Annette Doyle
School of Pharmacy & Biomolecular Sciences &
General Engineering Research Institute
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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.
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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
Intermediate filaments
(Green)
Double Membrane
Nucleus
Microtubules
(Blue)
Actin Cortex
(Red)
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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
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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
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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
Image actin filaments in the absence and
presence of bundling protein using:
Confocal microscopy
Absorption Assay
Transmission Electron Microscopy
(TEM)
AFM
Actin+ protein 5:1 ratio
Actin only
Bundles formed in presence of protein
Further image analysis will be carried out on images
by Munther Gdeisat and Gary Johnston
Absorbance
Absorbance
Absorbance
Absorbance
Absorbance
Absorbance
Actin
Actin + protein
Time (minutes)
Quantitative measurement of
polymerisation – l=300nm
Initial rate of polymerisation different in
the presence of protein
Polymerisation more stable in the
presence of protein
Actin+ protein 5:1 ratio
Actin only
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.
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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
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Images will be obtained using the confocal
and AFM
Image processing will be carried out by Dave
Burton, Munther Gdeisat and Gary Johnson
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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?
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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
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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
Mark Murphy
Dave Burton
Steven Crosby
Stephane Gross
Gary Johnston
Munther Gdeisat