The Human Nuclear Pore Complex
Figure 1. Canonical Nuclear Protein Import and Export Pathways. Reproduced from Tu and Musser.
Figure 2. Cartoon of the FG-Network. Reproduced from Tu et al.
We typically use narrow-field epifluorescence microscopy to image single molecules actively diffusing and interacting with NPCs in permeabilized cells (Pub16, Pub18, Pub19, Pub22, Pub29, and Pub30). We have recently established photoactivated localization microscopy (PALM) in the lab, and developed polarization PALM (p-PALM), for measuring rotational mobility (Pub33). We are currently building a high-resolution 3D multicolor PALM/p-PALM microscope, which will be used to probe the dynamics of the permeability barrier itself, as well as for determining the paths of particles traveling through it. This work will establish whether discrete transport pathways within the FG-network exist, and thus, whether transport efficiency is enhanced by minimizing interactions between import and export cargos. While the primary goal of the proposed work is to develop a comprehensive understanding of competing transport reactions and the potential implications for inhibition and regulation, the super-resolution microscopy technologies and algorithms developed are expected to comprise a necessary toolkit for the field as well as to have broad applicability.
The Escherichia coli Tat Machinery
Figure 3. Hairpin-Hinge Model of Tat Protein Transport. Adapted from Hamsanathan et al.
The goal of our project is to decipher the mechanism of Tat protein transport. Since the signal peptide is the part of the precursor protein that directly interacts with Tat machinery, we have probed the nature of signal peptide interactions with the receptor complex and the time evolution of signal peptide interactions (Pub34). We have established that the Tat machinery catalyzes insertion of a signal peptide hairpin into the membrane in an energy-independent manner, and that full translocation of the C-terminal end of the signal peptide requires a proton motive force. We postulate that this occurs by unhinging of the signal peptide hairpin (Hairpin-Hinge Model; Fig. 3). Our results indicate that the signal peptide's binding interactions and its membrane translocation are critical for directly promoting mature domain transport. We are currently developing single vesicle and single molecule fluorescence approaches to examine structural and dynamic properties of the Tat machinery, and to test hypotheses generated by our Hairpin-Hinge Model of transport.
FUS Liquid Droplet Formation
Figure 4. Liquid-liquid Phase Separation (LLPS). Reproduced from Brangwynne.
Figure 5. Maturation of FUS-GFP Droplets. Reproduced from Alberti and Hyman.
FUS (fused in sarcoma), a major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a nuclear DNA/RNA binding protein that regulates transcription, splicing, and mRNA transport. Arginine methylation defects and disease-causing mutations in FUS result in its accumulation in cytosolic membrane-less compartments called stress granules. In vitro, FUS undergoes LLPS forming spherical droplets, which convert into solid, fibrous aggregates over time. The goal of this project is to characterize the physicochemical properties of FUS droplets as a model for membrane-less organelles.
In vitro models suggest that LLPS between aqueous monomers and liquid-like droplets containing dense IDP assemblies occurs rapidly and reversibly, and is sensitive to ionic strength, temperature, and the charge state and concentrations of the component molecules (Fig. 4). LLPS readily occurs in vitro with purified components, and, at long incubation times, amyloid-like fibers form indicating conversion to a solid state (Fig. 5). The physicochemical properties required to form stable droplets are poorly understood, and novel tools are required to investigate the structural and functional properties of these liquid-like compartments. For our studies on the nuclear pore complex (NPC), whose IDP-rich permeability barrier bears similarities to membrane-less organelles, we developed polarization PALM (p-PALM) (Pub33), which is sensitive to the rotational mobility of the probe molecule, and thus sensitive to macromolecular crowding. Using p-PALM combined with the super-resolution technique PALM and particle tracking approaches, we will investigate droplets formed from the fused in sarcoma (FUS) protein by characterizing the mobility properties of individual probe-labeled FUS molecules within phases and transitioning between them.