Wash Shield which is an anti-fade mounting medium that

Wash is removed with 2ml of PBS for 2 x 1minutes,
using a Pasteur pipette. Approximately 500µl of the FITC-Phalloidin/DAPI/ Alexa
Fluor 568 goat anti-rabbit combined stain is added onto each coverslip. This is
left for an hour in the dark and at room temperature. For the remainder of the
protocol, the cells must continue to be kept in the dark. Each microscope slide
is labelled with group number, the date, cell line and name of the primary
antibody. 2ml of PBS is used to wash (for 2x2mins). The coverslips are mounted
using a soft set Vecta Shield which is an anti-fade mounting medium that
preserves fluorescence. The coverslips are fastened to the microscope slides
using nail varnish to seal around the perimeter. The stained slides are stored
in the dark and then can be viewed using the DSD confocal microscope.

A 1ml of FITC-Phalloidin/DAPI/Alexa Fluor 568 goat
anti-rabbit combined stain is prepared using 20µl of FITC-Phalloidin stock
solution, 979µl of DAPI stock solution and 1µl of Alexa Fluor 568 dye
conjugated to goat anti-rabbit antibody.

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HeLa cells, from an immortal human cell line, were
used for this experiment and were seeded onto sterile glass coverslips in a 6
well plate (at 1×105 per well). They are grown for 24-48 hours under
controlled cell culture conditions. The cells were then fixed by immersing the
coverslips in 4% formalin in PBS for a minimum of 30 minutes. A buffer,
consisting of 0.5% Triton-X 100 and 2% Goat Serum Albumin in phosphate buffered
saline, is used for 30 minutes to permeabilise and block cells. This step
permeabilises the cells by removing lipids from the cell membrane, therefore
allowing relatively large molecules (such as antibodies) to pass through into
the cell, while blocking the cells helps reduce the amount of non-specific
binding that occurs. The buffer is then removed. The primary antibody staining solution is prepared using
primary antibody anti paxillin in a 1:100 dilution for optimal high signal and
low background conditions. 200µl of the anti-paxillin is added
to each coverslip and incubated overnight at a constant temperature of 4°C.
Following this, the primary antibody is removed and cells are washed in PBS in
order to maintain a constant pH.

Materials
and Methods

 

 “In contrast to actin
microfilaments and microtubules, intermediate filaments aren’t directly
involved in cell movements” (Cooper 2000). Intermediate filaments are extremely
flexible fibres, with high tensile strength due to their coiled structure, that
play a structural role in the cell. They are composed of 6 classes of various
intermediate filament proteins. One type, the Lamins, forms the Nuclear Lamina
inside the inner nuclear envelope which provides a scaffold for the nucleus.
The remaining intermediate filament proteins are twisted into a coiled coil
structure which provides mechanical strength to the cell by extending across
the cytoplasm.

Microtubules are long, hollow cylinders, composed of polymers of the
globular protein tubulin. One end of microtubules is usually attached to the
microtubule-organizing centre (MTOC) which is the centrosome that is
responsible for their assembly. Microtubules play a key role in determining
cell shape and are also responsible for movements of the cell, including the
positioning of membrane organelles and intracellular transport; they are
responsible for separating chromosomes during cell division, as they can
rearrange themselves to form a mitotic spindle. Microtubules are also capable
of cilia formation which are motile ‘whip-like’ projections of the plasma
membrane.

Each major filament of the cytoskeleton is important for different
aspects of the cell’s properties. The actin microfilaments are two stranded
helical polymers of the protein actin and are arranged into a “variety of
linear bundles, 2D networks and 3D gels” (Alberts et al., 2015). Actin
filaments are highly concentrated in the cortex that underlies the plasma
membrane. They are involved in providing shape to the thin bilayer and forming
projections on the cell’s surface including lamellipodia and filopodia which
allow the cell to sense their external environment and are necessary for
whole-cell movement (locomotion). Actin filaments are also responsible for
cytokinesis resulting from their contraction, pinching the cell into two
daughter cells. Bundles of actin filaments, with myosin-II, together form
contractile stress fibres that are held together by crosslinking proteins. They
exert mechanical tension on the cell surface and stabilise the position of the
nucleus. They are usually anchored to focal adhesions which structurally link
the extracellular matrix to the actin cytoskeleton and, therefore, they play a
key role in coordinating cell migration and adhesion. Interactions of complexes
containing proteins regulate the focal adhesions such as integrins, paxillin
and vinculin, with vinculin being
the major connection to the actin cytoskeleton.

 

In this experiment, we performed a mixture of different stains on HeLa
cells which included FITC-phalloidin, DAPI and Alexa Fluor 568 stains that each
bind to specific regions. This allowed for the illustration of various cell
structures, formed from the assembly of cytoskeletal filaments, on a DSD
confocal microscope. Each stain has been selected to enable us to observe
certain structures. FITC-phalloidin selectively stains actin filaments
(F-actin) green and this therefore includes actin cytoskeleton features such as
stress fibres, lamellipodia and filopodia. DAPI emits blue fluorescence upon
binding strongly to AT-rich regions of DNA and will thus stain nuclei blue.
Alexa Fluor 568 is used to visualise focal adhesions which will stain red,
including proteins such as paxillin. This has proved to be very important in
determining the structure and function of the cytoskeleton.

The cytoskeleton is a cellular scaffold composed of 3 major filaments:
actin microfilaments, intermediate filaments and microtubules, which function
in cooperation with one another to maintain cell strength, shape and its
ability to move. The cytoskeleton is located within a cell’s cytoplasm and is
crucial for cells to function as it provides structural support and,
additionally, plays a role in the transport of organelles and cell growth,
division, and movement. The fluorescent staining of cells has enabled the
in-depth study, and therefore enhanced the knowledge, of cytoskeletal
structures as their major features can be visualised.