Cytoskeleton bundling and its role in cell mechanics

1. Cytoskeleton bundling and its role in cell mechanics Annette Doyle School of Pharmacy & Biomolecular Sciences & General Engineering Research Institute

2. Project Rationale • The proteins that interact and bundle actin cytoskeleton have been shown to have high levels in various cancers • Therefore we aim to determine if these proteins bundle actin and would therefore have an effect on the cytoskeleton's mechanical properties.

3. Cell Structure • The mammalian cell consists of a double membrane surrounding a viscous fluid (cytosol) which contains a central nucleus • The Structure and mechanical integrity of the cell is governed by a mesh-like structure known as the cytoskeleton which is dispersed throughout the cytosol • This cytoskeleton consists of 3 components: • Microtubules • Actin Filaments • Intermediate Filaments

4. Cell Structure Intermediate filaments (Green) Double Membrane Microtubules (Blue) Nucleus Actin Cortex (Red)

5. Cell Structure • The filaments interact with each other, the cell membrane, nucleus and cell substrate • It has been suggested that the mechanical integrity of the cells is mainly due to the actin filaments with the microtubules and intermediate filaments contributing less • In healthy cells the cytoskeleton is highly organised • In cancer cells the cytoskeleton becomes disorganised

6. Why do we want to examine cytoskeleton bundling? • Many diseases such as cancer, Alzheimer's disease, Parkinson’s disease, aging etc, are associated with changes in cellular structure, particularly with the structural element the cytoskeleton

7. Role of Cytoskeleton • No one is certain about the full role of the cytoskeleton. However evidence shows that it is involved in: • Underwriting the mechanical integrity of the cell. • Acting as a messaging conduit. • Influencing cell growth and division. • Cell adherence and differentiation. • Diseases change the cytoskeleton’s structure and may change the cell’s mechanical behaviour • Cytoskeleton bundling is associated with other proteins in the cell

8. Actin Filament Formation • Image actin filaments in the absence and presence of bundling protein using: • Confocalmicroscopy • Absorption Assay • Transmission Electron Microscopy (TEM) • AFM

9. Images of Actin Filaments-Confocal Actin+ protein 5:1 ratio Actin only • Bundles formed in presence of protein • Further image analysis will be carried out on images by MuntherGdeisat and Gary Johnston

10. Actin Filaments-Absorption Assay • Quantitative measurement of polymerisation – l=300nm • Initial rate of polymerisation different in the presence of protein • Polymerisation more stable in the presence of protein Absorbance Absorbance Absorbance Actin Absorbance Absorbance Absorbance Actin + protein Time (minutes)

11. Images of Actin Filaments-TEM Actin+ protein 5:1 ratio Actin only

12. Images of Actin Filaments-AFM Actin only Actin only • The AFM will be utilised to determine the size of actin filaments and bundles formed in the presence of the proteins.

13. Variables affecting actin bundling • The ratio of actin:protein may have an effect on the bundle formations • At present ratio of actin:protein is 5:1 • A range of ratio from 1:1 to 5:1 will be investigated • Images will be obtained using the confocal and AFM • Image processing will be carried out by Dave Burton, MuntherGdeisat and Gary Johnson

14. Effect of actin bundling in the Cell • Higher levels of protein in the cell may cause more actin bundling or bigger bundles in the cytoskeleton • As mention the cytoskeleton is involved in the mechanical integrity of the cell • Changes to the cytoskeleton in the form of bundles may affect the elasticity or ‘stiffness’ of cells • It has been reported that cancer cells are up to 70% less stiff than healthy cells • This raises the question Do bundles restrict the response of the cell to force?

15. Important factors to consider • The studies reported in the literature in regard to this actin bundling protein have been done from many species. • Therefore it is difficult to accept what is found for plants and animals in regard to the human protein. For e.g. the yeast form of protein is only 80% similarity to human forms • It has been reported that cations can cause actin bundling by electrostatic mechanisms and we will investigate the effect of buffer alone on actin bundling • Also solution crowdedness has been reported to cause actin bundling-need to ensure correct controls present in experiments

16. Future Work • We will continue to look at actin polymerisation and bundling using the different bundling proteins • Using yeast and mammalian cells models the level of protein in the cells will be increased • We will investigates if over expression of the bundling protein has an effect on actin bundling and if such bundling has any possible influence on changes in cell mechanics. • We will use AFM to measure the stiffness and combine AFM force measurements and confocal fluorescence imaging

17. Acknowledgments Mark Murphy Dave Burton Steven Crosby Stephane Gross Gary Johnston MuntherGdeisat