Gaczynska, Maria E.

Huang, Tim Hui-Ming

Osmulski, Pawel A.

Wlodyga, Karolina

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Sunday, February 8, 2015 141ais assumed that also in non-erythrocytes the membrane skeleton is mostly
composed of heterotetramers of a-II and b-II spectrin. Because of a-spectrins’
potential role as mechanotransducers, we investigate the effect of mechanical
stimulation on the spectrin-ankyrin B membrane skeleton of NIH 3T3 fibro-
blasts and murine C3H 10T½ cells. Using quantitative fluorescence micro-
scopy and biochemical techniques, the absolute changes in cellular spectrin
and ankyrin B content were determined. Before stimulation the spectrins
and ankyrin B make up more than 10% of cytosolic and membrane proteins.
Thus, they constitute a much more significant part of the non-erythroid cyto-
skeleton in these cells than previously assumed. Interestingly, both cell types
contain all four major isoforms of spectrin. However, while a-II spectrin is the
dominant a spectrin in 3T3, in 10T½ cells it is the ‘erythroid’ a-I spectrin.
Also, at least half of the a spectrins do not have b-spectrin counterparts
and thus cannot be part of the classical heterotetramere structure. Mechanical
stimulation decreases the overall spectrin amount by up to 60% with a-spec-
trins exhibiting the largest loss. In contrast, the number of ankyrin B copies
increases by almost 30%. Furthermore, the fraction of polyubiquitinated spec-
trin actually increases, which in part could account for the reduction in
a-spectrins and is in line with their proposed mechanically enhanced self-
ubiquitination activity.
701-Pos Board B481
Vimentin Intermediate Filament Mechanics in Cells under Shear Stress
Huayin Wu1, Mikkel H. Jensen2, Ming Guo2, David A. Weitz2.
1Applied Physics, Harvard University, Cambridge, MA, USA, 2Harvard
University, Cambridge, MA, USA.
Cells experience a wide range of mechanical stimuli depending on their envi-
ronment. Cellular responses to mechanical stress are thus an important aspect
of cell mechanics to understand. For example, cytoskeletal rearrangement
has been observed when shear stress is applied to cells. We are interested in
the contribute of this rearrangement to changes in local material properties
and mechanics. Using microrheological tools, we are able to better understand
these changes in the larger picture of cell mechanics.
702-Pos Board B482
Electomechanical Model for Non-Excitable Cells
Flori H. Yellin1, Varun K.A.C. Sreenivasan2, Brenda Farrell2, Sean X. Sun1.
1Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA,
2Otolaryngology-Head and Neck Surgery, Baylor College of Medicine,
Houston, TX, USA.
Mechanical and electrical properties of cells are crucial in regulating a
wide range of cellular processes, such as motility, cancer metastasis, and
embryogenesis. Cell size and membrane potential are both highly regulated
properties in eukaryotic, non-excitable cells. The proposed model explains
the interplay between mechanical, electrical, and biochemical properties of
a cell, and can have important implications for pathology and medicine.
The combination of osmosis-driven water flow, passive ion flux, ion pumping,
signaling, and cytoskeletal forces provide a mechanism for how cells control
their volume and membrane potential. For example, the model predicts that
cell volume increases during depolarization and decreases slightly during
hyperpolarization. Whole cell voltage clamp experiments provide a technique
for measuring cell volume while controlling membrane potential. Experiments
done on different eukaryotic, non-excitable cancer cell lines validate the
model.
703-Pos Board B483
The Role of Cellular Mechanical Properties in Microenvironment-
Dependent Behavior
Iris Pak, Manisha Kanthilal, Eric Darling.
Brown University, Providence, RI, USA.
Understanding the role of mechanics in cell behavior is necessary for effective
development of tissue engineering therapies. This study observed how cellular
mechanical phenotype influenced cell morphology and actin organization on
substrates coated with different extracellular matrix ligands. Inherent cell
stiffness, represented by Young’s modulus, for MG-63 (osteosarcoma) and
SH-SY5Y (neuroblastoma) cells was mechanically determined using atomic
force microscopy. These cells have dissimilar moduli when probed over the
nucleus (1.350.5 and 0.2650.15 kPa for MG-63 and SH-SY5Y cells, respec-
tively). To investigate the effect of substrate compliance, cells were cultured
on polyacrylamide gels ranging from 0.3 to 12 kPa and functionalized with
fibronectin, laminin, or collagen-1. We hypothesized that cells would spread
and attach more successfully to substrates with an elastic modulus equal to
or greater than that of the cell, provided physiologically relevant ligands
are present. Cellular responses were captured daily over 4 days using fluores-
cent microscopy. MG-63 cells showed two different organizational pheno-types: a multi-cell, spheroid-like structure and a spread monolayer. The
threshold substrate compliance required for MG-63 cells to organize into
either phenotype was designated by the cells’ own stiffness, most clearly
observed on collagen-1 coated gels. While SH-SY5Y cells exhibited similar
phenotypic organizations when plated on varying substrates, no mechanical
compliance threshold was observed. However, SH-SY5Y cells exhibited
greater tendencies toward forming spheroids when plated on fibronectin.
These morphological changes show a possible correlation between the cells’
characteristic mechanical phenotypes and their relationship to the microenvi-
ronment. Cells exhibiting less compliant phenotypes may have more substrate
stiffness-dependent behavior, whereas more compliant cell types may have
more ligand-dependent behavior. These findings indicate that cellular mecha-
nosensing is influenced not only by the mechanical and biological character-
istics of the microenvironment but also by the inherent mechanical phenotype
of cells comprising a population.
704-Pos Board B484
Characterizing Mechanical Forces during B Cell Responses
Katelyn M. Spillane, Pavel Tolar.
Immune Cell Biology, National Institute for Medical Research, London,
United Kingdom.
B cells are components of the adaptive immune response that produce high-
affinity antibodies that neutralize and provide immunity against infections.
Their responses are initiated when the B cell receptor (BCR) binds foreign
antigen, triggering intracellular signaling that leads to B cell activation. Current
evidence suggests that to elicit a full immune response involving development
into high-affinity memory and plasma cells, B cells need to acquire the antigen
from immune synapses with antigen presenting cells, and then internalize, pro-
cess and present it to receive T cell help. Recent studies from our group suggest
that B cells use contractile force to physically extract antigen from the present-
ing membrane prior to endocytosis. In addition, it was shown that pulling on the
BCR-antigen bond results in discrimination between high- and low-affinity in-
teractions. This suggests that B cells mechanically test the strength of BCR-
antigen bonds to actively regulate affinity discrimination, a process that is
important for the efficient development of high-affinity antibodies. To charac-
terize force generation in the B cell synapse, we combine live-cell imaging with
molecular force sensors. For the first time, we resolve the spatiotemporal dy-
namics and magnitude of mechanical forces in the B cell synapse, giving us
insight into how B cells use mechanical forces to actively regulate their re-
sponses to antigen binding.
705-Pos Board B485
Atomic Force Microscopy of Triple Negative Breast Cancer Cells: A Pre-
dictive Value of Mechanical Phenotype
Pawel A. Osmulski, Karolina Wlodyga, Tim Hui-Ming Huang,
Maria E. Gaczynska.
Molecular Medicine, UTHSCSA, San Antonio, TX, USA.
We applied Quantitative Nanomechanical Atomic Force Microscopy (QNM-
AFM) to test a response of physical phenotype of triple negative breast cancer
cells to treatment with proteasome inhibitors. Genetic screens identified
TNBCs as addicted to the activity of the ubiquitin-proteasome pathway, the
major intracellular venue of regulated protein degradation, served by the
essential protease, the proteasome. We investigated two types of proteasome
inhibitors: the drug bortezomib (Velcade) targeting active sites of the
enzyme and a novel allosteric inhibitor B1 interfering with stability of the
26S proteasome. Mechanical phenotype constitutes a sensitive indicator of
physiological status of cells. The most explored mechanical parameters are
cell elasticity (‘‘softness’’) and surface adhesiveness (‘‘stickiness’’). It has
been established that cancerous transformation makes cells softer and less
sticky. These differences are attributed to remodeling of the cytoskeleton
and altered expression of membrane proteins. Therefore, the physical pheno-
type should have a predictive value as the early indicator of the cells’ response
to a drug treatment and disease stage. Indeed, it has been shown that exposure
of cancer cells to anticancer drugs at least partially reverses their mechanical
phenotype resembling healthy cells. Here we found that subjecting breast
cancer cells to low doses of bortezomib increased their stiffness and adhesive-
ness about two times. Moreover, similar treatment with low doses of B1
induced almost a threefold increase of stiffness and a twofold increase in
cell adhesion. Surprisingly, much higher doses of the drugs were required
to inflict detectable changes in cells viability or morphology. The results point
at the extraordinary sensitivity of the mechanical phenotype to detect cells’
response to anticancer drugs. We are exploring the potential predictive value
of AFM-based cell surface studies in a search for effective drugs or drug
combinations.


Atomic Force Microscopy of Triple Negative Breast Cancer Cells: A Predictive Value of Mechanical Phenotype 

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Atomic Force Microscopy of Triple Negative Breast Cancer Cells: A Predictive Value of Mechanical Phenotype

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