As a fan of your blog, I was wondering if you could help me.. I've been given a presentation to do and my section is about tonofilaments in the case of stratified squamous epithelium and how they relate to the barrier function of the skin. Could you (maybe) give me a brief explanation of structure, etc? :)
What is a tonofilament?
Tonofilament is a fancy word for a keratin filament which is the ‘intermediate filament’ found in epithelial cells and hair.
Wait, back up, what is an intermediate filament?
The word intermediate filament is a non-specific term used to describe the filaments found in the cytoplasm of cells that are ‘intermediate’ in size to the common 7nm actin filament and 25nm microtubule.
Intermediate filaments can be made from a wide range of proteins that range in size from 9-11nm when they are assembled. There are 6 types of protein that intermediate filaments can be made from and each type is located in specific cells/regions of cells.
- Type I and Type I proteins are cytokeratins found in epithelial cells and hair.
- Type III proteins include desmins found in muscle, vimentins found in fibroblasts and endothelial cells and peripherins found in neurons
- Type IV proteins include the proteins found in neurofilaments along the axons of neurons
- Type V proteins are lamins that play a structural role in the nucleus of cells.
- Type VI proteins include nestin, a protein also found in nerve cells implicated in axon growth.
Seen as your presentation is on the intermediate filaments known as tonofilaments you need to be looking at those types of filaments made from cytokeratins – Type I and Type II.
OK, cool, but what is are cytokeratins?
Cytokeratins are structural proteins that have many different isoforms. There are in fact 54 recognized human keratin proteins that can be classified as the “epithelial keratins” found in epithelial cells or the “hair keratins” found in hair and nails. Each of the 54 keratins is either Type I (acidic) or Type II (neutral/basic) and they can be categorized as follows (Schweizer et al., 2006):
- Type I (acidic) human epithelial keratins - there are 17 numbered K9-K10, K12-20 and K23-28.
- Type I (acidic) human hair keratins - there are 11 numbered K31-32, K33a, K33b and K34-40.
- Type II (basic) epithelial keratins - there are 20 numbered K1-5, K6a, K6b, K6c and K71-K80.
- Type II (basic) hair keratins - there are 6 numbered K81-K86.
You are studying the keratins found in epithelial cells so you can probably disregard all those hair keratins.
What is the deal with the Type I (acidic) and Type II (basic) thing?
The Type I (acidic) and Type II (basic) epithelial keratin proteins are co-expressed by epithelial cells in specific pairs that can bind to each other to form acidic-basic heterodimers. These heterodimers associate to make alpha-helical keratin filaments (Lee et al., 2012: see image below). As we know from earlier, another name for a keratin filament is a tonofilament. Some tonofilaments can associate with each other to form thicker bundles called tonofibrils.
Seen as you are dealing with the epithelial cells of the skin you should probably think about the organization of the epidermis.
Right…erm…so how is the epidermis organized then?
The epidermis of the skin is a pretty cool specialized type of epithelium. Most stratified squamous epithelia play a purely protective role in the resistance of abrasive forces – such as in the esophagus, oral cavity or anal canal. Skin, however, is a little bit different. You have to investigate its features a little bit further to see why…
The majority of the cells in the epidermis are called keratinocytes (there are also some melanocytes in there that synthesize pigment, some Langerhan’s cells that are involved in antigen presentation and a handful of Merkel cells that are associated with the endings of sensory nerves for sensation).
Keratinocytes form four layers in regions of thin skin and five in regions where it is thick (like the palms of the hands and soles of the feet).
- Stratum basale- a region of dividing keratinocytes that replace the dead squames shed at the stratum corneum (surface)
- Stratum spinosum – tightly linked keratinocytes that provide mechanical integrity
- Stratum granulosum – contains keratohyalin grnaules and lamellar granules (Odland bodies).
- Stratum lucidum (not clearly seen ere and only in thick skin)
- Stratum corneum – a layer of enucleate squames (corneocytes).
That is just great, but how on earth is all of this related to the barrier function of skin?
The epidermal layers express different isoforms of keratin protein that are eventually organized to form thick compacted bundles within the cells in the stratum corneum.
1. Stratum basale
This is the layer of cells that proliferates to replace those cells shed at the surface. The turnover of skin is between 52 and 75 days. Basal keratinocytes are cuboidal to columnar in shape and may be impregnated with melanin, especially in individuals with pigmented skin. The cells manufacture Type I (acidic) K14 and Type II (basic) K5 keratins which assemble into K5/14 tonofilaments in the cytoplasm.
2. Stratum spinosum
The ‘prickle cell’ layer contains keratinocytes that are anchored to each other by desmosomes (cell junctions). This allows the epidermis to resist tensile forces. When the cells are prepared for histology they shrink apart except for where they are connected at these junctions resulting in a characteristic spiny appearance (looks more like a pale staining moat around them in light microscopy). These cells contain Type I (acidic) K10 and Type II (basic) K1 keratins organized into K1/10 tonofilaments and bundled into tonofibrils that are arranged concentrically around the nucleus and are anchored peripherally to the desmosomal plaques.
3. Stratum granulsoum
There are changes afoot in this layer. The nuclei are pyknotic (condensed chromatin) and starting to disintegrate. The basophilic (purple) granules seen in this layer are called keratohyalin granules and they contain a protein called profilaggrin. As the cells progress to the stratum corneum layer, profilaggrin is converted into filaggrin which binds to the K1 and K10 keratin proteins within the keratin cytoskeleton forming densely compacted bundles of tonofibrils within the cell (Manabe et al., 1991; Brown and McClean, 2012). This process facilitates the collapse and flattening of cells to form the ‘squames’ seen in the stratum corneum.
The stratum granulosum also contains small lamellar granules (aka Odland bodies) that fuse with the plasma membrane of the keratinocytes and release a hydrophobic glycophospholipid into the intercellular spaces around the keratinocytes. This secretion is important for creating the waterproofing barrier of skin.
4. Stratum lucidum (in thick skin only and is poorly characterized)
5. Stratum corneum
Composed of terminally differentiated keratinocytes. These cells are flat, metabolically inactive squames called corneocytes that are surrounded by a water repelling intercellular lipid matrix.
The corneocytes are composed of:
- tonofibrils embedded in a cytoplasmic matrix of filaggrin
- a cornified envelope of cross-linked proteins that are extremely resistant to disruption.
- a lipid envelope surrounding the cornified envelope replacing the plasma membrane of the keratinocyte.
The intercellular lipid matrix is composed of:
- the intercellular matrix composed of the lipid released from the Odland bodies.
So the corneocytes are like the bricks and the intercellular lipid as like a waterproof mortar that holds them all together?
Exactly! The entire structure forms a thick protein and lipid barrier that prevents water, microbes and molecules (with a molecular weight of great than 800-1000) from getting through. All impeded by the thick tonofibrils and lipid barrier. And on top of that it is dynamic! Constantly being shed (and therefore regularly getting rid of any microorganism that managed to stick to it or get past the initial layers) and replaced by new cells emerging from below.
What happens if something goes wrong with the process of cornification?
If there is a defect in the gene coding for the protein profilaggrin then this will never be able to form filaggrin and therefore the keratin filaments/tonofilaments in the keratinocytes do not get bundled up normally to form the core of the cornecytes and as a result the structure of the stratum corneum is affected. The result is ichthyosis vulgaris and the skin is scaly, dry and prone to cracking and infection (below).
If there is a defect in the gene coding for the Type II (basic) keratin protein K1 this results in a very rare disease called icthyosis hystrix (literally meaning scaly porcupine). The keratin proteins do not assemble correctly and the result is massive hyperkeratosis of the skin resulting in distinctive spiny scales (Sprecher et al., 2001). Edward Lambert suffered from the disease and became known as Suffolk, England’s very own Porcupine Man (below).
Hope this gives you a head start on your presentation!
Lee et al., 2012, Structural basis for heteromeric assembly and perinuclear organization of keratin filaments, Nat Struct Mol Biol. 19(7):707-15.
Schweizer et al., 2006, New concensus nomenclature for mammalian keratins, Journal of Cell Biology 14(2): 169
Brown, S.J . and Mclean, I.M., 2012, One remarkable molecule: filaggrin J Invet Dermatol 132 (3 Pt 2) 752-762
Manabe M et al., 1991, Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris. Differentiation, 48:43–50.