title: 4D imaging of protein aggregation in live cells.
abstract: One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances. The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state, target them for degradation by the ubiquitin-proteasome system, or direct them to protective aggregation inclusions. In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment. Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure. We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein (Ubc9(ts)) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9(ts) is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 ° C, or when the cell faces misfolding stress, Ubc9(ts) misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem.
authors.forename: Rachel
authors.initials: R
authors.lastname: Spokoini
authors.forename: Maya
authors.initials: M
authors.lastname: Shamir
authors.forename: Alma
authors.initials: A
authors.lastname: Keness
authors.forename: Daniel
authors.initials: D
authors.lastname: Kaganovich
mesh_terms: D005656:Fungal Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry; D015003:Yeasts / Q000737:chemistry / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9