Berg-Sørensen, Kirstine

Oddershede, Lene

Selhuber-Unkel, Christine

Yde, Pernille

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Intracellular Diffusion in Fission Yeast Cells Depends on Cell Cycle Stage 

Monday, February 22, 2010 369aliving cells at an unparalleled spatial and temporal resolution (50-100 nm, 4-8
min for a 30 mm scan). Furthermore, we were also able to map the elasticity of
a reconstituted actin network, which has not been achieved before. In combina-
tion with optical techniques this opens up a unique simultaneous view of the
mechanics of the living cell and the mechanical properties of its relevant mo-
lecular components.
Elasticity maps of a live HUVEC cell (30 mm scan)
1906-Pos
What is Measured By Passive Microbead Rheology?
Jay D. Schieber, Ekaterina Pilyugina.
Illinois Institute of Technology, Chicago, IL, USA.
It is often claimed that the dynamic modulus G* of a viscoelastic medium can
be measured by following the trajectory of a small bead subject to Brownian
motion. In the pioneering manuscript that introduced the idea [T. Mason and
D. Weitz, Physical Review Letters 74, 1250 (1995)], this equivalence between
the autocorrelation function and G* was assumed. Later work claimed that
a correspondence could be proven, but to our knowledge, the proof has never
been shown. We use here an analytic solution of the forces on a sphere under-
going arbitrary displacement in an arbitrary viscoelastic medium combined
with the fluctuation-dissipation theorem to derive what is actually measured
in the microbead rheology experiment. We find that a convolution of G* is in-
deed measured in the followed autocorrelation function. Under certain restric-
tions the autocorrelation function is a direct measurement of the dynamic mod-
ulus as is typically used. We examine experimental data published in the
literature and are unable to find any data where the restrictions do not hold.
Nonetheless, the results suggest that the technique could also be used at higher
frequencies, if proper analysis is made of the data.
1907-Pos
Intracellular Diffusion in Fission Yeast Cells Depends on Cell Cycle Stage
Christine Selhuber-Unkel1, Pernille Yde1, Kirstine Berg-Sørensen2,
Lene Oddershede1.
1Niels Bohr Institute, Copenhagen, Denmark, 2Danish Technical University,
Kgs. Lyngby, Denmark.
During the cell cycle, rearrangements of the cytoskeletal network play an
essential role, in particular for the success of cell division. In order to quan-
tify the influence of cytoskeletal rearrangements on the viscoelastic proper-
ties of the intracellular space, we studied the diffusion of endogenous lipid
granules within single fission yeast cells in the different stages of the cell
cycle. The position of the granules was tracked with optical tweezers at
nanometer and sub-millisecond resolution and the data were analyzed with
a power spectral analysis. We found that the majority of the lipid granules
underwent subdiffusive motion during all stages of the cell cycle, i.e. the
mean squared displacement of the granule is 2Dta with a<1. With our ex-
periments we have shown that a is significantly smaller during interphase
than during any stage of mitotic cell division and, surprisingly, we did
not find significant differences of a in the different stages of cell division.
These results indicate that the cytoplasm is more elastic during interphase
than during cell division and that its elasticity is relatively constant during
the stages of cell division.
1908-Pos
A Comparison of Single-Cell Elasticity of Osteogenic Cells Measured with
the Optical Stretcher and Holographic Microscope
Michael G. Nichols, Anya K. Burkart, Joy L. Chaput, Robert P. Thomen,
Kristina G. Ward, Semere M. Woldemariam.
Creighton University, Omaha, NE, USA.
While it has been known for some time that bone mass is remodeled in response
to mechanical stress, the identity of the primary mechanosensor has yet to be
clearly established. To determine if cellular elasticity may play a role in the
cell’s ability to detect a pressure variation, we have used the optical stretcher
to measure the elasticity of individual osteogenic cells. To determine cell elas-
ticity from measurements of cellular deformation, the optical pressure on the
cell surface is computed using a ray optics model which assumes a value for
the index of refraction of the cell. Previously we have estimated this value
from measurements of other eukaryotic cells, but the optical pressure varies
significantly with small changes in refractive index. Therefore, digital holo-
graphic microscopy is used to improve estimates of this critical parameter.
We consider the overall impact that a spatial variation in the index of refraction
can have on the determination of the optical stress, and compare single-cell
elasticity measurements of red blood cells, 2T3 osteoblast and MLO-Y4 oste-
ocyte cells.
* This work was supported by P20 RR016475 from the INBRE Program of the
National Center for Research Resources.Microtubule Motors-Kinesin-1
1909-Pos
An Atomic-Level Engine that Accounts for Kinesin Motility and Catalysis
Charles V. Sindelar1, Kenneth H. Downing2.
1Brandeis University, Waltham, MA, USA, 2Lawrence Berkeley National
Laboratory, Berkeley, CA, USA.
Kinesin motor proteins convert the energy of ATP hydrolysis into stepping
movement along microtubules. In this process, the microtubule can be consid-
ered as kinesin’s regulatory partner, responsible for activating the enzyme’s
functional behavior. In the absence of atomic resolution structures describing
the kinesin-microtubule complex, the mechanism of this activation has re-
mained unknown. We use cryo-electron microscopy to derive atomic models
describing the complete, microtubule-attached, kinetic cycle of a kinesin mo-
tor. The resolution of our reconstructions (~8A˚) enabled us to unambiguously
build crystallographically-determined conformations of kinesin’s key subcom-
ponents into the density maps. The resulting models reveal novel arrangements
of kinesin’s nucleotide-sensing switch loops and of its microtubule binding el-
ement known as the switch II helix. Based on these models, we present a de-
tailed molecular mechanism accounting for kinesin’s force generation cycle.
In this mechanism, the switch loops control a seesaw-like movement of the cat-
alytic domain relative to the switch II helix, which remains fixed on the micro-
tubule surface. Microtubules couple the seesaw movement to ATP binding by
stabilizing the formation of extra coils at the N terminus of the switch II helix,
which interact directly with the switch loops. Tilting of the seesaw to assume
the ATP-bound orientation in turn elicits a power stroke by the motor domain’s
force-delivering element known as the neck linker. This sequence of events ac-
counts for the essential mechanics of kinesin’s force-delivery cycle, and also
yields a new model for the catalytically active conformation of kinesin’s ances-
tral relative, myosin.
1910-Pos
Free Energy Changes During Kinesin’s Force-Generating Substep
Wonmuk Hwang1, Matthew J. Lang2, Martin Karplus3.
1Texas A&M University, College Station, TX, USA, 2MIT, Cambridge, MA,
USA, 3Harvard University, Cambridge, MA, USA.
We have previously suggested that Kinesin-1 generates force by transient
folding between the N-terminal cover strand and the C-terminal neck linker
domains into a beta-sheet, the so-called cover-neck bundle (CNB). Once
formed, the CNB has a conformational bias sufficient to move the neck linker
forward. Replica exchange molecular dynamics simulations have been per-
formed to elucidate the energetics of CNB formation with and without load.
Without load, the CNB state is weakly favorable compared to non-CNB states
by 0.85 kcal/mol at 300 K, which is in agreement with a previous experimen-
tal value based on electron paramagnetic resonance, 0.72 kcal/mol (Rice et al,
Biophys. J. 84:1844 (2003)), although the identity of the states involved is not
certain. In non-CNB states the mobile neck linker points mostly forward in the
ATP-like conformation of the motor head, so there is relatively little confor-
mational difference with the CNB-state. By contrast, when a 10-pN rearward
load is applied at the end of the beta9 part of the neck linker, a new local en-
ergy minimum appears for a rearward-pointing state. Compared to the CNB
state, the free energy of the rearward-pointing state is higher by 2.96 kcal/
mol at 300 K. This indicates that the CNB readily forms under applied load
and thus is able to move the neck linker forward. The significance of these
results for the mechanism by which kinesin-1 walks on microtubules will
be discussed.
1911-Pos
Neck-Linker Length is a Critical Determinant of Kinesin Processivity
Shankar Shastry, William Hancock.
Pennsylvania State University, University Park, PA, USA.
The kinesin neck-linker domain is a key mechanical element underlying proc-
essive kinesin motility. Not only is neck-linker docking thought to be the dom-
inant conformational change in the kinesin hydrolysis cycle, chemomechanical
communication between the two head domains must necessarily be transmitted
through the two neck-linker domains and their shared coil-coil. Hence, the
length of the neck-linker is expected to have a strong influence on kinesin
run length, a quantitative measure of processivity. Across different kinesin fam-
ilies, motors with longer neck-linker domains, such as Kinesin-2 are generally
less processive than Kinesin-1, which has the shortest neck-linker domain
among N-terminal kinesins. However, there is disagreement in the literature
as to whether artificially extending the Kinesin-1 neck-linker alters the motor
run length. Using single molecule TIRF analysis to visualize GFP-tagged mo-
tors in 80 mM PIPES buffer, we find that lengthening the Kinesin-1 neck-linker
by three amino acids results in a five-fold reduction in run length. Consistent


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Intracellular Diffusion in Fission Yeast Cells Depends on Cell Cycle Stage

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