Ganguly, Sujoy

Goldstein, Raymond E.

Palacios, Isa

Williams, Lucy

Elsevier - Publisher Connector 

Cytoplasmic Streaming in Drosophila Melanogaster 

Webnesday, February 29, 2012 697athese states is important for establishing basic molecular mechanisms. Tech-
niques such as a mean squared displacement (MSD) analysis average popu-
lations and thus cannot resolve multiple states of motion. By plotting non-
averaged rather than mean displacements of simulated trajectories, we
show that states of motion can be resolved and analyzed to determine the ki-
netics of transitions between dynamic states. We have applied this technique
to several different dynamic systems. First, we show that submaximal veloc-
ities of partially activated thin filament motility result from switching be-
tween an ‘‘on’’ and ‘‘off’’ state, and that the level of activation increases
with a decrease in the lifetime of the ‘‘off’’ state. Second, we show that sub-
maximal myosin-based actin sliding velocities observed at low myosin den-
sities result from a similar switching between ‘‘on’’ and ‘‘off’’ states. Next,
we use this technique to analyze the dynamics of myosin light chain kinase
(MLCK) moving on actin-myosin filaments in smooth muscle cells, where
the lifetime of the paused states could give potential insight into the kinetics
of smooth muscle phosphorylation by MLCK. Finally, we apply this tech-
nique to vesicle motility in 3T3 L1 to determine the kinetics of vesicle paus-
ing. In general, a non-averaged displacement analysis provides a simple
approach for determining the kinetics of transitions between dynamic states
of single molecules.
3530-Pos Board B391
Universal Optimality of Molecular Motors
Zhisong Wang.
National University of Singapore, Singapore, Singapore.
Abstract
Molecular motors capable of directional track-walking or rotation are abundant
in living cells, and inspired the emerging field of artificial nanomotors. Some
biomotors can convert 90% of free energy from chemical fuels into usable me-
chanical work, and the same motors still maintain a speed sufficient for cellular
functions. We have explored how this extreme efficiency-speed trade-off can
be achieved in a single-molecule system that is intrinsically prone to ubiquitous
thermal fluctuations. Specifically, we found that a regime of universal optimi-
zation unfamiliar in macroscopic engines exists for molecular motors. Like
Carnot cycle for heat engines, the universally optimized working cycle for mo-
lecular motors is infinitely slow. But when a deviation from the ideal conditions
reduces energy efficiency linearly from 100%, the speed is recovered exponen-
tially due to Boltzmann’s law. Experimental data on a biomotor indicate that
this universal optimization has been largely approached in living cells, under-
pinning the extreme efficiency-speed trade-off in biomotors. I shall also discuss
an experimental study by us towards achieving a similar artificial nanomotor in
engineered systems.
*Email: phywangz@nus.edu.sg
3531-Pos Board B392
Watching the Motions of the Cell Wall Synthesis Machinery and Underly-
ing Cytoskeleton in B. Subtilis
Ethan Garner, Wenqin Wang, Remi Bernard, David Rudner,
Xiaowei Zhuang, Timothy Mitchison.
Harvard University, Cambridge, MA, USA.
Rod-shaped bacteria elongate by the action of cell-wall synthesis complexes
linked to underlying dynamic MreB filaments, but how these proteins function
to allow continued elongation as a rod remains unknown. To understand how
the movement of these filaments relates to cell wall synthesis, we characterized
the dynamics of MreB and the cell wall elongation machinery using high-
resolution particle tracking in Bacillus subtilis. We found that both MreB
and the elongation machinery move in linear paths across the cell, moving at
similar rates (~20 nm / second) and angles to the cell body, suggesting they
function as single complexes. These proteins move circumferentially around
the cell, principally perpendicular to its length. We find that the motions of
these complexes are independent, as they can pause and reverse,and also as
nearby complexes move independently in both directions across one surface
of the cell. Inhibition of cell wall synthesis with antibiotics or depletions in
the cell wall synthesis machinery blocked MreB movement, suggesting that
the cell wall synthetic machinery is the motor in this system. We propose
that bacteria elongate by the uncoordinated, circumferential movements of syn-
thetic complexes that span the plasma membrane and insert radial hoops of new
peptidoglycan during their transit.3532-Pos Board B393
Quantitative Study of Tubulin Distrubution and Mitochondrial Dynamics
in Skeletal Muscle Cells
Marie Le Guen, Marcela Gonzalez-Granillo, Rita Guzun, Alexei Grichine,
Franc¸ois Boucher, Herve´ Dubouchaud, Yves Usson, Valdur Saks.
Universite´ Joseph Fourier, St Martin d’he`res, France.
The comparative studies of regulation of oxidative phosphorylation as a function
of mitochondrial organization in healthy and cancerous cardiac muscle cells
have revealed that the mechanisms of regulation of the energy metabolism de-
pend on the cellular organization (Biochim Biophys Acta, 2010, 1797: 678-97).
The aim of this study is to evaluate mitochondrial dynamics and organization of
mitochondria into intracellular energy units in adult skeletal muscle cells as
a function of the energy metabolic pattern and mitochondrial interactions
with cytoskeletal proteins. Among the latter, intracellular distribution of tubulin
isoform was evaluated in rat oxidative and glycolytic skeletal muscle cells by
Western blot analysis, immunofluorescence and functional methods. Investiga-
tion of mitochondrial dynamics in living skeletal muscle fibers was conducted
using fluorescent probe labelling analyzed by fluorescent confocal microscopy.
Fast scanning of cells was carried out using a line scanning LSM7 LIVE and
LSM710 confocal microscope. The fast and simultaneous line scanning in
two fluorescent channels synchronized with the piezo stage axial displacements
allow rapid 3D imaging of the mitochondrial dynamics referenced to the struc-
tural elements of sarcomers. The analysis of position of mitochondrial fluores-
cence centres, or virtual mass centres, is studied by applying the gradient
clustering algorithm.
Among the results obtained, we report specific tubulin isoforms distribution
profiles in oxidative and glycolytic muscles that could rely to their energy meat-
bolic pattern.
This study was performed in the framework of the ANR project SYBECAR,
France.
3533-Pos Board B394
Cytoplasmic Streaming in Drosophila Melanogaster
Sujoy Ganguly, Lucy Williams, Isa Palacios, Raymond E. Goldstein.
University of Cambridge, Cambridge, United Kingdom.
The persistent circulation of the cytoplasm, called cytoplasmic streaming, oc-
curs in a variety of eukaryotic cells. One context in which streaming occurs is
during the establishment of Drosophila body axes, when Kinesin-1 transports
the axes determinants and drives cytoplasmic streaming.AlthoughKinesin is es-
sential for flows, neither themechanism bywhichKinesin induces streaming nor
the impact of these flows on transport are known.We have succeeded in a precise
quantitative measurement of the statistical properties of streaming by Particle
Image Velocimetry. We have combined these measurements with an in vivo
study of the cytoplasm rheology, to calculate the energy dissipation due to
streaming. SinceKinesin is required for flowswe can relate the energy dissipated
to the work done on the fluid by Kinesin and determine the minimum number of
motors necessary to drive streaming. Furthermore we have performed these
measurements on mutants that effect Kinesin-1 motor activity and found re-
markable agreement between our in vivo measurements and in vitro studies.
3534-Pos Board B395
Structural Basis for Activation of the Cytoskeleton Regulatory Protein
IRSp53
David J.E. Kast, Yadaiah Madasu, Malgorzata Boczkowska,
Grzegorz Rebowski, Roberto Dominguez.
University of Pennsylvania, Philadelphia, PA, USA.
The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53)
is a ubiquitous regulator of the actin cytoskeleton that mediates filopodia and
lamellipodia formation under the control of Rho-family GTPases. IRSp53 is
comprised of an N-terminal IRSp53/MIM homology domain (IMD) that senses
negative membrane curvature and a central semi-CRIB motif followed by an
SRC homology 3 (SH3) domain that binds to proline-rich regions of a wide
range of actin regulators. Here, we present the crystal structure of a complex
between activated CDC42 and the CRIB domain of IRSp53. Isothermal titra-
tion calorimetry (ITC) experiments confirm the binding of this domain to
CDC42, and exclude the possibility of binding by inactive forms of CDC42
or activated forms of RAC1. Moreover, we demonstrate that the SH3 domain
also binds to the CRIB domain, suggesting a mechanism of regulation where
binding of CDC42 is required to release autoinhibition of the SH3 domain.


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Cytoplasmic Streaming in Drosophila Melanogaster

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