UC Berkeley

Fluorescence microscopy has allowed for decades of elegant and compelling biological discovery. However, the diffraction limit of light caps the spatial resolution of fluorescence microscopy to 250-300 nanometers. As the size of an average protein molecule is ~ 3 nm, diffraction-limited spatial resolution is often more than an order of magnitude larger than what is required to resolve nanoscale cellular structures and processes. With the advent of single-molecule localization microscopy (SMLM) and super-resolution microscopy (SRM), the spatial resolution of fluorescence microscopy has been improved more than 10-fold relative to conventional methods. Further, the fundamental principles of SMLM have engendered multiple other experimental methods to simultaneously probe a second informational domain, in addition to precise spatial information. For example, much of the work in this writing utilizes a functional SRM method known as single-molecule diffusivity mapping (SMdM), which will be overviewed in Chapter 1.3. We use SMdM to show that in the mammalian cell, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing vimentin constructs with differently charged linkers, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges are less screened or otherwise intensified. Additionally, we identify a key molecular player, DELE1, in relaying mitochondrial stress to the cytosol and triggering the integrated stress response. Then, using SMdM we corroborate the aforementioned finding and show that the intraorganellar diffusivity of both DELE1 and cytochrome c implies the presence of unique electrostatic interactions in the mitochondrial intermembrane space. Together, these studies represent some of the first applications of SMdM to study native cellular proteins rather than exogenous tracer proteins.