ABSTRACT filaments following fixation and staining of

ABSTRACTThe cytoskeleton, with its three majorcytoskeletal filaments are responsiblefor the cells spatial organisation and mechanical properties. The cytoskeletonhas a variety functions including, giving shape to cells, allowing for cellmovement, enabling movement of organelles within the cell, endocytosis, andcell division.

The protein filaments that come together to serve thesefunctions include the actin filaments,the microtubules and the intermediate filaments. Confocal microscopy is used tovisualise and observe the cytoskeletonand is particularly useful to view the actin filaments following fixation andstaining of HeLa cells using FITC-Phalloidin/DAPI/ Alexa Fluor 568 goatanti-rabbit combined stain. Dynamic structures associated with the actinfilament, such as filopodia, lamellipodia and stress fibres should be visible,as well as focal adhesions and thenucleus.   INTRODUCTIONInorder for cells to function they must beable to maintain correct shape, move in space, and rearrange their structure asthey grow, divide and adapt to environmental changes while maintaining contactwith each other. The structure that enables a cell to do so is called theCytoskeleton (Alberts et al, 2014), which is a network of filaments and tubulesthat interlink and extend through the cytoplasm; from the nucleus to the plasmamembrane (Hardin & Bertoni, 2015). Itwas in 1903 that Nikolai K. Koltsov proposed that the shape of cells were determined by a network of tubules whichhe called the cytoskeleton, and in 1929, Rudolph Peters suggested the idea of a protein mosaic that dynamicallycoordinated cytoplasmic biochemistry (Peters, 1963).

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It was originally thoughtthat the cytoskeleton structure was unique only to eukaryotes but this wasshown to not be the case in 1992 due to research findings that showed bacteriacontained homologues of both tubulin and actin which are the main components ofthe cytoskeleton (Wickstead & Gull, 2011). It is now agreed that thecytoskeleton is present in all domains of life which include archaea, bacteria and eukaryotes (Hardin & Bertoni,2015). The three cytoskeletal structures include: ActinFilaments – Also called microfilaments, theyunderlie the plasma membrane of animal cells to provide strength and shape toits lipid bilayer. It is composed primarily of actin and the transmembranefocal adhesions.

 Focal adhesions andadherens junctions are membrane-associated complexes that have a role asnucleation sites for actin filaments as well as being the cross-linkers betweenthe cell exterior, plasma membrane and actin cytoskeleton.Focal adhesions serve a structural function, allowing the linking of the ECM onthe outside with the actin cytoskeleton on the inside. It provides powerfulsignal transduction system which initiates signalling pathways in response toadhesion. Focal adhesions are associatedwith protein complexes containing vinculin, talin, a-actinin, paxillin, tensin,zyxin and focal adhesion kinase (FAK.) Actin filaments also form structures suchas the lamellipodia and filopodia through which cells interact with itsenvironment (Alberts et al, 2014; Bellis, Miller & Turner, 1995; Charras& Sahai, 2014; Mckayed & Simpson, 2013)Microtubules – These are important in building up andmaintaining the structure and shape of the cell. They have a role in a numberof cellular processes such as cell division and transportation.

They can form eukaryoticcilia and flagella, and act as substrates for motor proteins that can movealong the filaments to convert chemical energy into mechanical energy, whichallows for the facilitation of movement (Alberts et al, 2014; Janke, 2014)IntermediateFilaments – Important in providing the cellmechanical support. It also plays a role in the organisation of chromatin inthe nucleus of the cell, which is done by anchoring the chromatin to thenuclear lamina which lines the inner part of the nuclear membrane. They alsohave involvement in other processes of the cell such as migration, adhesion andtumour invasion (Leduc & Etienne-Manneville, 2015)Itshould be noted that depending on the organism or the cell type, there can bechanges in the structure, function and dynamic behaviour of the cytoskeleton(Wickstead & Gull, 2011; Alberts, 2017). This can also be the case withinone individual cell due to changes brought about by interactions with otherproteins (Hermann, Bär, Kreplak, Strelkov & Aebi, 2007)Regulatingof the dynamic behaviour and assembly of cytoskeletal filaments allows eukaryoticcells to build an enormous range of structuresfrom the three basic filament systems. (Alberts et al, 2014) StainingProtocol to visualise Cytoskeleton StructureIn order to view the cytoskeleton structure(in this case, the actin cytoskeleton), HeLa cells will be fixed ontocoverslips, transferred to a microscope slides and then stained to be viewed undera DSD confocal microscope which will outline structures such as stress fibres,lamellipodia and filopodia through fluorescence. The nucleus, as well as thefocal adhesions, should also be visible.

HeLa is part of an immortal cell line that isthe most common and widely used cell type in scientific research due to beingvery durable and prolific. The cell line was derived from cervical cancer cellstaken on February 8 1951, from Henrietta Lacks, who died of hercancer on October 4, 1951 (Capes-Davis et al, 2010; Rahbari et al, 2009;Syverton & Scherer, 1952).Tissue culture procedureThe cells are seeded onto sterile glasscoverslips in a 6 well plate at 1×105 per well (or optimum for cell line.). Thecells are then allowed to grow for 24-48 hours under the appropriate cellculture conditions. After this, it is fixed in 4% formalin in PBS for at least30 minutes.

Reagents and pre-practicalprotocolAfter fixation has taken place, apermeabilisation/blocking Buffer is usedto permeabilise and block the cells. The buffer used is 0.5% Triton-X 100 and2% Goat serum Albumin in phosphate buffered saline, and this is done for 30minutes. The permeabilisation/blockingbuffer is then removed and a 200µl measure of primary antibody anti paxillinmade up in a 1:100 dilution ratio with permeabilisation/blocking buffer isadded to each coverslip.

The cells are then left overnightat a temperature of 4°C. After this, the antibodies are then removed and thecells washed in PBS.1ml of FITC-Phalloidin/DAPI/ Alexa Fluor 568goat anti-rabbit combined stain:·        1µl Alexa Fluor 568 goatanti-rabbit·        979µl DAPI stock solution (DAPI stain is made up from a working concentration of 250ng/ml)·        20µl FITC-phalloidin stock solution (FITC-Phalloidinstock solution is 0.05mg/ml)Class Practical Protocol1.

      The cells are washed twice for one minute each with ameasure of 2ml PBS using a Pasteur pipette.2.      Then an approximate of 500µl FITC-Phalloidin/DAPI/ AlexaFluor 568 goat anti-rabbit combined stain is added onto each coverslip. This isthen 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 theremaining of the protocol.3.

      Each of the microscope slides that are to be used are labelled with the group number, date of thepractical, 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 of2ml of PBS twice for two minutes each.5.      The coverslips are then mounted using soft set VectaShield (This is an anti-fade mountant) and then fastened to the microscopeslides using nail varnish. The stained slides are then stored in the dark (Onceagain, this can be as simple as under an opaque box).6.      The final step is to simply use a DSD confocal microscopeview the microscope slides.

Different cellular structures will be observed withdifferent colours according to which of the three stain we have used that theyhave taken up. ·        The cytoskeletons observed shouldbe 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 DNAmaterial will show up as blue due to DAPI. RESULTSFigure1 – Showing a comparison, with focaladhesions.Figure 2 – Showing lamellipodiaFigure3 – Showing another comparison with focaladhesions.Figure 4 – Showing the filopodia     Figure5 – Showing the stress fibresFigure 6 – Showing overall structure of multiple cellsFigures1, 2 and 3, 4 are used here to compare side by side the focal adhesions andactin cytoskeleton fluorescence via staining and confocal microscopy. As youcan see in these comparisons, the red indicates focal adhesions due to AlexaFluor 568 stain. From our results, it suggests that the focal adhesions aremore prominent in the leading area of the cell.   Thegreen indicates the actin filament cytoskeleton (due to FITC-Phalloidin) whichis dispersed throughout the cell but is concentrated in the cortex. Apart fromfigure 1 and 2, the actin cytoskeleton structures are visible in all the otherfigures showing stress fibres, filopodia and lamellipodia.

We can see very wellfrom figure 5 that stress fibres (green) terminate in focal adhesions (thered). Figure 3 is very good at showing the filopodia projections and figure 2 highlightsthe lamellipodia. Figure 6 shows multiple cells with the different actinfilament structures. In all 6 figures, we can see the nucleus very clearly andthis is due to the DAPI stain. CONCLUSIONTherecent advances in various microscopy techniques and methodologies enable cell biologists to observe and visualisecomplex cellular processes. Imaging technologies allow us to view the cytoskeletonstructures and advance our understanding of its functions in the cell.  Fluorescent labelling and visualisation offer high sensitivity, specificity and the capabilityfor quantification.  This is particularlyrelevant for actin filaments as the dynamics and assemblies of the proteins arevery complex.

 The actin cytoskeleton isusually visualised with chemical toolssuch as fluorescently-labelledphalloidin. This method, however, has limited application in living cells. (Mckayed& Simpson, 2013)    Dueto the involvement in both direct (transduction of mechanical forces to thenucleus) and indirect (transduction of chemical signalling cascades to thenucleus) mechanotransduction, the observation of the cytoskeleton and focal adhesions within the cell is very important inthe field of research associated with drug treatments and medical materialtesting in vitro. (Dalby & Yarwood, 2007)REFERENCESAlberts,B., Johnson, A.

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