ABSTRACT fibres should be visible, as well as

ABSTRACT

The cytoskeleton, with its three major
cytoskeletal filaments are responsible
for the cells spatial organisation and mechanical properties. The cytoskeleton
has a variety functions including, giving shape to cells, allowing for cell
movement, enabling movement of organelles within the cell, endocytosis, and
cell division. The protein filaments that come together to serve these
functions include the actin filaments,
the microtubules and the intermediate filaments. Confocal microscopy is used to
visualise and observe the cytoskeleton
and is particularly useful to view the actin filaments following fixation and
staining of HeLa cells using FITC-Phalloidin/DAPI/ Alexa Fluor 568 goat
anti-rabbit combined stain. Dynamic structures associated with the actin
filament, such as filopodia, lamellipodia and stress fibres should be visible,
as well as focal adhesions and the
nucleus.

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INTRODUCTION

In
order for cells to function they must be
able to maintain correct shape, move in space, and rearrange their structure as
they grow, divide and adapt to environmental changes while maintaining contact
with each other. The structure that enables a cell to do so is called the
Cytoskeleton (Alberts et al, 2014), which is a network of filaments and tubules
that interlink and extend through the cytoplasm; from the nucleus to the plasma
membrane (Hardin & Bertoni, 2015).

It
was in 1903 that Nikolai K. Koltsov proposed that the shape of cells were determined by a network of tubules which
he called the cytoskeleton, and in 1929, Rudolph Peters suggested the idea of a protein mosaic that dynamically
coordinated cytoplasmic biochemistry (Peters, 1963). It was originally thought
that the cytoskeleton structure was unique only to eukaryotes but this was
shown to not be the case in 1992 due to research findings that showed bacteria
contained homologues of both tubulin and actin which are the main components of
the cytoskeleton (Wickstead & Gull, 2011). It is now agreed that the
cytoskeleton is present in all domains of life which include archaea, bacteria and eukaryotes (Hardin & Bertoni,
2015). The three cytoskeletal structures include:

Actin
Filaments – Also called microfilaments, they
underlie the plasma membrane of animal cells to provide strength and shape to
its lipid bilayer. It is composed primarily of actin and the transmembrane
focal adhesions.  Focal adhesions and
adherens junctions are membrane-associated complexes that have a role as
nucleation sites for actin filaments as well as being the cross-linkers between
the cell exterior, plasma membrane and actin cytoskeleton.
Focal adhesions serve a structural function, allowing the linking of the ECM on
the outside with the actin cytoskeleton on the inside. It provides powerful
signal transduction system which initiates signalling pathways in response to
adhesion. Focal adhesions are associated
with protein complexes containing vinculin, talin, a-actinin, paxillin, tensin,
zyxin and focal adhesion kinase (FAK.) Actin filaments also form structures such
as the lamellipodia and filopodia through which cells interact with its
environment (Alberts et al, 2014; Bellis, Miller & Turner, 1995; Charras
& Sahai, 2014; Mckayed & Simpson, 2013)

Microtubules – These are important in building up and
maintaining the structure and shape of the cell. They have a role in a number
of cellular processes such as cell division and transportation. They can form eukaryotic
cilia and flagella, and act as substrates for motor proteins that can move
along the filaments to convert chemical energy into mechanical energy, which
allows for the facilitation of movement (Alberts et al, 2014; Janke, 2014)

Intermediate
Filaments – Important in providing the cell
mechanical support. It also plays a role in the organisation of chromatin in
the nucleus of the cell, which is done by anchoring the chromatin to the
nuclear lamina which lines the inner part of the nuclear membrane. They also
have involvement in other processes of the cell such as migration, adhesion and
tumour invasion (Leduc & Etienne-Manneville, 2015)

It
should be noted that depending on the organism or the cell type, there can be
changes in the structure, function and dynamic behaviour of the cytoskeleton
(Wickstead & Gull, 2011; Alberts, 2017). This can also be the case within
one individual cell due to changes brought about by interactions with other
proteins (Hermann, Bär, Kreplak, Strelkov & Aebi, 2007)

Regulating
of the dynamic behaviour and assembly of cytoskeletal filaments allows eukaryotic
cells to build an enormous range of structures
from the three basic filament systems. (Alberts et al, 2014)

 

Staining
Protocol to visualise Cytoskeleton Structure

In order to view the cytoskeleton structure
(in this case, the actin cytoskeleton), HeLa cells will be fixed onto
coverslips, transferred to a microscope slides and then stained to be viewed under
a DSD confocal microscope which will outline structures such as stress fibres,
lamellipodia and filopodia through fluorescence. The nucleus, as well as the
focal adhesions, should also be visible.

HeLa is part of an immortal cell line that is
the most common and widely used cell type in scientific research due to being
very durable and prolific. The cell line was derived from cervical cancer cells
taken on February 8 1951, from Henrietta Lacks, who died of her
cancer on October 4, 1951 (Capes-Davis et al, 2010; Rahbari et al, 2009;
Syverton & Scherer, 1952).

Tissue culture procedure

The cells are seeded onto sterile glass
coverslips in a 6 well plate at 1×105 per well (or optimum for cell line.). The
cells are then allowed to grow for 24-48 hours under the appropriate cell
culture conditions. After this, it is fixed in 4% formalin in PBS for at least
30 minutes.

Reagents and pre-practical
protocol

After fixation has taken place, a
permeabilisation/blocking Buffer is used
to permeabilise and block the cells. The buffer used is 0.5% Triton-X 100 and
2% Goat serum Albumin in phosphate buffered saline, and this is done for 30
minutes.

The permeabilisation/blocking
buffer is then removed and a 200µl measure of primary antibody anti paxillin
made up in a 1:100 dilution ratio with permeabilisation/blocking buffer is
added to each coverslip.

The cells are then left overnight
at a temperature of 4°C. After this, the antibodies are then removed and the
cells washed in PBS.

1ml of FITC-Phalloidin/DAPI/ Alexa Fluor 568
goat anti-rabbit combined stain:

·        
1µl Alexa Fluor 568 goat
anti-rabbit

·        
979µl DAPI stock solution (DAPI stain is made up from a working concentration of 250ng/ml)

·        
20µl FITC-phalloidin stock solution (FITC-Phalloidin
stock solution is 0.05mg/ml)

Class Practical Protocol

1.      The cells are washed twice for one minute each with a
measure of 2ml PBS using a Pasteur pipette.

2.      Then an approximate of 500µl FITC-Phalloidin/DAPI/ Alexa
Fluor 568 goat anti-rabbit combined stain is added onto each coverslip. This is
then left for one hour in an area which is dark while at room temperature (e.g.
simply put an opaque box on top). The cells must be kept in the dark for the
remaining of the protocol.

3.      Each of the microscope slides that are to be used are labelled with the group number, date of the
practical, as well as the cell line and the name of the primary antibody used.

4.      The cells are then washed once again with a measure of
2ml of PBS twice for two minutes each.

5.      The coverslips are then mounted using soft set Vecta
Shield (This is an anti-fade mountant) and then fastened to the microscope
slides using nail varnish. The stained slides are then stored in the dark (Once
again, this can be as simple as under an opaque box).

6.      The final step is to simply use a DSD confocal microscope
view the microscope slides.

Different cellular structures will be observed with
different colours according to which of the three stain we have used that they
have taken up.

·        
The cytoskeletons observed should
be green, due to the use of FITC-Phalloidin.

·        
Focal adhesions will show up as red,
and this is the result of Alexa Fluor 568.

·        
Finally, the nucleus and DNA
material will show up as blue due to DAPI.

 

RESULTS

Figure
1 – Showing a comparison, with focal
adhesions.

Figure 2 – Showing lamellipodia

Figure
3 – Showing another comparison with focal
adhesions.

Figure 4 – Showing the filopodia

 

 

 

 

Figure
5 – Showing the stress fibres

Figure 6 – Showing overall structure of multiple cells

Figures
1, 2 and 3, 4 are used here to compare side by side the focal adhesions and
actin cytoskeleton fluorescence via staining and confocal microscopy. As you
can see in these comparisons, the red indicates focal adhesions due to Alexa
Fluor 568 stain. From our results, it suggests that the focal adhesions are
more prominent in the leading area of the cell.   

The
green indicates the actin filament cytoskeleton (due to FITC-Phalloidin) which
is dispersed throughout the cell but is concentrated in the cortex. Apart from
figure 1 and 2, the actin cytoskeleton structures are visible in all the other
figures showing stress fibres, filopodia and lamellipodia. We can see very well
from figure 5 that stress fibres (green) terminate in focal adhesions (the
red). Figure 3 is very good at showing the filopodia projections and figure 2 highlights
the lamellipodia. Figure 6 shows multiple cells with the different actin
filament structures. In all 6 figures, we can see the nucleus very clearly and
this is due to the DAPI stain.

 

CONCLUSION

The
recent advances in various microscopy techniques and methodologies enable cell biologists to observe and visualise
complex cellular processes. Imaging technologies allow us to view the cytoskeleton
structures and advance our understanding of its functions in the cell.  Fluorescent labelling and visualisation offer high sensitivity, specificity and the capability
for quantification.  This is particularly
relevant for actin filaments as the dynamics and assemblies of the proteins are
very complex.  The actin cytoskeleton is
usually visualised with chemical tools
such as fluorescently-labelled
phalloidin. This method, however, has limited application in living cells. (Mckayed
& Simpson, 2013)

 

 

 

 

Due
to the involvement in both direct (transduction of mechanical forces to the
nucleus) and indirect (transduction of chemical signalling cascades to the
nucleus) mechanotransduction, the observation of the cytoskeleton and focal adhesions within the cell is very important in
the field of research associated with drug treatments and medical material
testing in vitro. (Dalby & Yarwood, 2007)

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