MPI Campus Seminar: 3D architecture of the Nuclear Pore Complex visualized with light
MPI Campus Seminar
- Datum: 27.11.2019
- Uhrzeit: 12:00 - 13:00
- Vortragende(r): Mark Bates
- Department of NanoBiophotonics
- Ort: Max-Planck-Institut für biophysikalische Chemie (MPIBPC)
- Raum: Großer Semiarraum
- Gastgeber: S. Glöggler, A. Godec, A. Faesen, J. Liepe, S. Meek, A. Stein, M. Wilczek, S. Karpitschka, D. Zwicker
- Kontakt: stefan.gloeggler@mpibpc.mpg.de
3D architecture of the Nuclear Pore Complex visualized with light
Super-resolution fluorescence microscopy methods are capable of seeing biological structure at the nanometer scale, two orders of magnitude smaller than what is possible with a confocal microscope. Fluorescence images also have the advantage that only specific components of the cell may be labeled, making it easy to identify particular proteins. In my talk I will present the development of a microscope designed to achieve a 3D spatial resolution of 10 nanometers, in order to directly observe the sub-structure of macromolecular complexes in the cell. At this level of detail, it becomes possible to visualize the organization of large protein complexes, and we have applied this approach to study Nuclear Pore Complex (NPC) architecture. The combination of genetically engineered cell lines, small fluorescent tags, super-resolution 3D imaging, and computational particle alignment methods, have enabled us to obtain multicolor reconstructions of NPCs which may be compared with cryo-electron microscopy data. Our approach allows for the alignment and visualization of an arbitrary number of distinct structural elements in the NPC, and these results represent the first example of multicolor 3D light microscopy of a single protein complex at 10 nm resolution. Furthermore, as the number of aligned particles increases, individual fluorescent tags within the NPC may be localized with arbitrarily high precision. Finally, the specificity and high contrast of the fluorescence data reveals new insights into NPC structure which are not accessible by other methods, and our results suggest the existence of novel dynamic modes of the NPC which have not previously been observed.
Super-resolution fluorescence microscopy methods are capable of seeing biological structure at the nanometer scale, two orders of magnitude smaller than what is possible with a confocal microscope. Fluorescence images also have the advantage that only specific components of the cell may be labeled, making it easy to identify particular proteins. In my talk I will present the development of a microscope designed to achieve a 3D spatial resolution of 10 nanometers, in order to directly observe the sub-structure of macromolecular complexes in the cell. At this level of detail, it becomes possible to visualize the organization of large protein complexes, and we have applied this approach to study Nuclear Pore Complex (NPC) architecture. The combination of genetically engineered cell lines, small fluorescent tags, super-resolution 3D imaging, and computational particle alignment methods, have enabled us to obtain multicolor reconstructions of NPCs which may be compared with cryo-electron microscopy data. Our approach allows for the alignment and visualization of an arbitrary number of distinct structural elements in the NPC, and these results represent the first example of multicolor 3D light microscopy of a single protein complex at 10 nm resolution. Furthermore, as the number of aligned particles increases, individual fluorescent tags within the NPC may be localized with arbitrarily high precision. Finally, the specificity and high contrast of the fluorescence data reveals new insights into NPC structure which are not accessible by other methods, and our results suggest the existence of novel dynamic modes of the NPC which have not previously been observed.