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
Projects
The Human Nuclear Pore Complex
Nuclear pore complexes (NPCs) mediate cargo traffic between
the nucleus and the cytoplasm of eukaryotic cells (Fig. 1).
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
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.
Figure 1. Canonical Nuclear Protein Import and Export Pathways. Reproduced from Tu and Musser.
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.