School of Pharmacy & Biomolecular Sciences &

crookedjourneyΜηχανική

24 Οκτ 2013 (πριν από 3 χρόνια και 7 μήνες)

45 εμφανίσεις

Annette Doyle

School of Pharmacy &
Biomolecular

Sciences &
General Engineering Research Institute



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.


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

Nucleus

Double Membrane

Actin Cortex
(Red)

Microtubules
(Blue)

Intermediate filaments
(Green)


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


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


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


Quantitative measurement of
polymerisation


l
=300nm


Initial rate of polymerisation different in
the presence of protein


Polymerisation more stable in the
presence of protein

Time (minutes)

Absorbance

Absorbance

Actin +
protein

Actin

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.



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,
Munther

Gdeisat

and Gary Johnson



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?


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


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