The Role of Substrate Topography on Actin Organization and Dynamics

KEVIN BELNAP (BRIGHAM YOUNG UNIVERSITY) MIKE AZATOV (UNIVERSITY OF MARYLAND) ARPITA UPADHYAYA (UNIVERSITY OF MARYLAND)

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Pancreatic Tumor Associated Fibroblasts

Fibroblast cells are critical to the support of most tissues because they secrete the proteins necessary for the development of the extracellular matrix (ECM) during tissue repair [1].

Diseased fibroblasts can lead to serious health problems. Such is the case in diseases like cystic fibrosis [1].

In the case of human pancreatic cancer, fibroblasts associated with the tumor contribute to high tumor density and have been shown to increase cancer proliferation and metastasis in vivo [2].

[1] Hinz, Boris, et al. "The myofibroblast: one function, multiple origins." The American journal of pathology 170.6 (2007): 1807-1816.

[2] Hwang, Rosa F., et al. "Cancer-associated stromal fibroblasts promote pancreatic tumor progression." Cancer research 68.3 (2008): 918-926.

Why Actin?

   Actin is a filamentous protein found in the cytoplasm of cells and is critical for cell motility in eukaryotes. Actin forms the stress fibers involved in cell adherence [3].

By observing the actin in the cell, we can observe how the cell interacts mechanically with its environment.

We use an actin cross-linking protein called palladin to identify the actin stress fibers in the cell. These cells have been transfected with green fluorescent protein (GFP), which serves as a marker on the palladin.

[3] Cassimeris, Lynne, Vishwanath R. Lingappa, and George Plopper, eds. Lewin's Cells. Second ed. Boston: Jones and Bartlett Publishers, 2011. 558 59. Print. Image obtained from: http://www.asknature.org/strategy/02ce72d9b4af1f7e 31f4efb5fb8a69d4#.U-KmduNdX-s

Motivation

 Cells encounter more complex topography in vivo than that which they are exposed to on a smooth glass slide (e.g. the collagen fibers in tissue)  Does substrate topography affect cell morphology, actin filament structure and dynamics?

 We have developed a model to study this question using ‘nanopatterns’ fabricated on glass slides

Nanopattern Design

Cross-sectional View “Aerial” View Nanopatterns were fabricated by X. Sun in the Lab of Dr. John Fourkas (UMD) 5 μm

Microscopy and Imaging

Internal Reflection Microscopy Epifluorescent Microscopy ≈ 95 μm Brightfield Microscopy ≈ 65 μm

Data Analysis-Elliptical Modeling

≈150 μm

Cell Area is a Function of Pattern Width

Varies from Control: * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001

Cell Shape is a Function of Pattern Width

Varies from Control: * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001

≈148 μm

Modeling Stress Fibers

The Cells Become More Oriented to the Pattern as Pattern Width Increases Varies from Control: * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001

The Stress Fibers Become More Oriented to the Pattern with Increasing Pattern Width Varies from Control: * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001

Summary of Results

  As the ridge width of the nanopattern increases, the tumor associated fibroblast cells become significantly more elongated and the corresponding cell area decreases. As the ridge width increases, the cell and its stress fibers approach a parallel orientation to the ridges.

Future Directions: Dynamic Analysis

  A significant portion of this project was spent developing an analysis method for the dynamics of the stress fibers using pre-existing algorithms.

We have several methods of analysis that our program performs that we hope to apply to multi-frame images of the cells. These methods include: -Mapping the positional and motive behavior of the palladin spots as they approach the ridges.

-Measuring the angular direction of the tracked fibers relative to the ridges. -Measuring diffusivity and directional persistence of the particles. Since various force fluctuations within the ECM can lead to noise in stress fiber behavior, it is important to determine the cause of motion [4].

[4] Raupach, Carina, et al. "Stress fluctuations and motion of cytoskeletal-bound markers." Physical Review E 76.1 (2007): 011918.

Conclusions

  Our results from the static image analysis suggest that tumor associated fibroblasts exhibit mechanosensing of environmental topography. However, the signaling pathway that leads to the observed behavior remains unclear.

Future work will include analysis of multi-frame images in order to consider dynamics which may lead to clues about the underlying mechanisms of topographical sensation and cytoskeletal response.

Acknowledgements

    Thanks to Dr. John Fourkas and Xiaoyu Sun of the Fourkas Lab of the University of Maryland for fabricating the nanopatterns.

Thanks to Arpita Upadhyaya, Mike Azatov, Christina Ketchum, and King Lam Hui for experimental instruction.

TREND Program NSF