1. Imaging Structural Proteins
Annette Doyle
School of Pharmacy & Biomolecular Sciences & General Engineering Research Institute

2. Contents
Introduction –AFM, Fluorescence Microscopy, Cell Structure, Cytoskeleton and Cell Mechanics
Project Rationale
Methodology
Results
Summary & Advantage of AFM

3. Introduction
Understanding how cells function is critical to understanding disease
To understand how cells function requires us to understand how cells are organised structurally at the molecular level
To do this requires the use of imaging technology which enables us to image structures at the sub-nanometre level

4. Atomic Force Microscope
Images collected in intermitting contact mode (tapping mode)
The laser spot is aligned at the end of the cantilever on the opposite side of the tip and deflected onto a photo detector
In tapping mode the cantilever is oscillated at, or near, its resonance frequency at a set-point amplitude
Any changes from the set-point amplitude value of the oscillating cantilever are recorded and the image is constructed
Height and amplitude images are captured as the tip scans the sample

5. Fluorescence Microscopy
A Protein/Cell is stained with a probe specific to the protein of interest with a fluorescent conjugate
The conjugate when subjected to a laser at specific wavelength (excitation) emits light at a different wavelength (emission)
The emitted light rays are recorded and the image constructed
Multiple fluorescent probes can be used, but must ensure no cross over in the excitation or emission wavelengths

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

7. Cytoskeleton
Acts as a ‘skeleton’ providing the cell with shape, mechanical integrity and ability to move.
Consists of 3 types of filamentous protein
Actin
Microtubules
Intermediate Filaments
These extend throughout the cell forming a complex but organised network

8. Cytoskeleton
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
In healthy cells the cytoskeleton is highly organised
In pathological cell such as cancer cells the cytoskeleton becomes disorganised

9. Cytoskeleton Fluorescent image of MRC5 human lung fibroblast cells
Actin filaments (green), DNA (blue)
Actin stress fibres present, which play an important role in the cell function

10. Cell Mechanics
Mechanical properties are defined by the cytoskeleton and the volume of a cell
Much of the work is based on understanding the cell mechanics of normal cells and change, if any that occur in pathological cells
Higher levels of protein in the cell may cause more bundling or a change in orientation of the cells or even bigger bundles

11. Cell Mechanics
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 significantly less stiff than healthy cells
Most research is based on a complete cell but we are looking at the organisation and reorganisation of purified actin in the presence of a bundling protein

12. 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
Today, we will concentrate on the imaging to gain information about the structure and size of filaments

13. Methodology For initial experiments purified proteins were used
Actin was purified from rabbit muscle and the bundling protein from yeast cells
Actin filaments were imaged in the absence and presence of a bundling protein

14. Mica with Buffer
Mica substrate was imaged to ensure no artefacts are detected from buffer solution used to enable filament formation

15. Actin Filaments
Figure 1: 3D height image of actin filaments
Figure 2: Height image of unstable filaments – Monomeric actin
Actin filaments were imaged (fig 1) and correspond to the size quoted in the literature 7-8nm
Imaging actin filaments was difficult as they were unstable (fig 2)

16. Actin Filaments with Phalloidin
Fig 3: Confocal fluorescent image of actin filaments
Fig 4 & 5: AFM 3D height image of actin filaments
Phalloidin stabilises actin filaments
Filament size are slightly larger with phalloidin attached – 8-11nm
Actin monomers form a double helix filament

17. Actin Filaments with Phalloidin
Fig 6: 3D height image with phalloidin
Helical structure of actin filaments, size shown by upper graph, major cleft, approx 30nm
Fluorescent conjugate (indicated with arrow)
Size of conjugate measured and shown in lower graph, approx 1.2nm (reported to be between nm)

18. Actin with the bundling protein
Fig 8: 3D height image of actin and bundling protein bound along the filament
Fig 7: Amplitude image
It can be clearly seen that the bundling protein causes a change in organisation of actin, causing filaments to bundle together

19. Actin with the bundling protein
Fig 10: Amplitude image
Fig 9: Phase image
Phase imaging (Fig 9) provides nanometre scale information about the surface structure of molecules
Can distinguish between actin filaments and bundling protein
Shows bundling protein bound along the filament
Amplitude image (Fig 10) show the bundling protein between two filaments

20. Actin with the bundling protein and Phalloidin
Fig 12: 3D AFM height image of actin bundles
Fig 11: Confocal fluorescent image of actin bundles
Fig 13: AFM height image of actin bundles

21. Summary
AFM enables clear imaging of actin filaments and the binding of proteins to the filaments
Greater resolution that fluorescence microscopy
More information on the structure and size of actin filaments
The bundling protein binds to and re- organises actin filaments

22. Advantage of AFM imaging
AFM enables imaging at the molecular level, without interference of labelling molecules
Important that we can image the molecule involved in cell mechanics and investigate how they interact with each other

23. Acknowledgements Dr. Mark Murphy Dr. Steven Crosby
Professor David Burton
Dr. Francis Lilley
Professor Michael Lalor