Structural Studies of Cells with Superresolution Light Microscopy

心肌由肌原纤维中肌节的重复单元组成，肌节沿每个细胞的长度沿着排列。每个肌节长约2米，两端有一个Z盘。 在Z盘中，肌动蛋白丝以反平行组织排列，并通过-辅肌动蛋白交联，使得大约每19-20 nm有一个-辅肌动蛋白从Z盘的一侧移动到另一侧。此外，Z盘包含至少30种不同的蛋白质，这些蛋白质有助于肌肉肌节的整体结构、稳定性和组织以及信号传导。因此，Z盘是肌肉结构和功能的关键组织和信号中心。 Z盘的宽度很窄，大约100 nm，这意味着传统的光学显微镜没有足够的分辨率来定位其中的蛋白质。为了克服这一限制，我们开发了3-D光活化定位显微镜（PALM 1，2）来成像Z盘中的两种特定蛋白质; -辅肌动蛋白2和Lasp-2（LIM和SH 3含有蛋白质2）。- 辅肌动蛋白2形成反平行二聚体，其交联来自相邻肌节的相邻肌动蛋白丝。 Lasp-2结合-辅肌动蛋白2 3。重组DNA技术被用来产生腺病毒表达构建辅肌动蛋白和LASP融合到mEos 2，和纯化的腺病毒被用来表达这些标记的蛋白质在培养的胚胎小鼠或分离的成年大鼠心肌细胞。然后使用4%多聚甲醛将细胞固定10分钟并成像。 在成像之前添加的100 nm金基准点用于生成用于定位的测量点扩散函数2，并用于校正图像采集期间的漂移。将弱柱面透镜插入样本和相机之间的光路中，以从单个2D平面获得3D信息。 所得图像显示Z-圆盘内的mEos 2-β辅肌动蛋白的单个分子，定位精度为20 nm（X，Y）和Z中的50 nm，Z-圆盘的估计宽度为100 nm。分子沿Z圆盘的长度沿着分布不均匀（图10）。1)但显示出由较低密度区域分隔的具有较高密度的β-辅肌动蛋白簇。密度较低的区域可能代表肌原纤维之间的区域。我们还成功地获得了Z-盘中mEos 2-LASP单分子的图像，具有相似的定位精度（XY中为20 nm，Z中为50 nm）。这些分子的密度似乎低于mEos 2-辅肌动蛋白的密度，并且还显示出沿Z-盘的沿着长度的变化。 这些结果表明，PALM可用于定位Z盘内的特定蛋白质，并且两种不同的Z盘蛋白质在该狭窄结构内具有明显不同的分布或密度。 因此，这种方法具有很大的潜力，调查组织内的Z-光盘的组成蛋白质。 从表达GFP标记的肌球蛋白IIA的小鼠骨髓中分离出中性粒细胞。 我们正在使用由Hari Shroff（NIBIB）设计的定制结构照明显微镜（SIM）显微镜对肌球蛋白进行成像，并希望跟踪这种趋化细胞中单个肌球蛋白细丝的聚合和解聚。

Cardiac muscles are made up of repeating units of muscle sarcomeres, arranged end to end, along the length of each cell, in myofibrils. Each sarcomere is about 2 m long and bounded by a Z-disc at each end. In the Z-disc, actin filaments are arranged in an anti-parallel organization and are cross-linked by -actinin such that there is about one -actinin every 19-20 nm moving from one side of the Z-disc to the other. In addition, the Z-disc contains at least 30 different proteins, which contribute to the overall architecture, stability and organization of the muscle sarcomere and to signalling. Thus, the Z-disc is a key organizing and signalling centre for muscle structure and function. The narrow width of the Z-disc, about 100 nm, means that conventional light microscopy does not have enough resolution to localise proteins within it. To overcome this limitation, we have developed the use of 3-D Photoactivated Localisation Microscopy (PALM 1,2) to image two specific proteins within the Z-disc; -actinin 2 and Lasp-2 (LIM and SH3 containing protein 2). -actinin 2 forms an anti-parallel dimer that cross-links adjacent actin filaments from neighbouring sarcomeres. Lasp-2 binds to -actinin 2 3. Recombinant DNA techniques were used to generate adenoviral expression constructs for -actinin and LASP fused to mEos2, and purified adenovirus was used to express these tagged proteins in either cultured embryonic mouse or isolated adult rat cardiomyocytes. Cells were then fixed using 4% Paraformaldehyde for 10 minutes and imaged. 100 nm gold fiducials, added prior to imaging, were used to generate a measured point spread function 2 for localization, and for correcting drift during image acquisition. A weak cylindrical lens was inserted into the light path between the specimen and the camera to obtain 3D information from a single 2D plane. The resulting images show individual molecules of mEos2--actinin within the Z-disc to a localization precision of 20nm (X,Y) and 50nm in Z, with an estimated width for the Z-disc of 100 nm. The molecules are not evenly distributed along the length of the Z-disc (Figure. 1) but show clusters of -actinin with a higher density separated by regions of lower density. Areas of lower density could represent regions between myofibrils. We also successfully obtained images of single molecules of mEos2-LASP within the Z-disc to a similar localization precision (20nm in XY, and 50nm in Z). The density of these molecules appears to be lower than that for mEos2--actinin, and also shows variation along the length of the Z-disc. These results demonstrate that PALM can be used to localise specific proteins within the Z-disc, and that two different Z-disc proteins have an apparently different distribution or density within this narrow structure. This approach therefore has great potential for investigating the organisation of component proteins within the Z-disc. Neutrophils have been isolated from the bone marrow of mice expressing a GFP-tagged myosin IIA. We are imaging myosin with a custom built structure illumination microscopy (SIM) microscope designed by Hari Shroff (NIBIB) and hope to follow the polymerization and depolymerization of single myosin filaments in this chemotactic cell.