Transcript Effects of substrate topography on actin dynamics and

REU program sponsored by the
National Science Foundation
Award Number: PHY1156454
Effects of substrate topography
on actin dynamics and B cell
receptor clustering
Jacob Fondriest, Christina Ketchum, Arpita Upadhyaya
TREND 2013
Training and Research Experience in Nonlinear Dynamics
Actin and B lymphocyte Behavior
• B cells circulate through the body
in search of infection.
• Upon contact with a certain
antigen, the B cell spreads to
contact more antigen.
• Over time, the B Cell Receptors
(BCR) that contact the antigen
begin to form clusters.
• Actin plays a role in almost every
cell operation, and has been
shown to be linked to BCR
clustering.
BCR
clusters
Motivation
• How does substrate topography
affect actin dynamics and BCR
movement?
• What is the relationship between
the movements of the actin
cytoskeleton and the movements of
BCR clusters?
• Dr. Fourkas and Xiaovu Sun, from the
University of Maryland, provide
nanopattern substrates which
simulate the topography of antigen
presenting cells.
W
Y
X
Experimental Methods
•
We use B cells from mice, genetically modified with GFP and labeled with mbFab
AF546.
•
We use Total internal reflection fluorescence (TIRF) microscopy and to record
videos of B Cell actin and BCR dynamics on the nanopattern substrates.
3 µm
Overlay
Brightfield
Actin
BCR
Particle Image Velocimetry analysis using MATLAB
• Track actin and BCR as they
move.
• PIVlab creates a vector field
for every frame of the
video.
• Measure average velocity
and net flow rates and
directions
• Specify Regions of Interest
for analysis
On Ridge
Between
Ridges
3 µm
PIVlab - Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB
Developed by Dipl. Biol. William Thielicke and Prof. Dr. Eize J. Stamhuis
3 µm
Direction of Flow with respect to ridges
Actin
B Cell Receptors
• The direction of actin and BCR flow is mostly chaotic, but
slightly favors movement along the ridges.
Resultant displacement over time
Actin
BCR
Time Lapsed:
5 Minutes
• Over a longer period of time, the chaotic drift reveals much more clear paths.
• If we put particles in the center of the cell, these are the paths they might take.
Role of ridge spacing on actin velocity dynamics
3 µm ridges
5 µm ridges
Comparison by using a student’s T-Test reveals significant evidence that on 3
µm ridges, actin moves more quickly between the ridges than on the ridges.
The actin is unaffected on 5 µm ridges.
Actin and BCR line intensity profile comparison
Time Lapsed: 15 seconds
3 µm
3 µm
•
•
Actin and BCR clusters tend to aggregate near ridges, resulting in greater intensity values
at those areas.
Even between ridges there is apparent colocalization of actin and BCR.
Future Work
• Investigate the effects of more narrow nanopatterns on the dynamics
of actin and BCR clusters. Our research suggests narrower gaps will
have more profound effects on actin and BCR dynamics.
• Study the effect of nanotopography on B cell activation.
• Perturbation experiments to examine the role of various cytoskeletal
components (e.g. actin and myosin) on cell responses to
nanotopography.
• Improve Matlab software to streamline analysis and improve accuracy
Acknowledgements
• Christina Ketchum
• Dr. Arpita Upadhyaya
• Ming Zhang
• Mikheil Azatov
• Dr. John Fourkas and Xiaoyu Sun (Fourkas Lab)
• TREND Program
• NSF