The Musser Lab

  at the Texas A&M Health Science Center, College of Medicine



Overview

    We are primarily a biophysics lab that focuses on deciphering molecular mechanisms and dynamics via single molecule fluorescence (SMF) microscopy and particle tracking approaches.  Most diffusing molecules move hundreds of nanometers in milliseconds, even in the complex milieu of a cell.  Thus, the partitioning of proteins into various compartments is limited primarily by their interactions with encountered molecules rather than via inherent diffusive limits on molecular motion.  Our goal, therefore, is to decipher the rules for the sets of interactions required to migrate through a conduit (pore) within a lipid bilayer or between liquid phases.  The current spatial precision of SMF approaches is in the single digit nanometer range at ~1 kHz (millisecond time resolution), providing a powerful tool to address both structural as well as dynamical questions.

Projects

The Human Nuclear Pore Complex

NPC Import and Export

Figure 1.  Canonical Nuclear Protein Import and Export Pathways. Reproduced from Tu and Musser.

FG-Network

Figure 2.  Cartoon of the FG-Network. Reproduced from Tu et al.

    Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells (Fig. 1).  The ~50 nm diameter central channel of the NPC is occupied by a network of intrinsically disordered polypeptides containing phenylalanine-glycine (FG) repeat motifs, and hence is termed the FG-network (Fig. 2).  Soluble nuclear transport receptors (NTRs) bind to the FG motifs and carry cargos through the NPC by diffusion.  The precise mechanism by which NTRs allow cargos to overcome and migrate through the permeability barrier is unknown and hotly debated.  At steady-state, at least ~100 NTRs are asymmetrically distributed throughout the FG-network.  The heterogeneous and dynamic NTR/FG-network establishes a permeability barrier while simultaneously allowing for the translocation of import and export complexes of a wide range of sizes, affinities and surface properties.  With ~20 NTRs and opposing transport directions, multiple preferred paths through the ~50 nm diameter pore are both possible and likely.  The extent of pathway overlap and the possibility of dynamic regulation remains largely unexplored due to the absence of technological tools to dissect these pathways with the necessary spatial and temporal resolution.  This is the focus of current projects.
    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

NPC Import and Export

Figure 3.  Hairpin-Hinge Model of Tat Protein Transport.   Adapted from Hamsanathan et al.

    The twin-arginine translocation (Tat) protein transport system transports fully-folded and assembled protein complexes across the bacterial cytoplasmic membrane and the thylakoid membrane of plants. The Tat system is responsible for the export of numerous proteins important for bacterial virulence in humans, and its absence often leads to growth defects.  Since animals, including humans, do not contain homologues of Tat machinery proteins, inhibitors of Tat transport could potentially find use as novel antibiotics.
    The Tat system translocates proteins across membranes that maintain a proton motive force (pmf).  Consequently, the passage of large folded protein domains (> 100 kDa) is presumed to occur quickly or via a tightly sealed or gated channel.  Escherichia coli Tat transport requires a TatBC receptor complex, and TatA, which oligomerizes with the TatBC-substrate complex to form the translocation channel.  Importantly, the structural and oligomeric nature of the translocation pore and how leakage is prevented remain major unsolved problems.  Characterization of the translocation pore is particularly challenging because it disassembles in the absence of a pmf.
   
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

Phase
                  Separation

Figure 4.  Liquid-liquid Phase Separation (LLPS). Reproduced from Brangwynne.

FUS
                  Droplets

Figure 5.  Maturation of FUS-GFP Droplets. Reproduced from Alberti and Hyman.

    Membrane-less organelles are liquid-like compartments within cells typically characterized by high concentrations of intrinsically disordered proteins (IDPs) and RNA molecules. At least two dozen membrane-less compartments have been identified - examples include nucleoli, stress granules, and P bodies.  These structures are currently thought to arise from liquid-liquid phase separation (LLPS).  LLPS is the spontaneous partitioning into dense and dilute phases due to favorable interactions between the separating molecules. 
    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.