“In contrast to actinmicrofilaments and microtubules, intermediate filaments aren’t directlyinvolved in cell movements” (Cooper 2000). Intermediate filaments are extremelyflexible fibres, with high tensile strength due to their coiled structure, thatplay a structural role in the cell. They are composed of 6 classes of variousintermediate filament proteins. One type, the Lamins, forms the Nuclear Laminainside the inner nuclear envelope which provides a scaffold for the nucleus.
The remaining intermediate filament proteins are twisted into a coiled coilstructure which provides mechanical strength to the cell by extending acrossthe cytoplasm. Microtubules are long, hollow cylinders, composed of polymers of theglobular protein tubulin. One end of microtubules is usually attached to themicrotubule-organizing centre (MTOC) which is the centrosome that isresponsible for their assembly. Microtubules play a key role in determiningcell shape and are also responsible for movements of the cell, including thepositioning of membrane organelles and intracellular transport; they areresponsible for separating chromosomes during cell division, as they canrearrange themselves to form a mitotic spindle. Microtubules are also capableof cilia formation which are motile ‘whip-like’ projections of the plasmamembrane. Each major filament of the cytoskeleton is important for differentaspects of the cell’s properties.
The actin microfilaments are two strandedhelical polymers of the protein actin and are arranged into a “variety oflinear bundles, 2D networks and 3D gels” (Alberts et al., 2015). Actinfilaments are highly concentrated in the cortex that underlies the plasmamembrane. They are involved in providing shape to the thin bilayer and formingprojections on the cell’s surface including lamellipodia and filopodia whichallow the cell to sense their external environment and are necessary forwhole-cell movement (locomotion).
Actin filaments are also responsible forcytokinesis resulting from their contraction, pinching the cell into twodaughter cells. Bundles of actin filaments, with myosin-II, together formcontractile stress fibres that are held together by crosslinking proteins. Theyexert mechanical tension on the cell surface and stabilise the position of thenucleus. They are usually anchored to focal adhesions which structurally linkthe extracellular matrix to the actin cytoskeleton and, therefore, they play akey role in coordinating cell migration and adhesion.
Interactions of complexescontaining proteins regulate the focal adhesions such as integrins, paxillinand vinculin, with vinculin beingthe major connection to the actin cytoskeleton. In this experiment, we performed a mixture of different stains on HeLacells which included FITC-phalloidin, DAPI and Alexa Fluor 568 stains that eachbind to specific regions. This allowed for the illustration of various cellstructures, formed from the assembly of cytoskeletal filaments, on a DSDconfocal microscope. Each stain has been selected to enable us to observecertain structures. FITC-phalloidin selectively stains actin filaments(F-actin) green and this therefore includes actin cytoskeleton features such asstress fibres, lamellipodia and filopodia. DAPI emits blue fluorescence uponbinding 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 indetermining 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 functionin cooperation with one another to maintain cell strength, shape and itsability to move. The cytoskeleton is located within a cell’s cytoplasm and iscrucial 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 thein-depth study, and therefore enhanced the knowledge, of cytoskeletalstructures as their major features can be visualised.