Cobb, Garrett

Coraor, Juliana

Elbaum-Garfinkle, Shana

Melo, Ana M.

Rhoades, Elizabeth

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448a Tuesday, February 10, 2015Microtubules, Structure Dynamics, and
Associated Proteins
2250-Pos Board B387
Transformation of Taxol-Stabilized Microtubules into Inverted Tubulin
Tubules Triggered by a Tubulin Conformation Switch
Chaeyeon Song1, Miguel A. Ojeda-Lopez2, Daniel J. Needleman3,
Avi Ginsburg4, Phillip A. Kohl1, Youli Li1, Herbert P. Miller1,
Leslie Wilson1, Uri Raviv4, Myung Chul Choi5, Cyrus R. Safinya1.
1UCSB, Santa Barbara, CA, USA, 2Universidad Autonoma de San Luis
Potosi, San Luis Potosi, Mexico, 3Harvard University, Cambridge, MA, USA,
4The Hebrew University of Jerusalem, Jerusalem, Israel, 5KAIST, Daejeon,
Korea, Republic of.
Bundles of taxol-stabilized microtubules (MTs) - hollow protein nanotubes
comprised of assembled ab-tubulin heterodimers - spontaneously assemble
above a critical concentration of tetravalent spermine and are stable over
long times at room temperature. Here we report that at concentrations of sper-
mine several-fold higher the MT bundles (BMT) quickly become unstable and
undergo a shape transformation to bundles of inverted tubulin tubules (BITT),
the outside surface of which corresponds to the inner surface of the BMT tu-
bules. Using transmission electron microscopy and synchrotron small-angle
X-ray scattering, we quantitatively determined both the nature of the BMT-to-
BITT transformation pathway and the structure of the BITT phase. Inverted
tubulin tubules provide a platform for studies requiring exposure and availabil-
ity of the inside, luminal surface of MTs to MT-targeted drugs and MT-
associated proteins. (ref. Nature Materials 2014, 13, 195-203)
2251-Pos Board B388
Single-Molecule FRET Reveals an Extended Structure of Tau Bound to
Tubulin Heterodimers
Ana M. Melo1, Garrett Cobb1, Juliana Coraor1, Shana Elbaum-Garfinkle2,
Elizabeth Rhoades1.
1Molecular Biophysics and Biochemistry,Yale University, New Haven, CT,
USA, 2Chemical and Bioengineering, Princeton University, Princeton, NJ,
USA.
Tau is an abundant neuronal protein that promotes tubulin polymerization
and stabilizes microtubules (MTs) in the axons of neurons. The disruption
of its normal biological function as well as its aggregation and deposition
into neurofibrillary tangles has been proposed to contribute to several neuro-
degenerative disorders known as tauopathies, including Alzheimer’s disease.
Although tau´s interaction with MTs has been extensively studied, a detailed
structural characterization of the complex is lacking. This deficit is, in part,
due to the fact that tau is intrinsically disordered in solution and remains
largely so upon binding to MTs, making its structural characterization chal-
lenging. Here, we identified conformational changes in tau underlying its
binding to tubulin. To do so, we used intramolecular single-molecule Fo¨rster
Resonance Energy Transfer (smFRET) measurements of the full-length tau
protein upon binding to soluble tubulin heterodimers under non-assembly
conditions. Tau was labeled with donor and acceptor fluorophores at multiple
pairs of positions to cover all domains of interest. We observed that tau un-
dergoes significant structural rearrangements when binding to tubulin. Spe-
cifically, it results in a loss of long-range contacts between the two
termini and the microtubule binding region (MTBR) that characterizes tau´s
compact solution conformation, leading to an overall expansion of its struc-
ture. Moreover, we contrast the structures of two isoforms of tau which
differ in the number of MTBR (2N3R and 2N4R). From this work, we pro-
pose a topological model of the structure of tau bound to tubulin, which pro-
vides insight to the molecular mechanism of tau-mediated tubulin
polymerization into MTs.
2252-Pos Board B389
Statistical Mechanics Provides Novel Insights into Microtubule Stability
and Mechanism of Shrinkage
Ishutesh Jain1, Mandar M. Inamdar2, Ranjith Padinhateeri1.
1Biosciences and Bioengineering, Indian Institute of Technology Bombay,
Mumbai, India, 2Civil Engineering, Indian Institute of Technology Bombay,
Mumbai, India.
Microtubules are nano-machines that grow and shrink stochastically making
use of the coupling between chemical kinetics and mechanics of its constitu-
ent protofilaments (PFs). Understanding this coupling is crucial for under-
standing the stability and dynamics of the tubulin assembly, and for
predicting multi-scale effects induced by a number of regulating agents. In
this work we investigate the stability and shrinkage of microtubules taking
into account inter-protofilament interactions and bending interactions ofintrinsically curved PFs. Computing the free energy as a function of PF tip
position, we show that the competition between curvature energy, inter-PF
interaction energy and entropy leads to a rich landscape with a series of
minima that repeat over a length-scale determined by the intrinsic curvature.
Computing Langevin dynamics of the tip through the landscape and account-
ing for depolymerization, we calculate the average unzippering and shrinkage
velocities of GDP protofilaments and compare them with the experimentally
known results. Our analysis predicts that the strength of the inter-PF interac-
tion (Esm) has to be comparable to the strength of the curvature energy (E
b
m)
such that Esm -E
b
m z1kBT, and questions the prevalent notion that unzipper-
ing results from the domination of bending energy of curved GDP PFs. Our
work demonstrates how the shape of the free energy landscape is crucial in
explaining the mechanism of MT shrinkage where the unzippered PFs will
fluctuate in a set of partially peeled off states and subunit dissociation will
reduce the length.
2253-Pos Board B390
Molecular Dynamics Study of the Effect of Polyamine on Microtubule
Conformations
Chola Regmi, Shengfeng Cheng.
Physics, Virginia Tech, Blacksburg, VA, USA.
Microtubules (MTs) play important roles in cell division, intracellular protein
transport, and cell structure and movement in eukaryotic cells. MTs are
hollow supramolecular polymers made of alpha and beta tubulin hetero-
dimers. Microtubule assembly in cells require a proper orientation of tubulin
molecules to the MTs end. Experiments have shown that polyamines such as
spermine can facilitate the diffusion of tubulin dimers in the process of
nuclei growth or MT growth. The mechanism of this aggregation is the
bridging interaction of polyamines with highly negative C-terminal tail of
tubulin surface which helps tubulin together. Recent experiments also
show that with a high concentration of spermine, bundles of taxol-
stabilized MTs undergo a shape transformation to bundles of inverted tubulin
tubules. However, the molecular mechanism underlying the transformation is
unclear. We performed all atom molecular dynamics simulation of tubulin
clusters in presence of spermine in an explicit water solvent to study the
structural dynamics of alpha/beta tubulin and microtubule protofilament.
We discuss our results on the interaction between spermine and tubulin pro-
teins and the effect of spermine on conformational dynamics of MT
protofilaments.
2254-Pos Board B391
Molecular Basis for Age-Dependent Microtubule Acetylation
Antonina Roll-Mecak1, Agnieszka Szyk1, Alexandra Deaconescu2,
Jeffrey Spector1, Max Valenstein1, Niko Grigorieff3.
1National Institutes of Health, Bethesda, MD, USA, 2Brown University,
Providence, RI, USA, 3HHMI Janelia Farm, Ashburn, VA, USA.
Microtubules are decorated with chemically diverse and evolutionarily
conserved posttranslational modifications that mark them for specialized func-
tions in cells. Other than viruses and clathrin cages, microtubules are the only
known hollow polymers in eukaryotic cells. Acetylation on alpha-tubulin
Lys40 stands out as the only known tubulin posttranslational modification
located in the microtubule lumen. It marks stable, long-lived microtubules
and is required for polarity establishment and directional migration. Tubulin
acetyltransferase (TAT) presents a puzzle: its activity is stimulated by micro-
tubules, yet the luminal location of Lys40 hinders its access. TAT was pro-
posed to access its substrate by diffusing from microtubule ends, engaging
the Lys40 loop from the exterior of the microtubule or gaining access through
openings or defects in the microtubule. To elucidate substrate access by TAT
and uncover the molecular basis for the in vivo correlation between acetyla-
tion and microtubule age, we combined X-ray crystallography, electron mi-
croscopy, biochemical and single-molecule fluorescence analyses, and
modeling. We report the first crystal structure of TAT in complex with a bi-
substrate analog that together with structure-based functional analyses con-
strains the active enzyme to the microtubule lumen and reveal the structural
determinants for Lys40 loop recognition. Unexpectedly, acetylation by TAT
proceeds stochastically along the microtubule without a preference for ends
despite the luminal location of the acetylation site. Single-molecule fluores-
cence imaging reveals that TAT efficiently and stochastically scans the micro-
tubule, while modeling and kinetic analyses demonstrate that TAT catalytic
activity, rather than diffusion through the lumen, is rate-limiting. Consistent
with this, our 1.35A˚ X-ray complex crystal structure reveals that the TAT
active site is suboptimal for supporting the proton transfer required for acet-
ylation. Thus, by virtue of its preference for the microtubule and its low cat-
alytic rate, TAT alone can function as an enzymatic timer for microtubule
lifetimes.


Single-Molecule FRET Reveals an Extended Structure of Tau Bound to Tubulin Heterodimers 

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Single-Molecule FRET Reveals an Extended Structure of Tau Bound to Tubulin Heterodimers

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