Regulation of Subcellular Organization in Skeletal Muscle

在过去的一年中，我们已经成功地表达了编码与GFP连接的几种靶蛋白的cDNA到骨骼肌中。我们使用小鼠足垫的肌肉，指短屈肌（FDB）进行这些实验。FDB很容易获得; DNA可以被注射和电穿孔到肌肉中，而不需要打开皮肤。我们已经测试了编码形成微管核心的蛋白质（微管蛋白-GFP）、与微管相关的蛋白质（MAP 4-GFP，ensorimetric微管结合位点-GFP）和参与微管正末端尖端的蛋白质（EB 1-和EB 3-GFP）的构建体。我们已经能够获得大量的纤维表达的结构，更好的是，几个结构复制的内源性微管的模式。我们已经观察到，在肌细胞培养物中用于跟踪微管的构建体不一定在肌纤维中工作良好，反之亦然。 作为第一种方法，我们采用了cDNA注射后一周通过胶原酶解离肌肉获得的FDB的单纤维。将纤维涂布在适合于共聚焦显微镜的室上，并在涂布后24小时内观察。 在胶原酶分解肌肉后，对铺平板的单个FDB纤维进行延时记录，揭示了肌纤维微管网络的双重特征。有一个稳定的晶格，由不同极性的微管束组成，形成横向和纵向路径。沿着这个网络，单个的微管不断地移动，通常在一个方向上。微管开始倾斜运动时，通常会改变方向，当它们碰到一条晶格线，然后沿着这条线运动。我们提出与肌营养不良蛋白的相互作用（由Prins等人，2009）负责微管与晶格的对齐。肌营养不良形成横肋，肌纤维的中肋。在缺乏抗肌萎缩蛋白的mdx小鼠中，微管不形成正交晶格。这种稳定的微管晶格通过肌营养不良蛋白与肌纤维的其他细胞骨架系统相关联，可以为细胞提供结构支持。相反，沿着晶格沿着运动的非常动态的微管可能在蛋白质和细胞器的运输中发挥微管的传统作用。 我们已经观察到从诺考达唑处理中恢复期间和稳态时微管的成核，这只能在活纤维上观察到。成核通过形成短微管的星状体来表现，其类似于在增殖细胞如成肌细胞中观察到的那些。然而，成肌细胞中的成核从中心体开始，而肌纤维中的成核从多个高尔基复合体（高尔基元件）开始。肌纤维中的成核也与核膜相关，如肌管中的情况。与高尔基复合体的关联正在通过表达cDNA以敲除高尔基复合体蛋白来进一步测试，假设高尔基复合体蛋白参与该过程。 这种机制使肌纤维对微管组装具有非常局部的控制，并且似乎结合了成肌细胞和肌管中发现的成核机制，这是肌肉特有的“管理”系统，并且可能很好地适应了肌肉单个细胞的巨大尺寸。 我们对应用显微镜技术检测和分析肌病中肌肉缺陷的兴趣，加上我们与拉本博士在庞贝氏症方面的合作，使我们开发了用于定量肌肉异常的新软件。各种形式的显微镜检查揭示肌肉缺陷，如收缩蛋白的周期性变化，包含非收缩体等，然而，很难定量这些缺陷，因此，比较不同活检之间的疾病严重程度或肌肉健康的演变治疗，如酶替代疗法的情况下庞贝氏症。基于Matlab平台的新软件使用不同的滤波器和图像“纹理”的概念来量化肌肉缺陷，而无需事先假设缺陷的类型。我们正在测试其耐用性，并将其应用于庞贝氏症患者的样本。

During the past year we have successfully expressed cDNAs encoding several target proteins linked to GFP into skeletal muscles. We have used the muscle of the mouse footpad, the Flexor Digitorum Brevis (FDB) for these experiments. The FDB is easy to access; DNA can be injected and electroporated into the muscle without need to open the skin. We have tested constructs encoding proteins that form the core of the microtubules (tubulin-GFP), proteins associated with microtubules (MAP4-GFP, ensconsin microtubule-binding site-GFP) and proteins involved in the plus-end tip of the microtubules (EB1- and EB3-GFP). We have been able to obtain good numbers of fibers expressing the constructs and, better, several of the constructs reproduce the pattern of the endogenous microtubules. We have observed that constructs that are useful for tracking microtubules in muscle cell cultures are not necessarily working well in muscle fibers, and vice versa. As a first approach, we have taken single fibers of the FDB obtained by collagenase dissociation of the muscle one week after cDNA injection. The fibers are plated on a chamber suitable for confocal microscopy and observed within 24h of plating. Time-lapse recordings of single FDB fibers plated after collagenase dissociation of the muscle reveal the dual character to the microtubule network of muscle fibers. There is a stable lattice, composed of bundles of microtubules of different polarities, forming both transverse and longitudinal paths. Along this network, individual microtubules move constantly, generally in one direction. Microtubules starting their movement obliquely generally shift direction when they hit one of the lattice lines and then follow the line. We propose that the interaction with dystrophin (demonstrated by Prins et al., 2009) is responsible for the alignment of microtubules with the lattice. Dystrophin forms transverse ribs, the costameres of muscle fibers. In the mdx mouse which lacks dystrophin, microtubules do not form an orthogonal lattice. This stable microtubule lattice which, through dystrophin, would be associated with the other cytoskeletal systems of muscle fibers, may provide structural support to the cell. The very dynamic microtubules that course along the lattice may, in contrast, play the traditional roles of microtubules in protein and organelle transport. We have observed nucleation of microtubules during recovery from nocodazole treatment, and at steady-state, which could only be observed on live fibers. Nucleation is manifested by the formation of asters of short microtubules which resemble those observed in proliferating cells such as myoblasts. However, whereas nucleation in myoblasts starts from the centrosome, in muscle fibers nucleation starts from the multiple Golgi complexes (Golgi elements). Nucleation in muscle fibers is also associated with the nuclear membrane, as is the case in myotubes. The association with the Golgi complex is being further tested by expressing cDNAs to knock down Golgi complex proteins hypothesized to be involved in the process. This mechanism gives muscle fibers a very local control of microtubule assembly and appears to conjugate the nucleation mechanisms found in myoblasts and in myotubes, a "management" system unique to muscle and likely well adapted to the huge size of the individual cells of muscle. Our interest in applying microscopy techniques for the detection and analysis of muscle defects in myopathies, coupled with our collaboration with Dr. Raben on Pompe disease has led us into developing new software for the quantitation of muscle anomalies. Various forms of microscopy reveal muscle defects such as changes in the periodicity of the contractile proteins, inclusion of non-contractile bodies etc. It is however very difficult to quantitate these defects and therefore to compare the severity of disease between different biopsies or the evolution of muscle health following treatments such as enzyme replacement therapy in the case of Pompe disease. The new software based on the Matlab platform uses different filters and the concept of image "texture" to quantitate muscle defects without prior assumption of the type of defects. We are in the process of testing its ruggedness and applying it to samples from Pompe patients.