Biology of autophagy in skeletal muscles

自噬是一种蛋白质回收途径，细胞内容物可通过该途径被递送至溶酶体以产生可回收的营养成分。最近的研究表明，自噬在骨骼肌自噬中起着非常重要的作用。我们打算在麦吉尔大学建立一个研究项目，旨在研究骨骼肌中自噬的基本方面，并培养基础细胞生物学研究的研究生。该提案包括三个部分。 第一节：我们将评估线粒体活性氧（ROS）和通透性转换孔（PTP）作为信号转导器触发骨骼肌自噬诱导响应分解代谢刺激的作用。为此，我们将使用过表达过氧化物氧还蛋白3（Trx 3 +/+）（线粒体抗氧化酶）的小鼠和亲环蛋白D（Ppif-/-，线粒体PTP的必需组分）缺陷的小鼠。 将对Trx 3 +/+和Ppif-/-小鼠及其同窝野生型小鼠的自噬以及蛋白酶体降解途径和线粒体呼吸进行沿着检查。 第二节：我们将通过使用自噬体的荧光标记物来确定培养的骨骼肌细胞中自噬膜的形态学和生化特征。 我们将使用共聚焦和电子显微镜以及蛋白质组学方法来鉴定自噬的形成。 第三节：我们将利用生物信息学工具分析骨骼肌自噬基因的启动子，以确定调控骨骼肌自噬相关基因的转录因子。预测的转录因子的激活将使用专门的技术在肌细胞中验证，并且它们在自噬调节中的功能重要性将使用siRNA方法验证。

Autophagy is a protein recycling pathway whereby cellular contents can be delivered to the lysosome to yield recyclable nutrient components. Recent evidence suggests that autophagy plays a very important role in skeletal muscle autophagy. We intend to establish a research program at McGill University aimed at studying the basic aspects of autophagy in skeletal muscles and at training graduate students in basic cell biology research. The proposal consists of 3 sections. Section 1: We will evaluate the roles of mitochondrial reactive oxygen species (ROS) and permeability transition pore (PTP) as signal transducers triggering the induction of skeletal muscle autophagy in response to catabolic stimuli. To this end, we will use mice over-expressing peroxiredoxin 3 (Trx3+/+)(mitochondrial antioxidant enzyme) and mice deficient in cyclophilin D (Ppif-/-, essential component of mitochondrial PTP). Autophagy in Trx3+/+ and Ppif-/- mice and their littermate wild type mice will be examined along with proteasomal degradation pathways and mitochondrial respiration. Section 2: We will define morphological and biochemical characteristics of autophagy membranes in cultured skeletal muscle cells by using a fluorscent marker for autophagosomes. We will identify autophagy formation using confocal and electron microscopy as well proteomics approaches. Section 3: We will identify the transcriptional factors that regulate autophagy-related genes in skeletal muscles by using bioinformatics tools to analyze promoters of autophagy genes. Activation of predicted transcription factors will be verified in muscle cells using specialized technqiues and their functional importance in autophagy regulation will be verified with siRNA approach.