Murdaugh, Anne E.

Rodgers, Nicole C.

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The Effects of Medically Relevant Compounds on the Physical Properties of Biofilms 

388a Monday, February 17, 2014targeting riboflavin binding protein (RFBP), which acts as a model protein for
the riboflavin receptor. A single molecule force spectroscopy study using
Atomic Force Microscopy (AFM) was performed to examine the binding inter-
actions between riboflavin-conjugated dendrimers and RFBP. Using multiple
theoretical models, the apparent kinetic and thermodynamic parameters for
the riboflavin-conjugated dendrimer system were estimated.
1957-Pos Board B687
Atomic Force Microscopy of DNA-CTAB Aggregates
Adam Rimawi, Pamela M. St. John.
Chemistry, SUNY New Paltz, New Paltz, NY, USA.
Past studies of DNA-surfactant aggregates involving UV-spectroscopy and gel
electrophoresis show that when 20 to 100 base-length oligonucleotides interact
with the surfactant CTAB in specific ratios of surfactant to oligonucleotide
base, they form aggregates. The interaction between the oligonucleotide and
surfactant molecule appears to involve both the hydrophilic head and the hydro-
phobic tail of CTAB. We characterized these aggregates using an atomic force
microscope (AFM) by drying DNA-CTAB solutions on mica and silicon,
reconstituting the dried aggregates in buffer and imaging them in fluid. Topo-
graphical data was collected that showed consistent, almost hexagonal, regular
patterns of aggregates on the surface, and we studied the surface properties of
the aggregates using force spectroscopy with functionalized AFM tips. The
spring constants of the aggregates and information on the adhesion forces be-
tween the derivatized tips and the aggregate surfaces were obtained. Prelimi-
nary results show that tips functionalized with 1-dodecanethiol were found to
have a lower adhesion to the aggregate surface than tips functionalized with
mercaptoundecanoic acid implying that the surface of the aggregate is also
hydrophilic.
1958-Pos Board B688
Energetics and Kinetics of SNARE Zippering
Yongli Zhang.
Yale University, New Haven, CT, USA.
SNARE proteins drive membrane fusion by assembling into a four-helix bundle
in a zippering process. The energetics and kinetics of SNARE zippering have
not been well characterized, but are critical to understand the functions of
brains and other basic cellular processes, as well as their associated diseases.
We used high-resolution optical tweezers to observe in real time a long-
sought SNARE assembly intermediate in which only the membrane-distal
N-terminal half of the bundle is assembled. Our finding supports the zippering
hypothesis, but suggests that zippering proceeds through three sequential
binary switches, not continuously, in the N- and C-terminal halves of the
bundle and the linker domain. The half-zippered intermediate was stabilized
by externally applied force which mimicked the repulsion between apposed
membranes being forced to fuse. This intermediate then rapidly and forcefully
zippered, delivering free energy of 36 kBT to mediate fusion. Thus, the synap-
tic SNARE complex seems perfectly evolved for precise regulation of neuro-
transmitter release: Slow assembly of the membrane-distal half allows
control of vesicle priming by regulatory factors that can accelerate this step.
The discrete pause in the half-zippered state in the presence of a force load en-
ables clamping and thus regulation of release at this stage. Finally, the
membrane-proximal domain zippers at the diffusion-controlled rate limit to
allow neurotransmitter to be released.
1959-Pos Board B689
Dengue Virus Capsid Protein Interacts Specifically with Very Low-Density
Lipoproteins
Andre´ F. Faustino1, Filomena A. Carvalho1, Ivo C. Martins1,
Miguel A.R.B. Castanho1, Ronaldo Mohana-Borges2, Fabio C.L. Almeida2,
Andrea T. Da Poian2, Nuno C. Santos1.
1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa, Lisbon, Portugal, 2Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil.
Dengue virus (DENV) infects millions of people worldwide. With no specific
treatment available, understanding its replication mechanisms is highly
required to identify future therapeutic targets. In this study [1], mostly using
AFM-based force spectroscopy [2], but also DLS, NMR and computational
studies, we show that DENV capsid protein (C) binds specifically to very
low-density lipoproteins (VLDL) but not to low-density lipoproteins (LDL).
DENV C-VLDL binding is similar to DENV C interaction with lipid droplets
(LDs), host intracellular structures essential for viral replication [3]. As on the
DENV C-LDs binding, previously characterized by us [4-6], DENV C-VLDL
interaction is Kþ-dependent, involves the DENV C intrinsically disordered N-
terminus, and is inhibited by pep14-23, a novel peptide drug lead against
DENV [5,6]. As perilipin 3 (DENV C target on LDs [3]) is structurally similar
to the VLDL protein ApoE, this protein may be the DENV C ligand on VLDL,explaining the similarities between the interactions of DENVCwith VLDL and
LDs. These data suggest the possibly of lipoviroparticles formation, as it occurs
for other flaviviruses, such as the hepatitis C virus (HCV) [7]. This process may
potentially be targeted, for DENV life cycle inhibition.
[1] Faustino et al. (2013) Nanomedicine NBM, in press.
[2] Carvalho et al. (2012) IUBMB Life 64:465.
[3] Samsa et al. (2009) PLoS Pathog 5:e1000632.
[4] Carvalho et al. (2012) J Virol 86:2096.
[5] Martins et al. (2012) Biochem J 444:405.
[6] Patent WO/2012/15918.
[7] Bartenschlager et al. (2011) Trends Microbiol 19:95.
1960-Pos Board B690
Characterizing the Interaction of Desmosomal Cadherins at Single
Molecule Level
Omer Shafraz1, Sabyasachi Rakshit1, Molly Lowndes2, W. James Nelson2,
Sanjeevi Sivasankar1.
1Iowa State University, Ames, IA, USA, 2Stanford University, Stanford, CA,
USA.
Desmosomes are cell-cell adhesion complexes that are present in tissues that
resist mechanical stress. They are mainly composed of two adhesive proteins,
which are members of the cadherin superfamily of cell adhesion proteins, des-
mocollin (Dsc) and desmoglein (Dsg). However, the role of these proteins in
desmosomal adhesion is unclear. Here, we use the single molecule force spec-
troscopy with an Atomic Force Microscope (AFM-FS) to characterize the inter-
actions of type-2 isoforms of desmocollin (Dsc2) and desmoglein (Dsg2). We
show that Dsc2 forms Ca2þ dependent homophilic bonds by swapping a
conserved Tryptophan (Trp) residue between opposing binding partners;
mutating this Trp inhibits Ca2þ dependent homophilic binding. In contrast,
Dsg2 forms Ca2þ independent heterophilic bonds with Dsc2 via a mechanism
that does not involve Trp strand-swapping.
Previous studies suggest that desmosome formation requires the presence of
classical cadherins at the site of desmosome assembly. This suggests a
cross-talk between desmosomal and classical cadherins at cell-adhesion con-
tacts. We therefore used AFM-FS to test if Dsc2 and Dsg2 interact with
E-cadherin, a classical cadherin present in the epithelium. Our data shows
that while Dsc2 does not bind to E-cadherin in the presence of Ca2þ, Dsg2
forms Ca2þ independent complexes with E-cadherin. Using cadherin mutants
we show that the interactions between Dsg2 and E-cadherin occur via a pre-
viously uncharacterized binding interface that does not involve either Trp
strand-swapping or X-dimer formation (two well established classical cad-
herin binding mechanisms).
1961-Pos Board B691
Single Molecule Characterization of the Role of Divalent Ions in Prion
Protein Aggregation
Chi-Fu Yen, Sanjeevi Sivasankar.
Iowa State University, Ames, IA, USA.
Transmissible Spongiform Encephalopathies (TSEs) are a class of neurodegen-
erative disorders characterized by the accumulation of misfolded prion protein
aggregates in the brain. In these diseases, normal cellular prion protein (PrPC)
misfolds into an infectious scrapie isoform (PrPSc), which results in PrPC aggre-
gation and neurotoxicity. Metal ions are known to play an important role in pro-
moting PrPC misfolding and aggregation, though the underlying mechanisms
are poorly understood at molecular level.
Here we use single molecule Atomic Force Microscope-Force Spectroscopy
(AFM-FS) and Confocal Fluorescence Microscopy (CFM) to determine how
divalent metal ions mediate PrP aggregation in vitro. Using AFM-FS, we
show that divalent metal ions enhance the aggregation of misfolded PrP by
increasing the on-rate for aggregate formation; the lifetimes of aggregates
are however unaffected. Increase in association rate is most pronounced
when PrP is exposed to Cu2þ and less pronounced in the presence of Zn2þ,
Ni2þ and Mn2þ. Using CFM we show that binding of metal ions to PrP induces
a conformational change that makes the protein resistant to Proteinase-K diges-
tion.Finally, we show that enhanced PrP aggregation is eliminated upon
removing the octapeptide repeat, a major metal binding site on PrP.
1962-Pos Board B692
The Effects of Medically Relevant Compounds on the Physical Properties
of Biofilms
Nicole C. Rodgers1, Anne E. Murdaugh2.
1Rollins College, Winter Park, FL, USA, 2Physics, Rollins College, Winter
Park, FL, USA.
The adaptation of bacteria to form biofilms has been linked to many prob-
lems in the medical field including the rise of drug-resistant superbugs.
Exopolymeric substances (EPS), including carbohydrates, proteins, and
Figure 1: representative SCFS curves for rigid
NWs, flexible NWs and collagen-coated cover-
slips.
Monday, February 17, 2014 389apolysaccharides, secreted by the bacteria provide protection from harmful
chemical elements, such as chlorhexidine digluconate, commonly found in
medical grade soap. Much is known about the biological and chemical mech-
anisms of chlorhexidine digluconate as an antiseptic, whereas the physical
response of the bacterial biofilm to the chemical is poorly understood. In
this investigation, we used force spectroscopy to analyze the physical effects
of varied concentrations of chlorhexidine digluconate on a robust biofilm-
forming Escherichia coli strain. Preliminary data suggest a correlation be-
tween the stiffness (spring constant) of the biofilm and the concentration
of chlorhexidine digluconate.
1963-Pos Board B693
Increased Cytoskeletal Stiffness of Schlemm’s Canal Endothelial Cells in
Glaucoma
Rocio Vargas-Pinto, Mark Johnson.
Northwestern University, Evanston, IL, USA.
The elevated intraocular pressure characteristic of glaucoma has been associ-
ated with a decreased pore density in the cells of the inner wall of Schlemm’s
canal (SC). SC cells form these pores in response to a transcellular pressure
drop, facilitating the flow of aqueous humor across this endothelium. We
hypothesize that impaired pore formation in glaucoma is due to an elevated
stiffness of SC cells.
Atomic force microscopy (AFM) measurements of elastic modulus were per-
formed using pyramidal or spherical (4.5 or 10 mm) tips on SC cells isolated
from 6 healthy and 5 glaucomatous human eyes. Using finite element modeling
and AFM experiments with latrunculin-A (an F-actin depolymerizing agent),
we previously showed that sharp pyramidal tips characterize the cortex stiffness
while larger, spherical tips characterize the stiffness of the subcortical cytoskel-
eton. The geometry of an SC cell was reconstructed based on electron micro-
scopy images and used to model cell deformation under pressure (3-6 mmHg).
When probed with spherical tips, the modulus of glaucomatous cells
(1.3650.14 kPa) was significantly higher (p<0.02) than that of healthy cells
(0.8950.10 kPa). No significant difference was detected between healthy
and glaucomatous cells using sharp tips (7.4051.33 vs. 7.9950.97 kPa).
The higher modulus measured on glaucomatous cells using spherical tips sug-
gest that the altered stiffness is likely in the subcortical cytoskeleton and not in
the cell cortex. Preliminary studies using finite element modeling predict a
34-40% decrease in cell deformation solely due to the increased cell stiffness
measured on glaucomatous cells. This is consistent with our hypothesis that
cell deformation is likely a precursor to the pressure-driven pore formation pro-
cess and that increased cell stiffness may inhibit this process.
Acknowledgements: BrightFocus Foundation, NIH EY019696, NIH T32
EY007128.
1964-Pos Board B694
Cell Viscoelasticity as a Function of Substrate Stiffness
Rachel Brunetti1, Gawain Thomas2, Qi Wen2.
1Physics, Scripps College, Claremont, CA, USA, 2Physics, Worcester
Polytechnic Institute, Worcester, MA, USA.
While it is known that cells respond to the mechanical properties of their envi-
ronment, limited information is available regarding mechanosensing and the
means by which such stimuli are transduced. The cytoskeleton, a critical
component used by cells to sense substrate stiffness, determines the viscoelas-
ticity of cells. Information on cell viscoelasticity as a function of substrate stiff-
ness and vimentin levels can lead to a larger composite model of the
mechanism through which cells respond to external mechanical stimuli.
3T3 fibroblasts were grown on collagen-coated polyacrylamide gels, of which
the stiffness was controlled by varying both the concentrations of acrylamide
and bisacrylamide. An atomic force microscope (AFM) was used to perform
dynamic micromechanical tests. The force-mapping mode was employed to
obtain 24 by 24 maps of force curves. For each force curve, the AFM first in-
dents a cell with a 700 pN force and then applies a 10 Hz sinusoidal strain to the
cell. The storage, E’, and loss modulus, E’’, were calculated by fitting the sinu-
soidal portion of the applied force and resulting indentation to obtain the ampli-
tude and phase offset.
Both E’ and E’’ increase with substrate stiffness. While the increase in E’ is
consistent with literature, we found that the ratio of E’’ to E’ decreases as sub-
strate stiffness increases. This agrees with our hypothesis that cells become
more solid-like as the elastic modulus of the extracellular matrix increases.
Additionally, the value of E’ was higher in vimentin null cells, suggesting
that cells change their cytoskeletal composition to adapt to the loss of vimentin.
This may involve an increase in the concentration of actin filaments, which
contribute more to the elasticity than vimentin. Further experiments will inves-
tigate the potential link between vimentin expression and the mechanosensing
ability of the cells.1965-Pos Board B695
A Systematic, High Resolution Mapping of the Elastic Modulus of Mouse
Cartilage Matrix
Preethi L. Chandran1, Jessica Yuan1, Ferenc Horkey2, Edward Mertz3,
Emilios K. Dimitriadis1.
1BEPS, NIBIB, NIH, Bethesda, MD, USA, 2STBB, NICHD, NIH, Bethesda,
MD, USA, 3SPB, NICHD, NIH, Bethesda, MD, USA.
Hyaline cartilage cushions bone at articular joints and is responsible for elon-
gation and mechanical integrity of growing bones. The major components of
cartilage matrix are the collagen mesh and the highly charged proteoglycan
aggrecan entangled in and swelling the mesh. In growing cartilage, the compo-
sition and organization of these components varies across both growth plate and
articular cartilage and in the vicinity of chondrocytes. Mice are extensively
used as model system for a wide range of pathologies including arthritis, but
there are limited studies mapping matrix nanomechanics across different re-
gions. Nanomechanical studies, typically performed by indenting tissue sec-
tions, are subject to errors due to matrix collapse, surface roughness and
diffusional loss aggrecan loss from the section surface. Moreover, there are
often issues of data interpretation when the probe size and indentation depth
are comparable to the mesh and aggrecan sizes.
We performed a high-resolution (~1um) nanoindentation elasticity mapping of
extracellular matrix material of mouse growing cartilage across different re-
gions of both cartilage types in physiological solution. We used an AFM
with microsphere probes whose radii and indentation depth exceeded mesh
and molecular sizes. We mildly fixed the cartilage to prevent aggrecan loss.
We developed a new method for obtaining the Hertzian contact points on rough
surfaces. We applied large-strain indentations and force corrections to remove
the error due to surface roughness and to select the linear regime of the force
data. The latter greatly improved the consistency of our elasticity results. Tis-
sue collapse was partially corrected using the cartilage height mapping. Matrix
regions were extracted by correlation with optical images. The matrix elasticity
across the growing cartilage showed interesting difference between the two
cartilage types and correlations with previous studies of matrix composition
at the same locations.
1966-Pos Board B696
Cell Adhesion on Silicon Nanowires
Anna Murello1, Laura Andolfi2, Damiano Cassese1, Marco Lazzarino1,2.
1laboratorio TASC, IOM-CNR, Trieste, Italy, 2Consorzio per la Biomedicina
Molecolare (CBM), Trieste, Italy.
Understanding nanostructured materials - cell interactions is essential for the
development of innovative substrates for cell culture, differentiation and pre-
cise electrical stimulation. In this framework, silicon nanowires (SiNWs),
characterized by high aspect ratio and a broad range of mechanical, optical
and electrical properties, are interesting nanomaterials for innovative cellular
engineering. We investigated and quantified, using single cell force spectros-
copy (SCFS), the interaction of murine embryonic fibroblast with two me-
chanically different SiNW substrates, and two reference substrates, collagen
gel which is generally used for cell culture, and flat glass which has the
same chemical reactivity of SiNWs. We observed comparable adhesion values
on SiNWs substrates and on gel substrate, while flat glass generate a 10-times
weaker interaction. A closer analysis of the adhesion curves revealed significant
differences between soft and hard SiNW, suggesting that a different cell mem-
brane organization is developed depending on the mechanical and geometrical
properties of the substrate.
In conclusion SiNW sub-
strates are compatible with
cell engineering and thanks
to their high range of me-
chanical and geometric pro-
perties, a fine tuning of
cellular response can be
achieved.
1967-Pos Board B697
Characterization of Mechanotransensitivity of Articular Chondrocytes
Whasil Lee, Holly Leddy, Farshid Guilak, Wolfgang Liedtke.
Duke University Medical Center, Durham, NC, USA.
Articular cartilage is a protective soft connective tissue lining the ends of bones
in weight-bearing joints. It sustains numerous cycles of mechanical loading
during normal joint activity. Recent studies showed that mechanical factors
not only play a critical role in the pathology of cartilage, e.g. initiation of
degenerative joint disease such as osteoarthritis (OA); but also influence the
metabolic response of chondrocytes, the only cellular element of cartilage.
One of the early events implicated in chondrocyte mechanotransduction is a


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The Effects of Medically Relevant Compounds on the Physical Properties of Biofilms

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