Reverse structural biology: Cellular processes uncovered by electron microscopy

Abstract: This thesis contains two projects, both using electron microscopy as the primary technique. In the first project, nuclear envelope budding (NEB) is investigated as an alternate method of transport across the nuclear envelope. We show that NEB is a conserved aspect of eukaryotic cells grown under normal conditions, and that it is upregulated during various types of cellular stress. In the second part of the thesis, we investigate novel aspects of human flagellar structure using cryo-electron microscopy and tomography. We show that in the tip of human flagella, doublet microtubules can split into two complete singlet microtubules. These singlet MTs contain a helical structure called TAILS, which we hypothesize has a stabilizing effect on the microtubules. Using single particle analysis, we are attempting to identify the proteins that form TAILS by identifying protein domains in the structure. We also reconstructed doublet microtubules from near the tip region, and describe novel microtubule inner proteins, notably large bundles of filaments in the A-tubule.