deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (tirfm)解构的后期阶段波形蛋白装配的全内反射荧光显微镜(tirfm).pdfVIP

Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM) 1 1¤ 1 1 2 Stefan Winheim , Aaron R. Hieb , Marleen Silbermann , Eva-Maria Surmann , Tatjana Wedig , Harald 2 ¨ 1 ¨ 1 Herrmann , Jorg Langowski , Norbert Mucke * 1 Division Biophysics of Macromolecules, German Cancer Research Center, Heidelberg, Germany, 2 Functional Architecture of the Cell, German Cancer Research Center, Heidelberg, Germany Abstract Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several mm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed