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FRET analysis of actin-myosin interaction in contracting rat aortic smooth muscle

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We examined the interaction of smooth muscle myosin with α-actin and β-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from α-actin but not β-actin. Whole-cell FRET analysis confirmed these observations (α-actin-myosin -67%, β-actin-myosin -2%). Time course studies further showed that α-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in α-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, β-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of α-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of α-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.

Nous avons examiné l’interaction de la myosine du muscle lisse avec les isoformes α-actine et β-actine durant la contraction des cellules musculaires lisses A7r5 et la contraction du muscle lisse aortique de rat. Nous avons utilisé les techniques de microscopie confocale et de transfert d’énergie par résonance de fluorescence (FRET) analyse pour examiner les cellules A7r5 et les anneaux fibreux aortiques contractés avec le phorbol 12,13-dibutyrate. L’évaluation des images confocales des cellules musculaires lisses A7r5 contractées à l’aide du phorbol 12,13-dibutyrate a indiqué une dissociation significative de la myosine et de l’α-actine, mais pas de la myosine et de la β-actine. L’analyse du FRET des cellules entières a confirmé ces observations (α-actine-myosine -67 %, β-actine-myosine -2 %). De plus, les études de cours temporel ont montré que le complexe α-actine-myosine a augmenté significativement (40 %) moins de 1,5 min après l’ajout de phorbol 12,13-dibutyrate, pour ensuite diminuer la progression de la contraction. L’analyse du FRET des anneaux fibreux aortiques à différents intervalles de contraction a indiqué des augmentations significatives du complexe α-actine-myosine au début (79 %) et au plateau (67 %) du développement de la force, mais pas durant la période intermédiaire de développement lent de la tension (-4 %). Par comparaison, le complexe β-actine-myosine est demeuré stable, sauf durant le développement lent de la force où l’association a significativement diminué (-30 %). La fluorescence de l’Alexa 488 - phalloïdine a indiqué une augmentation de la teneur tissulaire en F-actine au début (21 %) et au plateau (62 %) du développement de la force dans les deux types de complexe. Les images du FRET ont montré le développement de granules (patches) corticaux et de câbles épais d’α-actine-myosine dans le tissu pendant tout l’intervalle de contraction. Les résultats démontrent le remodelage dynamique de la protéine contractile durant la contraction du muscle lisse vasculaire et donnent à penser que l’analyse du FRET pourrait être un outil puissant pour évaluer les associations protéine-protéine du tissu.
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Document Type: Research Article

Publication date: May 1, 2009

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