Fritsch, Anatol

Käs, Josef A.

Magin, Thomas

Seltmann, Kristin

Elsevier - Publisher Connector 

Keratins Significantly Contribute to Cell Stiffness and Impact Invasive Behavior 

574a Tuesday, February 18, 20142898-Pos Board B590
Myosin II Functions as a Direct Mechanosensor for Intercellular Invasion
during Cell-Cell Fusion
Ji Hoon Kim1, Yixin Ren2, Shuo Li1, Yee Kee2, Guofeng Zhang3,
Douglas Robinson2, Elizabeth Chen1.
1Molecular Biology and Genetics, Johns Hopkins University School of
Medicine, Baltimore, MD, USA, 2Cell Biology, Johns Hopkins University
School of Medicine, Baltimore, MD, USA, 3National Institue of Biomedical
Imaging and Bioengineering, Bethesda, MD, USA.
How cells sense and react to external mechanical stimuli is a fundamental
question in cellular biophysics. Here, we describe a previously unrecognized
mechanosensory response to a localized cellular protrusive force during cell-
cell fusion. Our previous studies of myoblast fusion in Drosophila embryos
and cell fusion in a reconstituted culture system revealed that cell-cell fusion
is an asymmetric process, in which one fusion partner (the attacking cell) ex-
tends invasive finger-like protrusions into the other (the receiving cell) to pro-
mote plasma membrane juxtaposition and fusion. Here, we demonstrate that
the Rho-Rok-Myosin II (MyoII) pathway is specifically activated in the
receiving cell in response to the invasive force from the attacking cell. Dis-
rupting the function of this pathway renders less cortical resistance in the
receiving cell and defects in cell-cell fusion, despite deeper invasion of the
attacking cell. Increasing the cortical tension in the receiving cell by overex-
pressing an actin crosslinker significantly rescued such fusion defect. We
show that MyoII accumulates to the cell cortex earlier than its upstream
biochemical regulators Rok and Rho in response to applied force, and that
MyoII is required for the steady-state accumulation of Rok and Rho. Further-
more, the motor domain of MyoII is indispensible for its cortical accumula-
tion triggered by intercellular invasion. These results strongly suggest that
MyoII functions as a direct mechanosensor that feeds back to its upstream
regulators, and that the mechanosensory function of MyoII is mediated by
its binding to actin filaments under mechanical stress. This newly discovered
mechanosensory system, consisting of a well-defined source of protrusive
force and the corresponding activation of the Rho-Rok-MyoII pathway in
the neighboring cell, highlights a central role of MyoII in the mechanosen-
sory response that ultimately leads to cortical tension generation in animal
cells.
2899-Pos Board B591
Electromechanical Model for Eukaryotic Cells
Florence H. Yellin1, Brenda Farrell2, Varun K.A.C. Sreenivasan2,
Sean X. Sun1,3.
1Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA,
2Otolaryngology-Head and Neck Surgery, Baylor College of Medicine,
Houston, TX, USA, 3Biomedical Engineering and Johns Hopkins Physical
Science Oncology Center, Johns Hopkins University, Baltimore, MD, USA.
Electromechanics is important in many cellular processes, including motility,
cancer metastasis, wound healing, and embryogenesis. Experiments show
that cells placed in direct current electric fields are able to sense the field and
direct their motion. Other experiments show that cell volume changes are
important in invading cancer cells. A computational model is necessary to un-
derstand how cells respond electro-mechanically to electro-mechanical
changes in their environment. The model proposed in this study considers
how ion flux and water flux across the cell membrane enable a cell to regulate
its size, internal pressure, and membrane voltage. This model also studies how
active ion pumps, voltage gated channels, mechanosensitive channels, and wa-
ter transport allow a cell to change its size when its membrane voltage is fixed
during a voltage clamp experiment. Specifically, the model predicts cell vol-
ume increases during hyperpolarization and decreases during depolarization.
Preliminary voltage clamp experiments suggest that the predicted size changes
are observed in eukaryotic cancer cells, which validates our model.
2900-Pos Board B592
Molecular Mechanisms Underlying the Inside-Out Signaling through
Focal Adhesions
Hengameh Shams, Mohammad R.K. Mofrad.
Bioengineering, UC Berkeley, Berkeley, CA, USA.
Cell adhesion is key to many important processes such as cell differentiation
and migration, which all involve bi-directional signaling across the plasma
membrane. At sites of adhesion a large assembly of macromolecules, called
focal adhesions, function together in order to orchestrate complex signaling
events. It is not yet clear how molecules in focal adhesions communicate
with one another causing the signals to be transmitted through their interaction.
Cells sense and respond to both chemical and mechanical signals suggesting
that individual focal adhesion molecules should act as mechanosensors. Me-
chanical forces from the extracellular matrix (ECM) are detected by integrin
receptors on the plasma membrane and transmitted to actin cytoskeletonthrough focal adhesions, which is called out-side-in signaling. The reverse pro-
cess is also possible in which acto-myosin forces are applied to the ECM. Some
proteins play key roles in regulating focal adhesions and their function may
shed light on the order of events in a certain signaling pathway. Talin, alpha-
actinin and filamin are among a few molecules that directly bind to both actin
and integrin, and thus focal adhesions are largely affected by their function. It
has been shown that talin plays a significant role in integrin activation, which is
essential for initiating focal adhesion formation, while filamin inhibits integrin
activation. The role of alpha-actinin is somehow controversial, i.e. it is not yet
clear whether it inhibits or promotes activation since both observations have
been made. We use all-atom molecular dynamics simulations to investigate
the role of each of these molecules both in isolation and in competition with
one another. Our results reveal how the presence of one molecule would affect
the interaction of others suggesting possible cooperative functions in some
cases.
2901-Pos Board B593
Quantitative Determination of Cell Wall Mechanical Properties using
Microfluidics
Amir Sanati Nezhad1, Muthukumaran Packirisamy1, Anja Geitmann2.
1Concordia University, Montreal, QC, Canada, 2University of Montreal,
Montreal, QC, Canada.
Certain cell types have the intriguing ability to forcibly invade a neighboring
tissue or a solid growth matrix. The purpose of this invasive growth activity de-
pends on the cell type and ranges from establishing contact between remote lo-
cations in the organism (neurons), procuring nutrients and water (fungi, root
hairs), to the delivery of gametes (pollen tubes). To invade a tissue or solid ma-
trix, these cells exert significant penetrative forces generated either by the cyto-
skeleton or the hydrostatic turgor pressure. Using a microfluidic device we
quantified the penetrative forces generated by pollen tubes, the fastest growing
plant cells. The tubes were guided through microscopic gaps made of elastic
polydimethylsiloxane (PDMS) material. Depending on the size ratio between
tube and gap, the tubes either deformed the gap walls completely, became
deformed themselves while passing, or stalled. Within a narrow range of size
ratios, the tubes successfully passed the gap but subsequently burst raising
the question whether sperm cell release in planta is triggered mechanically.
Based on the deformation of the PDMS-gaps the extrusive force exerted by
the elongating tubes was determined using reverse engineering and finite
element modeling.
2902-Pos Board B594
Assessing the Influence of Electric Cues and Conductivity on Pollen Tube
Growth via Lab-On-A-Chip Technology
Carlos G. Agudelo1, Muthukumaran Packirisamy1, Anja Geitmann2.
1Concordia University, Montreal, QC, Canada, 2University of Montreal,
Montreal, QC, Canada.
Pollen tubes are believed to react to a combination of chemical, mechanical, and
electrical cues during its journey through the pistil in order to achieve fertiliza-
tion. Despite extensivework dedicated to the subject it is still not clear how these
exogenous guidance signals work or how they are processed internally. Using
Lab-on-a-chip (LOC) technology, we assessed the influence of electric fields
on pollen tube growth at the microscale. Microelectrodes were integrated into
the LOC in order to enable the application of electric fields in a controlled
manner.Due simulation of the LOCelectrical configuration and characterization
of the pollen growth medium conductivity were carried out. DC and AC electric
fields were applied to batches of Camellia japonica pollen grains under various
conditions. Results show that pollen tube growth is increasingly degraded as the
applied DC electric field increases. Furthermore, germination is completely in-
hibited for sufficiently strong fields. AC electric fields, however, had a restoring
effect as growth is promoted as frequency increases beyond 100 mHz, which
suggests a significant role of the medium conductivity in enabling cell growth.
Interestingly, no sign of pollen tube orientation was found under any tested con-
dition,weakening themuch debated argument for electrotropism in pollen tubes.
When exposed to a highly localized field, pollen tubes did not deviate. This work
suggests that both strength and frequency of an applied electric field influence
pollen tubes, and most likely living cells in general, in a much more subtle
way rather than being a macro scale exogenous guiding signal.
2903-Pos Board B595
Keratins Significantly Contribute to Cell Stiffness and Impact Invasive
Behavior
Josef A. Ka¨s, Anatol Fritsch, Kristin Seltmann, Thomas Magin.
University of Leipzig, Leipzig, Germany.
Cell motility and cell shape adaptations are crucial during wound healing,
inflammation and malignant progression. These processes require the remod-
eling of the keratin cytoskeleton, to facilitate cell-cell and matrix adhesion.
Tuesday, February 18, 2014 575aHowever, the role of keratins for biomechanical properties and invasion of
epithelial cells are only partially understood. Here, we address this issue in
murine keratinocytes lacking all keratins upon genome engineering. In
contrast to prediction, keratin-free cells show an about 60% higher cell de-
formability even for small deformations. This is compared to less pronounced
softening effects for actin depolymerization induced via latrunculin A. To
relate these findings with functional consequences, we use invasion and
three-dimensional growth assays. These reveal higher invasiveness of
keratin-free cells. Re-expression of a small amount of the keratin pair K5/
K14 in keratin-free cells reverses the above phenotype for the invasion but
does not with respect to cell deformability. Our data shows a novel role of ker-
atins as major player of cell stiffness influencing invasion with implications for
epidermal homeostasis and pathogenesis. This study supports the view that
downregulation of keratins observed during epithelial-mesenchymal transition
directly contributes to the migratory and invasive behavior of tumor cells. (see
K. Seltmann et al., PNAS, in press).
2904-Pos Board B596
Dry Mass and Cell Cycle Follow-Up from Quantitative Phase Imaging
Julien Savatier1, Sherazade Aknoun1,2, Pierre Bon1, Lamiae Abdeladim2,
Benoit Wattellier2, Serge Monneret1.
1Aix Marseille Universite´, CNRS, Centrale Marseille, Institut Fresnel,
Marseille, France, 2Phasics S.A., Palaiseau, France.
During cell cycle, a cell doubles all its components and divides into two cells.
Cell cycle is often studied with fluorescent labeling, by flow cytometry. Here,
we propose a simple method to analyze cell cycle and dry mass fluctuations us-
ing quantitative phase imaging.
The technique is based on a quadri-wave lateral shearing interferometry
(QWLSI) wave front sensor [1]. It provides a quantitative measurement of
the optical path difference (OPD = Dnthickness) in nm. This measurement,
when integrated over the cell surface, is directly proportional to the cell dry
mass [2], giving direct information on the cell growth. No labeling is needed.
It is self-referenced and can be plugged on any microscope with classic objec-
tives, a white light and a camera port. Since it is achromatic, it can be used in
near IR for long live cell imaging. It can easily be combined with fluorescence
for simultaneous correlative microscopy. Automated segmentation of cells is
easy due to the absence of halo or artifacts. It is fast (camera frame-rate limited)
and sensitive (diffraction-limited in X and Y,5 0.5 nm in OPD,5 0.6 pg for a
570 pg cell).
We established criteria integrating both dry mass and morphological parame-
ters to identify different cell cycle stages and growth rate of haploid and diploid
yeasts, as well as four different mammalian cell lines, under different condi-
tions. We studied them by time lapse and population snapshot imaging. The
method is robust to record cellular division processes and effects of drugs on
cell growth.
[1] P Bon, G Maucort, B Wattellier and S Monneret, ‘‘Quadriwave lateral
shearing interferometry for quantitative phase microscopy of living cells’’, Op-
tics Express, 17 (15): 13080-13094 (2009).
[2] R Barer, ‘‘Interference microscopy and mass determination’’, Nature, 169:
366-367 (1952).
2905-Pos Board B597
How Embryonic Cartilage Grows: Insights Gained fromQuantitative Live
Imaging
Vikas Trivedi1, Yuwei Li2, Thai V. Truong2, David Koos2,
Chuong Cheng-Ming2, Rex Moats3, Scott E. Fraser2.
1California Institute of Technology, Pasadena, CA, USA, 2University of
Southern California, Los Angeles, CA, USA, 3Childrens Hospital Los
Angeles, Los Angeles, CA, USA.
One central question in development biology is how individual cell behav-
iors contribute to tissue morphogenesis. Growth plate cartilage contains
morphologically distinct populations of chondrocytes in different zones dur-
ing much of its growth phase, indicating a link between regulated cell behav-
iors and tissue elongation. To gain insights into this process, we develop a
robust avian embryonic metacarpal culture system, and employ time-lapsed
live 2-photon laser scanning microscopy to observe the cartilage growth.
Quantitative analysis of the cellular displacements during the tissue growth
reveals that cells in the proliferative and prehypertrophic zones, though
morphologically distinct, display similar displacement trajectories, contrib-
uting in a linearly additive fashion to the unidirectional tissue growth. Our
analysis rules out cell division and convergence-extension as the driving
mechanisms for tissue elongation; rather, anisotropic matrix deposition andcell volume enlargement are responsible for sculpting the directional tissue
growth.
2906-Pos Board B598
Optical Measurement of Biomechanical Properties of Human Red Blood
Cell using Digital Holographic Microscopy: Malaria and Sickle Cell
Diseases
Youngchan Kim, HeeSu Byun, YongKeun Park.
department of physics, KAIST, daejeon, Korea, Republic of.
Pathophysiological aspect of several hematologic diseases is largely deter-
mined by biomechanical properties of red blood cells (RBCs) and their hemo-
dynamical properties in circulatory system. Here we present the biomechanical
properties of individual RBCs from patients with sickle cell disease and RBCs
infected with malaria-inducing parasites Pf. falciparum. Using laser digital ho-
lographic technique, we non-invasively quantify membrane fluctuation in
RBCs at the nanometer and millisecond scale, which is analyzed with the math-
ematical model to retrieve four important mechanical properties of RBCs;
bending modulus, shear modulus, area expansion modulus, and cytoplasmic
viscosity. We find significant alterations in the mechanical properties of
RBCs in several pathophysiological states, ranging from depletion of
Adenosine-5’-triphosphate (ATP)3, different osmotic pressures4, malaria infec-
tions5,6,7, and sickle cell diseases9.
References
1. G.A. Barabino, M.O. Platt, and D.K. Kaul, Annual review of biomedical en-
gineering 12, 345 (2010).
2. YK Park, CA Best, K Badizadegan et al., Proceedings of the National Acad-
emy of Sciences 107 (15), 6731 (2010).
3. Y. Park, C. A. Best, T. Auth et al., Proceedings of the National Academy of
Sciences of the United States of America 107 (4), 1289 (2010).
4. Y. Park, C. A. Best, T. Kuriabova et al., Physical Review E 83 (5) (2011).
5. Y. Park, M. Diez-Silva, G. Popescu et al., Proc. Natl. Acad. Sci. USA 105
(37), 13730 (2008).
6. R. Chandramohanadas, Y. Park, L. Lui et al., PLoS One 6 (6), e20869 (2011).
7. M. Diez-Silva, Y. Park, S. Huang et al., Scientific Reports (2012).
8. H.S. Byun, T.R. Hillman, J.M. Higgins et al., Acta Biomaterialia (2012).
9. Youngchan Kim, John M. Higgins, Ramachandra R. Dasari et al., Journal of
Biomedical Optics 17 (4), 0405011 (2012).
2907-Pos Board B599
The Nanoscale Organization of Focal Adhesion Signaling Complexes can
Reflect Changes in Cellular Contractility and Motility
Matthew G. Rubashkin1, Christopher Dufort1, Patrick Oakes2,
Matthew Paszek1, Guanqing Ou1, Johnathan Lakins1, Michael Davidson3,
Margaret Gardel2, Valerie Weaver1.
1Surgery, University of California, San Francisco, San Francisco, CA, USA,
2Physics, University of Chicago, Chicago, IL, USA, 3Florida State
University, Tallahassee, FL, USA.
Focal adhesions are the conduits through which cells receive and interpret me-
chanical signals. It is not known if nanoscale protein organization is altered to
accommodate changes in mechanical inputs from the cytoskeleton and extra-
cellular matrix components. We hypothesized that the relative position of spe-
cific focal adhesion proteins could correspond to the engagement of a physical
protein clutch for different adhesion functions. To this end we employed Scan-
ning Angle Interference Microscopy to determine the 3D organization of pro-
teins comprising focal adhesions with a precision of ~5nm. We found paxillin,
FAK, vinculin, talin, and zyxin to be stratified in distinct layers over a vertical
range of 60 nm. We then compared nascent versus focal adhesions at the cell
leading edge, and found that paxillin localized ~7nm towards the cell mem-
brane in developing adhesions. We inhibited intracellular contractility to see
how adhesion architecture dynamically responds to changes in mechanical
input, and observed that paxillin and zyxin, but not vinculin, undergoes a
marked increase in height of >15nm. Conversely, vinculin without a force
dependent auto-inhibition domain, T12; undergoes dramatic reorganization at
the nanoscale after contractility inhibition. Overexpression of vinculinT12 re-
sulted in increased intramolecular forces as seen in a vinculinT12 FRET tension
sensor, targeting of vinculin to an architecture that corresponded with talin and
actin engagement, but not changes in cellular traction. When we reduced
cellular motility through overexpression of a constitutively active Rac1 mutant,
adhesions at the lamella-lamellipodia border had different vinculin architecture
than other adhesions in the cell. Our results suggest that elimination of vinculin
force dependent auto-inhibition can dictate focal adhesion architecture and


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Keratins Significantly Contribute to Cell Stiffness and Impact Invasive Behavior

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