Bourniquel, Aude

Fisher, Jay

Kleckner, Nancy E.

Prentiss, Mara

Witz, Guillaume

Elsevier - Publisher Connector 

Organization and Dynamics of the Living E. Coli Nucleoid at High Resolution in Space and Time 

16a Sunday, February 26, 2012to wildtype cells. Moreover, the distinct effect of condensin I and condensin II is
also discussed. Our studies provide a quantitative analysis of the effect of con-
densin on mitotic chromosome condensation.
79-Plat
Organization and Dynamics of the Living E. Coli Nucleoid at High
Resolution in Space and Time
Jay Fisher, Aude Bourniquel, Guillaume Witz, Mara Prentiss,
Nancy E. Kleckner.
Harvard University, Cambridge, MA, USA.
Living fluorescently-labeled E.coli nucleoids were imaged at ~75nm and>2sec
resolution. Non-replicating single-chromosome nucleoids are fat helicoidal el-
lipsoids. They are denser centrally than peripherally and have a strong intrinsic
tendency to split longitudinally into two, sometimes more, subellipsoids. Nu-
cleoid density distributions and splitting patterns fluctuate on <5s time scales,
dynamically modulating morphology. Analogous helicoids occur throughout
the cell cycle. Thus: the E.coli nucleoid is physically coherent object and can-
not be modeled as a randomly ordered entropic polymer. Diverse evidences
suggest that the nucleoid is stiff, longitudinally and radially, and is trapped
within the cell cylinder by radial confinement. We propose that: (i) the nucleoid
self-assembles into longitudinal bundles via association of short negatively-
supercoiled plectoneme segments; (ii) bundling is opposed by inter-segment re-
pulsion; and (iii) the shape and mechanical properties of bundles underlie the
shape and mechanical properties of the helicoid. Nucleoid shapes and bundle
patterns were examined throughout the cell cycle. Replication initiates in the
helicoid groove towards its old-pole end. Early in replication, straight fingers
of newly-synthesized material dynamically protrude and retract, with protru-
sions curving around the cell periphery. Overall, replication-linked sister seg-
regation is seen to comprise three analogous cycles. In each cycle, a single
fat helicoid develop. Then, concomitant with loss of programmed inter-sister
snaps, the shape evolves into two thinner spatially-separated helicoids. We pro-
pose that genomic contiguity biases longitudinal bundling to give individual-
ized sister shapes (‘‘sister individualization’’) and that sisters are driven into
an end-to-end relationship, suitable for clean segregation, by the mechanical
stress of radial confinement.This scenario explains chromosome dynamics in
diverse bacteria. Conversely, bacteria may occur as rods, spirals and spheres
because these are the three shapes that promote clean symmetrical sister sepa-
ration. Implications for eukaryotic chromosomes can be envisioned.
80-Plat
Mechanical Stress on Double Stranded DNADrives Homology Recognition
Efraim Feinstein, Claudia Danilowicz, Mara Prentiss.
Harvard University, Cambridge, MA, USA.
RecA performs a homology search that compares a single stranded DNA
(ssDNA) to a double stranded DNA (dsDNA) and accurately recognizes homol-
ogous sequences in a background containing non-homologous and near-
homologous sequences. This recognition process can occur in the absence of
ATP hydrolysis, indicating that it must be thermodynamically reversible and
free energetically favorable. In this poster, we examine themechanism bywhich
RecA performs comparisons over sequences that are much longer than an indi-
vidual protein while avoiding kinetic trapping in regions with local accidental
homology.Wepresent amodel ofRecAbound toDNA that uses a simplifiedver-
sion of its known structure to calculate the distributionofmechanical stress in the
protein/DNA complex. In addition, we use computer simulations to model the
dynamic polymerization process.We propose that the lattice mismatch between
the more strongly and weakly bound strands of DNA results in a mechanical
stress that increases non-linearly with the number of bound base pairs. The dis-
tribution of stress combined with an iterative homology search may explain sev-
eral previously unexplained features of the structure and function of RecA. The
model is consistent with mechanical data obtained frommagnetic tweezer mea-
surements of polymerization of RecAon double strandedDNA (dsDNA) and the
extension of RecA-ssDNA filaments on dsDNA. RecA’smechanism can also be
instructive for artificial self-assembled systems.
Platform: Muscle: Fiber & Molecular Mechanics
& Structure
81-Plat
Subnanometer Structure of the Actin/Myosin/Tropomyosin Complex
Elmar Behrmann1, Krishna Chinthalapudi2, Sarah M. Heissler2,
Pawel A. Penczek3, Hans G. Mannherz4, Dietmar J. Manstein2,
Stefan Raunser1.
1MPI of Molecular Physiology, Dortmund, Germany, 2Hannover Medical
School, Hannover, Germany, 3Houston Medical School, Houston, TX, USA,
4Ruhr University, Bochum, Germany.Actins and myosins play a central role in life as they form the molecular motors
that transform chemical energy into movement. Without them, we would be
confined to a very static existence.
To fully understand how the interplay between actin and myosin leads to the
conversion of the energy stored in ATP into mechanical energy, one needs
structural information. Therefore, we have set out to determine the complex
structure of the three major muscle proteins (actin, myosin and tropomyosin)
using cryo electron microscopy and iterative helical real-space reconstruction.
Using state of the art instruments and reconstruction algorithms we have deter-
mined a sub-nanometer resolution electron density map of the complex. With
the help of electron-density guided flexible fitting we have subsequently refined
the structure to obtain a quasi-atomic resolution structure of the full complex
which enabled us, for the first time, to visualize interactions between actin
and myosin that have been postulated by computational methods beforehand.
In addition, we could also identify a novel interaction between myosin and
tropomyosin.
82-Plat
Structural Model of the Pre-Powerstroke State of the Actomyosin
Complex
Yang Zhenhui, Boglarka Varkuti, Anna Rauscher, Miklos Kepiro,
Andras Malnasi-Csizmadia.
Eotvos University, Budapest, Hungary.
We created a structural model of the ADP.Pi pre-powerstroke state of Dictyos-
telium myosin motor domain complexed with actin trimer by an in silico pro-
tein docking procedure followed by a long timescale molecular dynamics
relaxation. Furthermore, we also modeled the ADP.Pi down lever state of my-
osin motor domain and the rigor complexes using 1q5q (apo structure of Dic-
tyostelium myosin motor domain) or squid myosin S1 (3I5G) atomic structures
and actin trimer model. During the molecular dynamics of the apo myosin
(1q5q ) complexed with actin trimer the actin binding cleft of myosin motor
domain spontaneously closed and the relaxed actomyosin rigor structure fits
well with the structural rigor model determined by EM. The analysis of the
ADP.Pi pre-powerstroke actomyosin complex showed that actin spontaneously
induces significant conformational changes in the myosin motor domain. Most
strikingly, actin further closes the closed switch 2 loop coupled with a further
up movement of the lever. Interestingly, if the interaction between the N-termi-
nal region of actin and myosin activation loop is interrupted by a single muta-
tion (K520N) this conformational change does not occur upon actin binding.
Furthermore, actin binding rearranges correlating movements of different my-
osin motor regions which effect was significantly reduced in the mutant.
Recently we shoved experimentally that the interaction between actin and
the activation loop is responsible for channeling the enzymatic pathway of ac-
tomyosin into the effective powerstroke path. The further closure of switch 2
induced by actin indicates that cocking is induced by actin mainly through
the actin binding of activation loop. In order to test the indicated conforma-
tional change we have produced a myosin motor domain containg a FLASH
and REASH probes located at the N and C-terminus, respectively, to sensitively
follow the lever movement upon actin binding by FRET.
83-Plat
The Two-Dimensional Kinetics of Binding and Unbinding are Both
Regulated by Myosin’s Actin-Binding Loop
Amy M. Clobes, William H. Guilford.
University of Virginia, Charlottesville, VA, USA.
Binding of myosin to actin is a multi-step, ionic strength-sensitive process for
which the kinetic properties of bond formation and rupture have been studied in
solution. However, the solution (three dimensional) environment is a poor
model of the pseudo-two dimensional geometry in which myofilament proteins
interact in vivo. We therefore investigated the rate of actomyosin bond forma-
tion and rupture in a two-dimensional setting using dynamic force spectroscopy
at physiological and reduced ionic strength. We previously showed that catch
bond rupture is slowed at physiologic ionic strength (145 mM KCl) when com-
pared to low ionic strength (25 mM), and that bond lifetime is maximal at the
isometric force generated by a single myosin molecule. In the current study we
confirmed this result and sought to determine whether this difference is mir-
rored in the rate of actomyosin bond formation. A laser trap was used to mea-
sure the time to bond formation (tb) between actin filaments and nucleotide-free
heavy meromyosin (HMM) over a range of compressive loads. Two dimen-
sional on-rates were determined from tb and were increasingly force-
dependent with decreasing ionic strength. We examined the effect of ionic
strength on the initial long-range ionic interaction constituting the first step
of actomyosin binding by targeted tryptic digestion of HMM’s actin-binding
loop. Our data at physiologic ionic strength suggest that the actin-binding
loop contributes to the decreased 2D rate and load-dependence of bond forma-
tion. This is matched by reduced rates of actomyosin unbinding. These data


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Organization and Dynamics of the Living E. Coli Nucleoid at High Resolution in Space and Time

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