25.9: Studying the Cytoskeleton
The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary shadowing or metal replica, the cytoskeletal filaments are fixed using chemicals such as glutaraldehyde or formaldehyde, followed by dehydration of the samples using alcohol to a critical point of drying and then embedded into a resin, followed by thin sectioning. The sample is positively or negatively stained with heavy metal salts such as platinum and observed under the electron microscope.
However, electron microscopy techniques only produce static images and cannot help in studying the dynamic structure of the cytoskeletal filaments and their functions. A better alternative to understanding the cytoskeleton's structure and function is to study it using fluorescence microscopy. This technique has helped researchers analyze the molecular processes of living cells in real-time through an approach known as live-cell imaging. A commonly used protocol for live-cell imaging involves synthesizing fluorescently labeled proteins within a cell as fusion proteins with GFP or green fluorescent protein. Alternatively, protein subunits of cytoskeletal structures such as purified actin or tubulin can be covalent to a small fluorescent dye in vitro and injected into a living cell. These fusion or tagged proteins are then observed under a fluorescent microscope to study their dynamic behavior in the cell.
