1. 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 REU program sponsored by the National Science Foundation Award Number: PHY1156454

2. 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

3. Motivatio n 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 X Y

4. 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. Overlay Brightfield ActinBCR 3 µm

5. 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 PIVlab - Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB Developed by Dipl. Biol. William Thielicke and Prof. Dr. Eize J. Stamhuis Between Ridges On Ridge 3 µm

6. Direction of Flow with respect to ridges The direction of actin and BCR flow is mostly chaotic, but slightly favors movement along the ridges. Actin B Cell Receptors

7. Resultant displacement over time 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. Time Lapsed: 5 Minutes ActinBCR

8. 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. Role of ridge spacing on actin velocity dynamics 3 µm ridges5 µm ridges Between ridges On ridges

9. Actin and BCR line intensity profile comparison Time Lapsed: 15 seconds Actin and BCR clusters tend to aggregate near ridges, resulting in greater intensity values at those areas. Even between ridges there is definitive colocalization of actin and BCR. Intensity charts confirm B cell receptors tend to form clusters, whereas actin is more evenly distributed throughout the cell. 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. 3 µm

10. 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

11. Acknowledgeme nts Christina Ketchum Dr. Arpita Upadhyaya Ming Zhang Mikheil Azatov Dr. John Fourkas and Xiaoyu Sun (Fourkas Lab) TREND Program NSF