RESUMO
The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.
Assuntos
Macrófagos/química , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Actinas/química , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Fluorescência , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Fagocitose , Células RAW 264.7RESUMO
Expression of all of the isoforms of the subunits of AMP-activated protein kinase (AMPK) and AMPK activity is increased in skeletal muscle of hyperthyroid rats. Activity of AMPK in skeletal muscle is regulated principally by the upstream kinase, LKB1. This experiment was designed to determine whether the increase in AMPK activity is accompanied by increased expression of the LKB1, along with binding partner proteins. LKB1, MO25, and downstream targets were determined in muscle extracts in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 wk, and in rats given 0.01% propylthiouracil (PTU; an inhibitor of thyroid hormone synthesis) in drinking water for 4 wk (hypothyroid group). LKB1 and MO25 increased in the soleus of thyroid hormone-treated rats vs. the controls. In other muscle types, LKB1 responses were variable, but MO25 increased in all. In soleus, MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC were elevated in soleus and gastrocnemius of hyperthyroid rats. Thyroid hormone treatment also increased the amount of phospho-cAMP response element binding protein (CREB) in the soleus, heart, and red quadriceps. Four proteins having CREB response elements (CRE) in promoter regions of their genes (peroxisome proliferator-activated receptor-gamma coactivator-1alpha, uncoupling protein 3, cytochrome c, and hexokinase II) were all increased in soleus in response to thyroid hormones. These data provide evidence that thyroid hormones increase soleus muscle LKB1 and MO25 content with subsequent activation of AMPK, phosphorylation of CREB, and expression of mitochondrial protein genes having CRE in their promoters.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipertireoidismo/enzimologia , Hipotireoidismo/enzimologia , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Antitireóideos , Western Blotting , Proteínas de Ligação ao Cálcio , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Modelos Animais de Doenças , Estimulação Elétrica , Hipertireoidismo/induzido quimicamente , Hipotireoidismo/induzido quimicamente , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Propiltiouracila , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Tiroxina , Fatores de Transcrição/genética , Tri-IodotironinaRESUMO
5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle.
