6.1: Overview of the Cytoskeleton and Intermediate Filaments

Learning Goals

• Compare and contrast the structure and function of the three types of cytoskeletal elements: actin filaments, intermediate filaments, and microtubules.
• Identify the different types of cytoskeletal elements in micrographs.
• Discuss different types of intermediate filaments, and correlate their structure with the strength of the assembled, functional cytoskeletal element.

Introduction

The cytoskeleton is a filamentous network that extends throughout the cell. There are three different types of cytoskeletal filament that exist. All known eukaryotic cells have both actin filaments and microtubules (Figure 06-01). The third type, intermediate filaments (Figure 06-02), is no less important in the cells that have them; however, they are really only found in the cells of vertebrates. Collectively, all three of these filaments have vital roles to play in virtually every function in the cell. Not only do they help give the cell its shape, but they also help resist mechanical stresses; act as an anchor for many proteins and organelles; provide tracks for transport of vesicles, organelles, and other cargo; and aid in the locomotion of many cell types. They also help with the division of both the cytoplasm and the DNA during mitosis.

Figure 06-01: Two mouse fibroblast cells in interphase. Actin filaments (purple web), microtubules (yellow lines), and nuclei (green blobs in center) are labeled with fluorescent molecules. This image won first place in the Nikon 2003 Small World photo competition. Torsten Wittmann (2011), CIL:240. CIL. Dataset. doi.org/10.7295/W9CIL240. This image is in the public domain.

Even cell types that were previously thought to lack a cytoskeleton have now been shown to have proteins that fulfill these roles. A very good example of this are bacteria, which were originally thought to have no cytoskeleton at all, but we now know have a cytoskeleton-like network that functions similarly. Another example is plant and insect cells, which do not have intermediate filament genes in their genomes, yet they both still have a nuclear lamina. To build it, they use genetically unrelated proteins, which perform the function of nuclear lamins (we call them lamin-like proteins). As such, it is safe to say that the cytoskeleton is (another) essential component of all living cells. Cells simply cannot survive without it.

Figure 06-02: Mouse (Mus musculus) fibroblast cells, labeled to show the mitochondria (red squiggles) and intermediate filaments (green lines). Adapted from Linda Parysek and Trudy Aebig (2010), CIL:2, Mus musculus, fibroblast. CIL. Dataset. doi.org/10.7295/W9CIL2. This image is in the public domain.

The features of each of the types of cytoskeletal filaments that we list here will be explained in further detail throughout the rest of the chapter. For now, here is a quick summary, in order of filament size (see also Video 06-01):

1. Microtubules (MT)—described in Topic 6.2
   • The largest of the three types (~25 nm in diameter).
   • Made of repeating subunits called tubulin. Tubulin itself is a dimer, made of two smaller subunits, called alpha- and beta-tubulin.
   • Go through rounds of polymerization and depolymerization (dynamic instability).
   • Involved in a variety of cell functions.
     • In all cells, they are involved in mitosis, cargo trafficking, and maintenance of cell shape.
     • In animal cells, they radiate outward from the center of the cell and act as “highways” for vesicular transport.
     • In plants and algae, they play an important role in the secretion of cellulose, which happens at the plasma membrane. Thus, they are found in the cell cortex in plants (meaning that the microtubules are found directly underneath the plasma membrane).
     • In fungi and other protists, the arrangement of the cytoskeleton is more variable.
     • Both cilia (singular cilium) and flagella (singular flagellum) are specialized structures that depend on microtubules for function.
2. Intermediate filaments (IF)—described later in this topic (6.1)
   • Their diameter is “in between” the other two types of cytoskeletal filaments (~10 nm).
   • They are a family of proteins classified into five major types (e.g., keratins, vimentin, neurofilaments, and nuclear lamins). These types describe the subunit that they are made from.
     • Generally, only one or two subtypes are expressed in each cell types (i.e., nuclear lamins plus keratin, for example).
     • A single intermediate filament is always made from the same kind of subunit. They do not mix and match.
   • They do not undergo dynamic instability, but they are assembled and disassembled as needed.
   • They help with anchoring cells that are not motile, resist mechanical strain in tissues, and are vital to the maintenance of cell shape. Nuclear lamins are very important in the maintenance and organization of the nucleus.
3. Actin filaments (AF)—described in Topic 6.3
   • Also known as microfilaments.
   • The smallest of the three types (~7 nm in diameter).
   • Made of repeating subunits, also called actin.
     • Monomers of actin are sometimes referred to as g-actin to differentiate from the filament form, which is call f-actin.
   • Like microtubules, they also undergo dynamic instability.
   • In animal cells, they are heavily involved in cellular locomotion but also cellular adhesion and shape, cytokinesis, cargo trafficking, and muscle contraction. As a result, actin filaments tend to remain close to the plasma membrane, in the cell cortex.
   • In plant and algae cells, the cellular arrangement is different. They are found throughout the cytoplasm (not just underneath the plasma membrane). As such, they are involved more heavily in cargo trafficking and maintenance of cell shape (plant cells are not motile). They also help produce the phenomenon known as cytoplasmic streaming.
   • In other cells, once again, the arrangement is more variable.

Video 06-01: A general overview of intermediate filaments, microtubules, and actin filaments in animal cells.

Intermediate Filaments (IFs)

As mentioned, intermediate filaments get their name from the fact that they are “intermediate” in size between actin and microtubules. Their importance in the cell is often overshadowed by the focus on actin and microtubules when discussing the cytoskeleton and by the fact that both plants and insects do not appear to carry any genes in this family. This discovery initially implied they were “less important” than the “universally essential” filaments. However, we have since discovered that even though the genes for intermediate filaments are not always present, there are often other proteins that are able to functionally fill this role. For example, there is always a structure that resembles the nuclear lamina in all nuclei, and it functions similarly, suggesting that it plays a vital cellular role. In addition, in the cell types that contain additional intermediate filaments (like skin or neurons), they play a key role, helping to maintain shape and resist mechanical strain. Thus, intermediate filaments, or intermediate filament-like proteins, are an essential cytoskeletal element for all cells.

Intermediate filaments (IFs) are a genetic family of proteins rather than a single highly conserved type. Within one filament, only one type of protein subunit is present. Each intermediate filament type is tissue specific, which means that you can tell cell type based on what kind of IF it expresses. For example, neurons express neurofilaments, but skin cells do not.

Some of the most well-known types of intermediate filaments include the following:

Making Connections: IFs

Can you speculate how this might be useful in cancer diagnoses? Imagine that you found keratin, which is normally expressed in skin and hair, in an abdominal tumor. What type of cancer does the patient have?

• Keratin: Found in epithelial cells and their derivatives, hair, nails, and horn. They help hold skin cells to each other and to the underlying membrane and provide resistance against mechanical tension (also made famous by shampoo commercials promoting their effects on keratin).
• Vimentin: Found in intracellular fibers of connective tissue cells, including muscle. They help hold tissues together and provide strength.
• Neurofilaments: Found in neurons. They help form and support the characteristic (and extreme!) shape of a nerve cell.
• Lamins: Components of the nuclear lamina. We discussed these previously in Chapter 3. They form a meshwork beneath the nuclear envelope and help maintain the integrity of the nuclear envelope.

Intermediate filaments are considered less dynamic compared to the other filaments (i.e., actin and microtubules). They do not undergo dynamic instability, which is a process of rapid switches between growth and shrinkage that is key to microtubule and actin function. However, they are still able to assemble and disassemble as the cell requires. A perfect example of this is the breakdown of the nucleus during mitosis, which is driven by the phosphorylation and breakdown of the nuclear lamina, which we saw in Chapter 3 and will discuss again later in this chapter.

Structure of Intermediate Filaments

Regardless of the specific subunit used to make it, all intermediate filaments are somewhat similar and follow the same pattern of assembly. Like all three of the cytoskeletal filaments, intermediate filaments are formed when smaller subunits, known as monomers, come together in a particular way to form a longer, filamentous polymer. So when we refer to the specific types of intermediate filament (i.e., keratin, vimentin, etc.), we are referring to the monomer being used as the building block.

All of the intermediate filament monomers are what are known as filamentous proteins, meaning that they are long and threadlike (Figure 06-03). They have a large central region that is a long alpha helix, which makes up the bulk of the filament. On either end are the “head” and “tail” region, which are specific to each of the different monomer types (keratin, vimentin, etc.). It is these head and tail regions that help the intermediate filament carry out its specific function. For example, the nuclear lamins will require sites that can bind to the chromatin, whereas keratin, which is not in the nucleus, does not require this feature.

Figure 06-03: Intermediate filament assembly. Monomers coil together into dimers with the heads and tails on the same side. Then two dimers bind into an antiparallel (head-to-tail) tetramer. Groups of tetramers assemble into a ropelike structure. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

In order to build a filament (Figure 06-03), two monomers first come together to form a dimer. The dimers then assemble with each other in an antiparallel arrangement, meaning that they are oriented in the opposite direction from each other. This forms a tetramer. The tetramers then assemble to form the long filament. Since the ends of the monomers are staggered compared to each other in the filament, this forms a long, ropelike structure that doesn’t stretch and is difficult to break.

Like many protein complexes, the intermediate filament subunits undergo self-assembly in order to spontaneously form the final filament. This means that energy is not required to facilitate this assembly process (this is different from actin and microtubule assembly).

Making Connections: SNAREs

SNAREs, which you encountered when learning about vesicle transport in Chapter 4, also use the coiled-coil strategy to come together.

Remember from Chapter 2 that the alpha helix is a secondary structure, commonly used in protein folding. Thus, the amino acid sequence of the IF subunit is such that the alpha helix takes shape on its own. The formation of the dimers and tetramers is also facilitated by the amino acid sequence. Chemical analysis of the alpha helix shows that they have a line of nonpolar amino acids on one side of the alpha helix. This facilitates the formation of a structure known as a coiled coil (Figure 06-04). This is a very common structural strategy, as it ensures precise alignment of subunits, and the nonpolar interactions are difficult to break in the aqueous environment of the cell.

Figure 06-04: The arrangement of the polypeptides in a coiled coil. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Examples of Intermediate Filaments in Cells

Example 1

The Nuclear Lamina

Since all eukaryotic cells have a nucleus, the nuclear lamina is easily the most important example of intermediate filament function. We have already discussed this function back in Chapter 3, when we examined the structure of the nucleus in detail. The nuclear lamina is vital to the proper organization and function of the nucleus throughout the cell cycle. All Eukaryotes that have a nucleus will also have a nuclear lamina of some kind.

In interphase, the assembled nuclear lamins form a latticework just underneath the nuclear envelope (Figure 06-05). Not only does this help provide structure to the nuclear envelope, but it also helps with the organization of the chromatin. The chromosomes are attached to the nuclear envelope and the underlying lamina. This attachment helps maintain the regional organization of the chromatin, thus facilitating gene expression.

Figure 06-05: The nuclear lamina and its role in the nucleus. (A) Close-up, labeled fluorescent micrograph of the nucleus of a cell derived from the Henrietta Lacks (HeLa) cell line. The nuclear lamina is a blue circle (labeled) that lies directly underneath the (unlabeled, not visible) nuclear envelope. Adapted from Sui Huang, Thomas J. Deerinck, Mark H. Ellisman, and David L. Spector (2010), CIL:9233, Homo sapiens, cervical carcinoma of Ms. Henrietta Lacks (doi.org/10.7295/W9CIL9233) and shared under a CC BY-NC-SA 3.0 license. (B) Schematic of the interphase nucleus showing the location and function of the nuclear lamina. This image was created by Heather Ng-Cornish and is shared under a CC BY-NC-SA 4.0 license.

Not only is the nuclear lamina attached to the nuclear envelope and internal components of the nucleus, but it also maintains attachments to the cytoskeleton on the exterior of the nucleus (Figure 06-05). This attachment can help maintain the location of the nucleus within the cell (and allow it to be moved if necessary). A variety of proteins facilitate connections between the nuclear lamina, on the inside of the nucleus, and the cytoplasmic cytoskeleton. In some cases, specialized transmembrane proteins form complexes spanning the inner and outer membranes of the nucleus.

During mitosis, destabilization of the nuclear lamina (via phosphorylation of the lamins) is what drives the disassembly of the nuclear envelope (Figure 06-06). Once mitosis is over, the lamins are dephosphorylated, and the nuclear lamina reforms. Since it’s attached to both the membrane and the chromosomes, the result is that everything ends up back where it’s supposed to be.

Figure 06-06: The breakdown and reformation of the nuclear lamina during mitosis. Lamins are phosphorylated at the beginning of mitosis, which triggers the nuclear lamins to depolymerize. This results in the breakdown of the nuclear envelope as well. This is reversed in anaphase when the lamins are dephosphorylated. The nuclear lamins polymerize and the nuclear envelope reforms. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

To further illustrate the importance of these nuclear lamins, here are a few examples of genetic diseases that result from mutations in lamin proteins. In one disease known as Hutchinson-Gilford progeria syndrome, children appear to age at a highly accelerated rate and often die in their early teens (Prokocimer et al., 2013). Emery-Dreifuss muscular dystrophy is caused by a few different genes, but one of them is a nuclear lamin, and others include proteins that are also involved with maintaining the shape of the nuclear envelope (Maggi et al., 2021).

Example 2

Neurofilaments and Cell Shape

As you likely know, neurons have a very distinct shape, characterized by the axon that projects out one side of the cell. Axons can be extremely long—the longest axon in the human body is the sciatic nerve, which runs from the base of your spinal cord to your big toe, so it’s about 1 m long. However, larger animals likely have much longer axons than that. (The blue whale is thought to have a dorsal root ganglion that is 25–30 m long!!!)

Cells do not easily make this kind of extreme shape…it’s a long way from the sphere that is considered to be the most energetically favorable shape for a cell! Thus, the cytoskeleton, including both microtubules and intermediate filaments, is required to maintain this structure. Neurons express a type of intermediate filament known as a neurofilament. Both neurofilaments and microtubules can be seen in the cross section of a nerve axon in Figure 06-07. If either the neurofilaments or the microtubules are disrupted, the axon will lose its integrity and retract, and the cell will die. Many kinds of dementia are the result of this kind of loss of shape due to impaired cytoskeleton function.

Figure 06-07: TEM cross section of a frog neuron at the edge of a neuromuscular junction. At the top, a Schwann cell can be seen (part of the myelin sheath of a neuron). At the bottom, the muscle cell is visible, with its highly ordered actin ready to initiate muscle contraction. Don W. Fawcett, John Heuser, and Tom Reese (2011), CIL:36010, Anura, peripheral neuron, muscle cell, myelinating Schwann cell. CIL. Dataset. doi.org/10.7295/W9CIL36010. Shared under a CC BY-NC-ND 3.0 license.

Example 3

Desmosomes and Hemidesmosomes

As was mentioned earlier, not all cells appear to require cytosolic intermediate filaments in high numbers, but in the cell types that have them, they are key to proper function. Our skin and the cells lining our gastrointestinal tract are perfect examples of this.

The role of our skin is not just to keep all of our internal bits on the inside but also to protect us from the world around us. It forms a flexible yet impenetrable barrier to pathogens, like bacteria, viruses, and other toxins. Thus, even if we twist, push, or pull on our skin, we cannot penetrate it. Breaking the skin barrier requires a sharp object that can cut through the cells and expose the tissues underneath. (We do not recommend trying this…)

In order for our skin to resist everything that we put it through, the cells need to be much stronger than what a plasma membrane can provide on its own. Intermediate filaments, specifically keratin, are used to help our skin stay strong. The keratin filaments pass from one side of the cell to the other and bind to large protein plaques in the plasma membrane. These plaques, known as desmosomes (Figure 06-08), bind to similar plaques in adjacent cells, which are attached to that cell’s keratin. The keratin + desmosome complex transmits mechanical stress along the ropes instead of through the membrane. Desmosomes act like the rivets in your jeans that hold the different pieces of fabric together. They also provide an anchor point for the keratin filaments. Not only do desmosomes attach cells to each other, but they also bind to the membrane layer underneath the skin (called the basal lamina). In this case we call them hemidesmosomes.

Figure 06-08: Desmosomes. (A) Schematic of the structure of desmosomes, showing the attachment plaque and the intermediate filaments (keratin) that connect desmosomes through the cytoplasm. (B) Electron micrograph showing two desmosomes in a cell membrane and the keratin filaments leading away on either side of the desmosome. Dr. Robin Young compiled the full figure from the following sources: Panel A is by Mariana Ruiz. Panel B is adapted from Wai Yan Lam and Marian Rice (2011), CIL:27225, Fundulus heteroclitus, epidermal cell. CIL. Dataset. doi.org/10.7295/W9CIL27225. Both images are in the public domain.

Once again, we can see the importance of intermediate filaments by examining the diseases that result from genetic mutations in keratin. Epidermolysis bullosa is a very painful disease in which the skin blisters and tears at the slightest touch. Even soft clothing and the act of eating can cause ruptures and blisters in the skin. The children born with this disease are often referred to as “butterfly children,” as their skin is incredibly fragile (like the wings of a butterfly). It is caused by several different mutations affecting the strength of the attachment of the skin, including more than one mutation in keratin genes. Like the other diseases we’ve seen in this section, there is no cure. However, regeneration of skin through stem cell therapy and grafting has shown promise in recent years.