Compression induced formation of Actin bundle networks in Adherent Cells

Burhan Saif Addin

Ming Guo, Weitz LabHarvard University

ES 298rMar 23, 2012

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Outline

• Introduction and Motivation • Background Information• Project Idea• Results and Discussion• Conclusion

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Introduction

• What is the Cytoskeleton (CSK) :– 3D protein scaffold.– Determine structure, shape, organization, .... – More than 8% of Human Genome encodes for cytoskeleton– Cytoskeleton mechanics are not well understood and the search for universal

laws , if any, is in progress. – Contain three major skeletal proteins: actin, intermediate filaments, and

microtubules.

• What is role of Actin Filaments :– Cell motility and adhesion – Cell mechanical response and modulation.

en.wikipedia.org/wiki/Cytoskeleton

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Motivation: Why study cells actin networks?

• How life evolved ?• Morphogenesis ?• How cytoskeleton work ? Cell Motility, etc ?• How Cells sense and process information ? • To understand Cell Mechanics and stiffness:– Physics not well understood. Are there universal laws ?– Understand Diseases. For example: Malaria and Cancer.– Cells signaling and gene expressions.

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Adherent Cells feel and respond to the stiffness of their substrate

“Tissue Cells Feel and Respond to the Stiffness of Their Substrate” Discher et al, Science, 18 Nov 2005.

Esoft~ 1kPa Estiff~ 30-100kPa

Stress versus strain illustrated for different soft tissues

Mechanism for Cellular response to substrate stiffness are not well understood. Possible Mechanisms:

-Molecular pathways.-Volume change ?

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How do we know that reducing the volume of the cell induce actin formation ?

• In actin solution droplets, Huber et al showed that increased ion concentration and actin concentration led to highly ordered and regularly spaced actin bundle networks without the need for crosslinkers or motors.

• In this work, we compress the cell by increasing the osmotic pressure of the

cell’s medium by adding PEG (suck water out of cell) to the solution. Kas et al. Biophysics Society Meeting 2012, San Diego. “Counterion Induced formation of regular actin bundle networks”. Hubber et al. Soft Materials. March 2012

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Cells on Glass without compression

Images are by confocal fluorescence microscopy

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Cells on Gel Before Compression

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Compressed Cells on Gel (10% PEG solution)

Volume decrease might be the mechanism that induce actin networks formation thus cells stiffness.

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Actin’s Dye Fluorescence Intensity across the lines in the left figure which are cell’s cross-sections. .

Green line is ~31 μm)

Rough Results:Without compression:0.28 peaks/umWith compression:1.72 peaks/um

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Possible Problems with this method of dye intensity quantization

• Reproducibility.• Highly dense Interlinked actin filaments. • Unlabeled actin filaments might not be

uniform*.

*Polar patterns of driven filaments. Nature. Volker Schaller et al. 24 June 2010.

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Other method for quantifying actin bundles networks ?

We attempted to quantify the Actin based on intensity.How do you quantify this ?

“Counterion Induced formation of regular actin bundle networks”. Hubber et al. Soft Materials. March 2012

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Conclusion

• Volume change in cell can change Actin concentration or ion concentration, and hence induce actin polymerization.

• Perhaps the substrate stiffness effect is not the only reason for actin polymerization.

• Is actin polymerization due to volume change or due substrate or chemical signaling ? This remain to be investigated.

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Acknowledgment

• Ming Guo

• Weitz Lab Members

• Prof. David Weitz

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Additional Slides

Cell Volume decreases with increasing Substrate Stiffness

softer stiffer

Mung Gou and Dave Weitz, unpublished Data

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Cells on Glass before compression

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Compressed Cells on Gel