Finkenstaedt-Quinn, Solaire

Hanson, Rachel N.

Haynes, Christy L.

Johnson, Jolene L.

Olson, Sara J.

Elsevier - Publisher Connector 

Sunday, February 16, 2014 171aassembly may lead to kinetically trapped networks [1], thus giving rise to in-
ternal stress. To investigate this phenomenon local probing of the stress field is
necessary, which is achieved by means of diffraction limited UV-laser abla-
tion: We produce small cuts inside in vitro networks of fluorescently labeled
actin and its crosslinkers, or keratin. Analysis of simultaneously acquired
confocal and bright field images of the system’s reaction reveal relaxation
properties and show anisotropies in the viscoelastic behavior of these
networks.
[1] Schmoller et al.: Internal stress in kinetically trapped actin bundle networks.
Soft Matter, 2008, 4, 2365-2367.
864-Pos Board B619
Investigating the Relationship between Cellular Mechanics and Beta
Amyloid in Alzheimer’s Disease
Nicole M. Shamitko-Klingensmith, Jonathan W. Boyd, Justin Legleiter.
West Virginia University, Morgantown, WV, USA.
The beta amyloid (Ab) peptide was first implicated in the formation of am-
yloid plaques, a characteristic of Alzheimer’s disease (AD), nearly 30 years
ago. Misfolding of the Ab peptide into nonnative conformations promotes
the formation of aggregates such as oligomers, annular aggregates and fibrils
that can impair cellular function. Although these aggregates are known to be
toxic, the exact mechanism of their cytotoxicity remains unclear. As aging is
associated with AD, we explored how cytoskeletal degradation, associated
with the aging process, modulates a cell’s ability to cope with exposure to
exogenous Ab. In this study, hypothalamic neurons, of the GT1-7 cell
line, were treated using a variety of cytoskeletal altering drugs. The me-
chanics of the altered neurons were examined by atomic force microscopy-
based techniques (force-distance curve and force volume analysis) and the
altered neurons were then exposed to the Ab peptide to determine how the
cytoskeletal network affects peptide binding and toxicity. Binding studies
were performed using fluorescence activated cell sorting and toxicity was
examined using a variety of biochemical assays. Finally, the topography
and mechanics of the the aged neurons exposed to Ab were examined to
determine the impact of Ab binding. This research gives a mechanistic un-
derstanding of AD pathology in relation to the cytoskeleton and examines
the potential for cytoskeletal stabilization as a therapeutic intervention for
combating AD.
865-Pos Board B620
TIRF and Model Blood Vessels Combined to Elucidate the Role of the
Cytoskeleton in Platelet Activation
Rachel N. Hanson1, Sara J. Olson1, Solaire Finkenstaedt-Quinn2,
Christy L. Haynes2, Jolene L. Johnson3.
1Math and Physics, St. Catherine University, St. Paul, MN, USA, 2Chemistry,
University of Minnesota, Minneapolis, MN, USA, 3Math and Physics, St
Catherine University, St. Paul, MN, USA.
Platelets are cell fragments that play a central role in hemostasis and throm-
bosis. Typically platelets circulate as thin flat disks; however upon encoun-
tering a damaged vessel wall they are recruited to the exposed subendothelial
matrix where they adhere and activate. The platelets interact with the exposed
matrix proteins via specific adhesive glycoproteins found on the surface of the
platelet. Activated platelets have extensive pseudopodia due to a change in the
assembly of cytoskeletal proteins such as actin and tubulin. The rearrangement
of the cytoskeleton also corresponds with the secretion of granule content lead-
ing to the amplification of the platelet stimulation and the formation of a stable
platelet-fibrin plug. A popular model assumes that the granules are drawn to the
center of the platelet before release occurs, indicating the importance of the
cytoskeletal components that drive this centralization leading to the eventual
clot formation.
In this study we will examine cytoskeletal protein arrangements in granule
release using TIRF to study the activated platelets that aggregate on the protein
coatings along a microfluidic channel. These protein coatings will include pro-
teins found in damaged vessel walls such as collagen and fibrinogen. TIRF uses
an induced evanescent wave created at the interface of a glass and to selectively
excite fluorescent molecules located near the interface. Here we will describe
the microfluidic channels we used to model blood vessels. By controlling which
proteins are coated on the inside of the channels we are able to visualize platelet
activation in a more native setting. We will also discuss methods that were used
to stain cytoskeletal proteins to reduce the premature activation of the platelets.
A better understanding of these models will lead to a greater understanding clot
formation.866-Pos Board B621
Reconstituted Active Actin Networks in Confinement
Carina Pelzl, Katharina Henneberg, Andreas R. Bausch.
Physics, Technische Universita¨t Mu¨nchen, Garching, Germany.
Giant unilamellar vesicles (GUVs) are a great model system to explore the
functional mechanisms of the constantly rearranging cytoskeletal network in
a bottom-up approach. Here, we developed a reconstituted active actin network
consisting of actin filaments, crosslinking proteins and myosin-II motor fila-
ments in confinement. In this minimal model system, the key parameters are
tightly controlled, which leads the way to gain a better understanding of the
basic physical principles underlying the complex cytoskeletal dynamics. By
means of quantitative fluorescence microscopy and image analysis, the inter-
play between force generation by molecular motors and the stabilization of
the network by crosslinking proteins is identified to be responsible for the high-
ly dynamic structure formation process.
867-Pos Board B622
Force-Dependent Mechanical Properties of Dendritic Actin Networks
Tai-De Li1, Peter Bieling2, Dyche Mullins3, Daniel Fletcher1.
1BioEngineering, UC Berkleey, Berkeley, CA, USA, 2BioEngineering/
Cellular and Molecular Pharmacology, UC Berkeley/UCSF, Berkeley/San
Francisco, CA, USA, 3Cellular and Molecular Pharmacology, UCSF, San
Francisco, CA, USA.
Branched actin networks generate protrusive forces required for cell motility
and movement of sub-cellular structures. While biochemical knowledge of
dendritic actin network assembly continues to advance, relatively little is
known about how the mechanical properties of networks respond to physical
stimuli during growth. By combining surface micropatterning with AFM and
TIRF microscopy, we are able to measure mechanical properties of in vitro re-
constituted branched networks that grew under different counter forces in a
biochemically defined environment and simultaneously measure protein den-
sities at the force-generating surface. Our measurements show that 1) the elas-
ticity of the network increases more strongly than actin density with increasing
counter force, 2) the force to plastically deform the network also increases with
the increasing counter force, 3) the networks growing under larger counter
forces show stronger stress stiffening and softening, while networks growing
under small counter forces do not. These results indicate that the structure of
actin networks is permanently altered by different counter forces experienced
during growth, giving rise to different network mechanical properties. Our
AFM-TIRF measurements provide new insight into the role of force in the as-
sembly and mechanical properties of dendritic actin networks.
868-Pos Board B623
Athermal Fluctuations of Probe Particles in Active Cytoskeletal Network
Irwin Zaid1, Heev L. Ayade2, Daisuke Mizuno2.
1Physics, Oxford University, Oxford, United Kingdom, 2Physics, Kyushu
University, Fukuoka, Japan.
A reconstituted active cytoskeletal networks consisting of an actin filament
network coupled to myosins (motor proteins) have been shown to display
rich in dynamical and mechanical behaviors that is often in contrast to pas-
sive, equilibrium system. The motor proteins, which spontaneously generate
forces, kept the active cytoskeletal network out of equilibrium. The athermal
fluctuations observed in the network are linked to the active force generation
by motor proteins which give more relevant information including the inter-
action with the surrounding materials. In prior studies, only the second
moment–also referred to as the mean square displacement or power spectral
density–of the athermal fluctuations has been investigated. In equilibrium
where the Gaussian statistics are implied, second moment analysis supplies
all the necessary information to characterize the fluctuation. There is no
reason a priori to expect Gaussian statistics in non-equilibrium systems.
Indeed, the full displacement distribution of the athermal fluctuations in
active cytoskeleton recently probed using video microrheology is found to
be far from Gauss. No theoretical model has been found yet that describes
the non-Gaussian signature of the distribution. Therefore, in this study, we
examine the non-equilibrium statistics and dynamics of the active network
by analyzing the athermal fluctuations using a new theoretical model devel-
oped by I. Zaid, et al. The model, which is based on Le´vy statistics, incorpo-
rates the thermal and athermal fluctuations and assumes that a single myosin
acts as a force dipole. In our results, it was found out the full displacement
distribution follows truncated Le´vy statistics distribution. Sum action of mul-
tiple motor proteins, which drives the probe particle, only slowly converges
to Gauss distribution because of the 1/r2 spatial decay of the motor impacts.


TIRF and Model Blood Vessels Combined to Elucidate the Role of the Cytoskeleton in Platelet Activation 

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TIRF and Model Blood Vessels Combined to Elucidate the Role of the Cytoskeleton in Platelet Activation

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