The Musser Lab

  at the Texas A&M Health Science Center, Naresh K. Vashisht 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.  For protein translocation systems, our goal is to decipher the rules for the sets of interactions required to migrate through a conduit (pore) within a lipid bilayer.  For condensates, our goal is identify changes the physical properties and sub-structural elements that are present in distinct phase states.  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

    Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells (Fig. 1).

NPC Import and Export

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

  The 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.  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 unresolved.  At steady-state, up to ~100 NTRs are asymmetrically distributed throughout the FG-network.  The heterogeneous and dynamic NTR/FG-network establishes a permeability barrier while simultaneously providing pathways for the translocation of import and export complexes of a wide range of sizes, affinities and surface properties.  A key question is how the massive level of bi-directional traffic occurs concurrently and efficiently (i.e., without getting clogged). Multiple preferred paths through the ~60 nm diameter pore are 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.  We have recently constructed a high-resolution 3D multi-color microscope that generates PALM/STORM-type pointillistic images using astigmatism imaging and that can be used for millisecond-scale 3D tracking.  This instrument is being used to probe the structure and dynamics of the permeability barrier, 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

    The Tat transport system is found in the cytoplasmic membrane of most bacteria, and is mechanistically unique:  it transports folded proteins (up to ~6 nm diameter) across an energized membrane without causing the collapse of ion gradients.  The bacterial Tat machinery is a potential antibiotic target due to the absence of a homologous system in humans and animals and because it has important roles in virulence, cell division, cell wall integrity, and motility.  Biochemical and biophysical studies have provided a wealth of information about Tat signal peptides, the protein cargos, the oligomerization of protein components of the transport system, and the energetic requirements of transport.  However, the specific molecular interactions and conformational changes/rearrangements required for successful Tat protein translocation remain unknown or largely unsettled. Deciphering the Tat transport mechanism with molecular-scale biochemical and biophysical detail has been challenging, primarily for two reasons.  First, the oligomerization state of the Tat proteins depends on the transmembrane proton motive force (pmf), which explicitly requires a membrane environment and which is only transiently maintained without continuous energy input.  Thus, while it is considered well-established that a core receptor complex consisting of TatB and TatC in Escherichia coli recruits TatA to form the active translocon in the presence of a pmf, the structural organization and composition of the active translocon structure remains unknown.  And second, the inability to definitively link outcomes to discrete molecular species and interactions is a frustrating consequence of numerous ensemble studies to date.  To address these challenges, we are taking advantage of a single vesicle fluorescence microscopy approach to determine fundamental structural and functional properties of the Tat transport system.  By using inverted membrane vesicles (IMVs) as individually addressable reaction chambers, we are identifying the minimal number of TatA molecules required for transport, determining the magnitude of the electric field component of the pmf required for transport, and characterizing kinetic transitions between various intermediates during transport.    


FUS Condensates

    Biomolecular condensates (BMCs) are typically highly heterogeneous and can contain hundreds of different types of proteins and RNAs.  These complex mixtures can consist of different phase states and sub-compartments each with distinct physical properties. Solidified aggregates containing proteins that initially phase separate into liquid assemblies are hallmarks of devastating neurological diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson’s and Alzheimer’s diseases.  The transformation from liquid condensate to solid aggregate is termed phase maturation.  Characterization of the physical, structural, dynamical, and functional properties of cellular coacervates and other crowded environments remains challenging due to the dearth of appropriate tools to investigate their complexity and heterogeneity on the nanoscale.  We are using a multitude of single molecule fluorescence (SMF) approaches to characterize the properties of condensates containing the fused in sarcoma (FUS) protein.  A novel approach that we have developed, single molecule rotational diffusion microscopy (SiMRoD; formerly termed polarization-PALM, or p-PALM) measures rotational diffusion, which is expected to decrease during formation of solid aggregates.  3D astigmatism imaging microscopy provides structural and dynamic information on the millisecond timescale at high spatial resolution.  A key strategy for obtaining thousands of individual measurements within single coacervates is the use of spontaneously blinking dyes.  We find that even simple coacervates exhibit complex biophysical readouts indicating heterogeneity and/or sub-structures.