presente trabalho teve por objetivo principal avaliar o potencial do MVaf como agente antitumoral, através de abordagens de terapia gênica em modelo animal. Foram geradas linhagens estabilizadas e com expressão controlada pelo sistema Tet-ON onde a expressão de EGFP ou EGFP-MVaf é induzida com a adição de doxiciclina. Essas linhagens foram testadas quanto à porcentagem de morte por apoptose e ativação de caspases. Tumores foram induzidos em camundongos C57BL/6 por inoculação subcutânea de células tumorigênicas positivas ou não para a expressão de EGFP-MVaf. Também foram utilizadas linhagens de fibroblasto embrionário murino selvagem (MEFs WT) e nocautes para os fatores Bim/Bmf e Bax/Bak (MEFsBim-/-,Bmf-/-; MEFsBax-/-,Bak-/-) para estudos do mecanismo de ação do fragmento da miosina Va. Observou-se que a adição de butirato de sódio potencializa a expressão de EGFP-MVaf e, conseqüentemente, o efeito pró-apoptótico desse fragmento e que essas células são mais sensíveis aos quimioterápicos etoposídeo e taxol, apresentando maior susceptibilidade à apoptose. Verificou-se que a expressão de EGFP-MVaf em células de tumores de melanoma induzidos em camundongos C57BL/6J dificulta o crescimento desses tumores. Quanto ao estudo com MEFs, observou-se que células nocautes para os fatores pró-apoptóticos Bim/Bmf e Bax/Bak são menos susceptíveis à morte induzida pelo fragmento da miosina Va. Indução da expressão de MVaf desencadeia a liberação da proteína proapoptótica Smac (fusionada ao repórter fluorescente Cherry) do espaço intermembranas da mitocôndria para o citoplasma sugerindo que a morte apoptótica induzida por MVaf requer a permeabilização da membrana mitocondrial externa (MOMP). Concluindo, os dados apresentados aqui nos permitem propor o MVaf como uma molécula promissora para o desenvolvimento de novas abordagens terapêuticas contra o câncer. / Myosin Va is a motor protein involved in the transport and positioning of vesicles, organelles and mRNA. Additionally, myosin-Va has been implicated in the sequestering of a proapoptotic factor, Bmf, to the actin cytoskeleton. Research in our laboratory demonstrated that a fragment of myosin Va (MVaf), which corresponds to the binding site of DLC2-Bmf, is capable to induce intense apoptosis in melanoma and carcinoma cells in vitro. Here, our goal was to assess the potential of MVaf as antitumor agent, through gene therapy approaches in animal models. We generated Tet-ON controlled B16-F10 melanoma cells whose expression of EGFP or EGFP-MVaf is induced with the addition of doxycycline. These cells were tested for apoptotic death and activation of caspases, and were used to induce tumors in C57BL/6J mice by subcutaneous inoculation. We also used cell lines of murine embryonic fibroblasts, wild-type (MEFs WT) and knockouts for the proapoptotic proteins Bim/Bmf or Bax/Bak (MEFsBim-/-,Bmf-/-, MEFsBax-/-,Bak-/-), to study the mechanism by which MVaf induces apoptosis. We observed that addition of sodium butyrate to the cultures enhances the EGFP-MVaf expression and, consequently, the pro-apoptotic effect of this fragment. Treated cells were more sensitive to the chemotherapeutic drugs etoposide and taxol, showing a higher susceptibility to apoptosis. Moreover, in vivo induction of EGFP-MVaf expression retards growth of B16-F10 melanoma tumors in mouse model. As for the study with MEFs, we observed that cells knockout for the proapoptotic factors Bim/Bmf or Bax/Bak are less susceptible to death induced by MVaf than wild-type MEFs. Accordingly, we showed that MVaf expression triggers release of the proapoptotic protein Smac (tagged with the fluorescent protein Cherry) supporting the involvement of the mitochondrial outer membrane permeabilization (MOMP) in the MVaf-induced apoptotic death response. In conclusion, these data lead us to propose MVaf as a promising molecule for the development of new therapeutic approaches against cancer.

"Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering. "

Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of immature progenitor cells into the scar area may compensate for the cell loss and provides a new therapeutic avenue for infarct treatment. Premature myoblasts derived from skeletal muscle are one of the best candidates for this therapeutic purpose, because biopsies used for autologous cell therapy can be accessed easily, the isolated myoblasts can proliferate well in vitro, and the skeletal and cardiac muscles are structurally and functionally similar. In this study we investigated the survival and differentiation of the implanted skeletal myoblasts in the non-cryoinjured myocardium and the myocardial scar, using a syngeneic Lewis rat model. A therapeutic dose of 4x106 skeletal myoblasts/animal was implanted into the non-cryoinjured and scar tissue, and the fate of the implant was monitored at 12, 28 and 56 days after implantation by immunohistochemistry. We detected fast myosin heavy chain (fMHC) expression at each time point but significantly fewer positive cells in the scar than in the non-injured tissue. This was consistent with the staining patterns of slow myosin heavy chain (sMHC) and myogenin that overlapped with fMHC positive areas. Although the implanted myoblasts differentiated into skeletal muscle cells, they did not transdifferentiate into cardiac muscle, demonstrated by the absence of cardiac troponin I expression. During this analysis we developed a model, which could be useful to test new strategies for myoblast implantation (dosage, genetic modification, new injection technique etc.) designed to promote better engraftment of cultured myoblasts in the myocardial scar.

Cardiac muscle contraction is responsible for pumping blood throughout the body. The cyclical, ATP-hydrolysis dependent interaction of the myosin motor protein with filamentous actin drives muscle contraction. During this process the α-helical neck region of myosin acts as a lever arm, transmitting contractile force between thick and thin filaments by amplifying small conformational changes in the myosin motor domain. The resulting relative displacement of thick and thin filaments causes muscle shortening. The regulatory light chain (RLC) of myosin mechanically supports the lever arm by binding to the myosin heavy chain neck region; this is a crucial interaction in maintaining myosin's ability to produce force and motion. We investigated the role of N-terminal modifications of the RLC in modulating actomyosin contractility at the molecular level. Phosphorylation of the RLC is a naturally occurring post-translational modification of the RLC N-terminus that is important for cardiac function and has been shown to enhance contractility at the cellular level. In contrast, genetic mutations of the RLC that lead to familial hypertrophic cardiomyopathy (FHC) disrupt cardiac function and trigger remodeling of the cardiac muscle structure. We studied two FHC-linked mutations, N47K and R58Q, located in the N-terminus of the RLC in close proximity to the phosphorylation site. Using in vitro motility assays we examined how RLC modifications affect the mechanochemical properties of cardiac β-myosin. We found that the FHC mutations reduced myosin force and power generation, in contrast to RLC phosphorylation which increased myosin force and power for WT and mutant myosins. Phosphorylation of mutant RLC resulted in a restoration of the mutation-induced decreases in contractility to WT dephosphorylated levels. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the fundamental contractile FHC-induced phenotype.

A miosina Va é uma proteína motora envolvida no transporte e posicionamento de vesículas, organelas e mRNA. Além disso, postulou-se que a miosina-Va atua no seqüestro do fator pró-apoptótico, Bmf, no citoesqueleto de actina. Pesquisas realizadas em nosso laboratório demonstraram que um fragmento da miosina Va (MVaf), que corresponde ao sítio ligante de DLC2-Bmf, é capaz de induzir intensa apoptose em células de melanoma e de carcinoma in vitro. O presente trabalho teve por objetivo principal avaliar o potencial do MVaf como agente antitumoral, através de abordagens de terapia gênica em modelo animal. Foram geradas linhagens estabilizadas e com expressão controlada pelo sistema Tet-ON onde a expressão de EGFP ou EGFP-MVaf é induzida com a adição de doxiciclina. Essas linhagens foram testadas quanto à porcentagem de morte por apoptose e ativação de caspases. Tumores foram induzidos em camundongos C57BL/6 por inoculação subcutânea de células tumorigênicas positivas ou não para a expressão de EGFP-MVaf. Também foram utilizadas linhagens de fibroblasto embrionário murino selvagem (MEFs WT) e nocautes para os fatores Bim/Bmf e Bax/Bak (MEFsBim-/-,Bmf-/-; MEFsBax-/-,Bak-/-) para estudos do mecanismo de ação do fragmento da miosina Va. Observou-se que a adição de butirato de sódio potencializa a expressão de EGFP-MVaf e, conseqüentemente, o efeito pró-apoptótico desse fragmento e que essas células são mais sensíveis aos quimioterápicos etoposídeo e taxol, apresentando maior susceptibilidade à apoptose. Verificou-se que a expressão de EGFP-MVaf em células de tumores de melanoma induzidos em camundongos C57BL/6J dificulta o crescimento desses tumores. Quanto ao estudo com MEFs, observou-se que células nocautes para os fatores pró-apoptóticos Bim/Bmf e Bax/Bak são menos susceptíveis à morte induzida pelo fragmento da miosina Va. Indução da expressão de MVaf desencadeia a liberação da proteína proapoptótica Smac (fusionada ao repórter fluorescente Cherry) do espaço intermembranas da mitocôndria para o citoplasma sugerindo que a morte apoptótica induzida por MVaf requer a permeabilização da membrana mitocondrial externa (MOMP). Concluindo, os dados apresentados aqui nos permitem propor o MVaf como uma molécula promissora para o desenvolvimento de novas abordagens terapêuticas contra o câncer. / Myosin Va is a motor protein involved in the transport and positioning of vesicles, organelles and mRNA. Additionally, myosin-Va has been implicated in the sequestering of a proapoptotic factor, Bmf, to the actin cytoskeleton. Research in our laboratory demonstrated that a fragment of myosin Va (MVaf), which corresponds to the binding site of DLC2-Bmf, is capable to induce intense apoptosis in melanoma and carcinoma cells in vitro. Here, our goal was to assess the potential of MVaf as antitumor agent, through gene therapy approaches in animal models. We generated Tet-ON controlled B16-F10 melanoma cells whose expression of EGFP or EGFP-MVaf is induced with the addition of doxycycline. These cells were tested for apoptotic death and activation of caspases, and were used to induce tumors in C57BL/6J mice by subcutaneous inoculation. We also used cell lines of murine embryonic fibroblasts, wild-type (MEFs WT) and knockouts for the proapoptotic proteins Bim/Bmf or Bax/Bak (MEFsBim-/-,Bmf-/-, MEFsBax-/-,Bak-/-), to study the mechanism by which MVaf induces apoptosis. We observed that addition of sodium butyrate to the cultures enhances the EGFP-MVaf expression and, consequently, the pro-apoptotic effect of this fragment. Treated cells were more sensitive to the chemotherapeutic drugs etoposide and taxol, showing a higher susceptibility to apoptosis. Moreover, in vivo induction of EGFP-MVaf expression retards growth of B16-F10 melanoma tumors in mouse model. As for the study with MEFs, we observed that cells knockout for the proapoptotic factors Bim/Bmf or Bax/Bak are less susceptible to death induced by MVaf than wild-type MEFs. Accordingly, we showed that MVaf expression triggers release of the proapoptotic protein Smac (tagged with the fluorescent protein Cherry) supporting the involvement of the mitochondrial outer membrane permeabilization (MOMP) in the MVaf-induced apoptotic death response. In conclusion, these data lead us to propose MVaf as a promising molecule for the development of new therapeutic approaches against cancer.

A Tropomiosina (Tm) está diretamente envolvida no processo de regulação da contração muscular, que é controlado por um mecanismo alostérico que envolve Ca2+, troponina (Tn), actina (Ac) e miosina. A Tm é uma proteína flexível, de estrutura \"coiled-coil\", constituída de duas α-hélices com 284 aminoácidos cada uma. A molécula de Tm faz interações do tipo \"cabeça-cauda\" com outra molécula de Tm através da sobreposição de aproximadamente 8 a 15 resíduos da extremidade N- terminal de uma molécula com 8 a 15 resíduos da extremidade e-terminal da outra molécula de Tm. Desta maneira, em baixas forças iônicas, formam-se filamentos lineares através de um processo de polimerização. A estabilidade de regiões específicas da Tm pode ser importante para a sua função no controle da regulação da contração muscular. Além disso, a Tm pode ser usada como um modelo relativamente simples e de ocorrência natural para entendermos as interações intra- e intermoleculares que governam a estabilidade das \"coiled-coils\". Assim sendo, nós produzimos oito fragmentos recombinantes de Tm (Tm143-284(50HW), Tm189-284(50Hw), Tm189-284, Tm220-284(50HW), Tm220-284, Tm143-235, Tm167-260 e Tm143-260) e um peptídeo sintético (Ac-Tm215-235) com a finalidade de investigar as estabilidades conformacionais relativas das diferentes regiões derivadas da metade C-terminal da proteína, a qual é conhecida por sua interação com o complexo troponina. Experimentos de ultracentrifugação analítica mostram que os fragmentos que incluem os últimos 24 resíduos da molécula (Tm143-284(50HW), Tm189-284(50HW), Tm220-284(50HW), Tm220-284) estão completamente dimerizados a 10 µM (concentração do dímero em 50 mM de tampão fosfato, pH 7,0; 100 mM de NaCI; 0,5 mM de DTT e 0,5 mM de EDTA, 10°C), enquanto que fragmentos que não possuem o e-terminal nativo (Tm143-235, Tm167-260 e Tm143-260) se encontram em equilíbrio monômero-dímero nestas condições. A presença de trifluoroetanol promove uma diminuição na razão [θ]222/[θ]208, observada por dicroísmo circular, em todos os fragmentos e induz a formação de trímeros estáveis apenas para aqueles contendo os resíduos 261-284. Estudos de desnaturação por uréia, acompanhados por dicroísmo circular e fluorescência, mostram que os resíduos 261-284 da tropomiosina são muito importantes para estabilidade da metade C-terminal da molécula. Além do mais, a ausência desta região promove um aumento na cooperatividade do desenovelamento induzido por uréia. Os experimentos de desnaturação por temperatura e por uréia mostram que o fragmento Tm143-235 é relativamente instável quando comparado com outros fragmentos de mesmo tamanho. Nós identificamos alguns fatores que podem estar contribuindo para a particular instabilidade desta região, incluindo repulsões inter-hélices entre resíduos em posições g e e\' da repetição heptapeptídica, um resíduo carregado na interface hidrofóbica da \"coiled-coil\" e por fim, uma grande fração de resíduos β-ramificados localizados em posições d. Sabe-se que a não acetilação do N-terminal da molécula de Tm, bem como a ausência de alguns resíduos na sua extremidade C-terminal, fazem com que a Tm deixe de sofrer polimerização. Entretanto, trabalhos anteriores realizados em nosso laboratório haviam mostrado que um fragmento de Tm recombinante (ASTm1-260), contendo a fusão dipeptídica Ala-Ser (que é conhecida por restaurar a capacidade de polimerização de Tm não acetiladas no N-terminal), polimerizava-se mais do que a proteína recombinante de tamanho integral (ASTm), apesar da deleção dos últimos 24 aminoácidos C-terminais. Para investigar com mais detalhes a natureza da interação cabeça-cauda, nós construímos dois fragmentos que compreendem a metade N-terminal da molécula de Tm, ASTm1-142 e nfTm1-142, o primeiro deles contendo uma fusão dipeptídica AS no N-terminal e o segundo com o N-terminal não acetilado, sem a fusão dipeptídica. Estes dois fragmentos foram empregados em ensaios de interação cabeça-cauda, realizados através de ensaios de desnaturação térmica acompanhados por dicroísmo circular, juntamente com três fragmentos da região C-terminal da Tm. Dois dos fragmentos e-terminais acabam na posição 260 (Tm167-260 e Tm143-260) e um deles termina na posição 284 (Tm220-284), que corresponde ao C-terminal nativo da proteína. Os resultados mostram que ocorre uma interação cabeça-cauda entre o fragmento N-terminal ASTm1-142 e todos os fragmentos C-terminais utilizados neste estudo. As moléculas recombinantes de Tm que terminam na posição 260 são, de fato, capazes de fazer interações cabeça-cauda independentemente do fato de elas se apresentarem instáveis nas condições estudadas. Inclusive, após a interação, ocorre um aumento considerável na estrutura a-hélice, o que se dá preferencialmente nos fragmentos C-terminais. / Tropomyosin (Tm) participates in the process of muscle contraction, which is controlled by an allosteric mechanism involving Ca2+, troponin (Tn), actin (Ac) and myosin. Tm is a coiled-coil flexible molecule composed by two α-helices with 284 amino acids each. Tm molecule performs head-to-tail interactions with another Tm molecule through the overlap of approximately 8 to 15 N-terminal residues of one molecule with 8 to 15 C-terminal residues of the other molecule. Thus Tm forms linear filaments in low ionic strengths, which is characteristic for a polymerization process. The stability of specific regions of Tm may be important to its function in controlling the regulation of muscle contraction. Besides, Tm can be used as a relatively simple model and of natural occurrence to understand the intra- and intermolecular interactions that govern the stability of \"coiled-coils\". We therefore produced eight recombinant fragments of Tm (Tm143-284(50HW), Tm189-284(50HW), Tm189-284, Tm220-284(50HW), Tm220-284, Tm143-235, Tm167-260 and Tm143-260) and one synthetic peptide (Ac-Tm215-235) to investigate the relative conformational stabilities of different regions derived from the C-terminal end of the molecule, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that fragments comprising the last 24 residues of the molecule (Tm143-284(50Hw), Tm189-284(50HW), Tm220-284(50Hw), Tm220-284) are completely dimerized at 10 µM (dimer concentration in buffer containing 50 mM phosphate, pH 7.0, 100 mM NaCI, 0.5 mM DTT and 0.5 mM EDTA, 10ºC), whereas the fragments that do not posses the native C-terminal portion (Tm143-235, Tm167-260 and Tm143-260) present a monomer-dimer equilibrium at the same conditions. Trifluoroethanol promoted a decrease in the ratio [θ]222/[θ]208 for all fragments (observed by circular dichroism), and induced the formation of stable trimers for the fragments comprising the residues 261-284. Urea denaturation studies, followed by circular dichroism and fluorescence, show that residues 261-284 of Tm are very important for the stability of the C-terminal half of the molecule. Still, the absence of this region promotes an increase in the cooperativity of unfolding induced by urea. Urea and temperature denaturation experiments showed that the fragment Tm143-235 is relatively unstable when compared to other fragments of the same size. We identified some factors that may be contributing to the particular instability of this region, including interhelix repulsions between residues in positions g and e\' of the heptad repeat, a charged residue in the hydrophobic interface of the coiled-coil and a great fraction of (β-branched residues located at d positions. It is known that the non-acetylation of the N-terminus of the Tm molecule, as well as, the absence of some residues in the C-terminus of the molecule cause Tm to loose its ability to undergo polymerization. Former works performed in our laboratory showed that a fragment of recombinant Tm (ASTm1-260) with the dipeptide fusion Ala-Ser (which is known by its ability in restores the polymerization of non-acetylated Tms), despite the absence of the C-terminal 24 amino acids, polymerizes to a much greater extent than the corresponding full length recombinant protein. To better investigate the nature of the head-to-tail interaction we constructed two fragments that comprise the N-terminal half of the Tm: ASTm1-142 and nfTm1-142, the former containing the dipeptide AS (Ala-Ser) fusion to its N-terminus, and the latter presenting a bare non-acetylated N-terminus without the dipeptide fusion . These two fragments were employed in thermal denaturation assays followed by circular dichroism to test their head-to-tail interaction along with three fragments comprising Tm\'s C-terminal region. Two of the C-terminal fragments ends at position 260 (Tm167-260 and Tm143-260), whereas one ends at position 284 (Tm220-284). Results show that there is a head-to-tail interaction between the N-terminal fragment ASTm1-142 and all other e-terminal fragments employed in this study. Recombinant Tm molecules ending at position 260, are capable of performing head-to-tail interactions, regardless of their stability under the studied condition. Upon interaction, an increase in the a-helix content occurs preferentially in the e-terminal fragments.

Uma série de mutantes recombinantes da tropomiosina com a sonda fluorescente 5-hidroxitriptofano inserida em várias posições ao longo da seqüência primária vêm sendo utilizados em nosso laboratório para o estudo em nível molecular do controle do processo de contração muscular. Estes mutantes têm sido produzidos em cepas de bactéria auxotróficas para triptofano em meio mínimo ao qual adicionamos 5-hidroxitriptofano, um análogo do aminoácido triptofano. Esta sonda fluorescente apresenta a vantagem de absorver energia numa faixa de comprimentos de onda não absorvida pelo triptofano (temos usado com sucesso o comprimento de onda de 312 nm para excitar seletivamente o 5-hidroxitriptofano). Desta forma, o ambiente da sonda pode ser estudado em situações nas quais outras proteínas que possuam triptofanos, como, por exemplo a actina, estejam presentes. Procedemos a uma caracterização dos mutantes utilizados quanto à sua ligação à actina na ausência e presença de troponina (+/- Ca2+) e na presença de acrilamida por meio de ensaios de co-sedimentação com actina. O comportamento funcional dos diversos mutantes foi investigado pela análise da regulação da atividade Mg2+ -ATPásica da acto-S1 miosina destes mutantes na ausência e na presença de troponina (+/- Ca2+). Estudos da estabilidade destes mutantes da tropomiosina por meio de desnaturação por temperatura seguida por dicroísmo circular foram realizados para analisarmos o efeito das mutações na estabilidade global da molécula. Ensaios de desnaturação por uréia seguidas por fluorescência também foram realizados para analisarmos a estabilidade da molécula próxima à região da sonda fluorescente. O presente estudo compreendeu ainda a análise da supressão de fluorescência pelos supressores extrínsecos acrilamida e iodeto para diversos mutantes da tropomiosina na presença e ausência de outras proteínas do filamento fino, o que permitiu a obtenção de informações sobre o grau de exposição da sonda fluorescente nas diversas situações estudadas bem como sobre o ambiente eletrostático ao redor das sondas nestas diferentes situações. Estes resultados permitiram a obtenção de um modelo para a ligação da tropomiosina ao filamento de actina na presença de troponina (+/- Ca2+). Este modelo propõe que as bandas α do padrão de repetição α/β inicialmente proposto por Mclachlan e Stewart (1975 e 1976) se liguem ao filamento na ausência de íons cálcio. A introdução de cálcio no sistema é capaz de induzir uma rotação e um deslocamento na molécula de tropomiosina com relação ao filamento de actina. Nesta situação, seriam as bandas β da tropomiosina as responsáveis pela ligação desta molécula ao filamento de actina. / Several recombinant mutants of tropomyosin with the fluorescent probe 5-hydroxytryptophan inserted at several positions along the primary sequence are being used in our laboratory for studying the control of the muscular contraction process at a molecular level. These mutants are produced in bacterial strains auxotrophic to tryptophan in minimal medium to which 5- hydroxytryptophan, a tryptophan analogue, is added. This fluorescent probe has the advantage of absorbing light in a range of wavelengths not absorbed by the amino acid tryptophan (we have been successfully using the wavelength of 312 nm to selectively excite the 5-hydroxytryptophan). The environment of the probe can thus be studied even when other tryptophan containing proteins, such as actin, are present. We have characterized the mutants as to their ability to bind to actin in the absence and in the presence of troponin (+/- Ca2+) and in the presence of acrylamide through actin co-sedimentation assays. The functional behavior of the several mutants constructed was assessed by analyzing their ability to regulate the acto-S1 myosin Mg2+-ATPase activity in the absence and in the presence of troponin (+/- Ca2+). Studies on the stability of these tropomyosin mutants by thermal denaturation followed by circular dichroism were performed to analyze the effect of the mutations on the molecule\'s global stability. Urea denaturation assays followed by fluorescence were also performed to allow us to investigate the stability of the molecule in the vicinity of the fluorescent probe. The present study also involved the analysis of the fluorescence quenching by the extrinsic quenchers acrylamide and iodide for divers tropomyosin mutants in the presence and in the absence of other thin filament proteins. This allowed us to obtain information on the degree of exposure of the fluorescent probe in the several conditions studied as well as on the electrostatic microenvironment surrounding the probes in these different situations. These results enabled us to propose a model for the binding of tropomyosin to the actin filament in the presence of troponin (+/Ca2+). In this model the α-bands from the α/β repetition pattern initially proposed by Mclachlan and Stewart (1975 and 1976) would be binding the filament in the absence of calcium ions. The introduction of calcium to the system would induce a rolling motion in and a displacement of the tropomyosin molecule with relation to the actin filament. In this situation tropomyosin\'s β-bands would be responsible for the binding of this molecule to the actin filament.

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system.