Module 7_ The Cytoskeleton - BMSC6010E Fundamental Cell Biology Fall 2023 PDF

10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology VBDI 6010E: Fundamental Cell biology Module 7: The Cytoskeleton 7.1. Introduction The function of the cytoskeleton is to help the cell maintain its structure and organization. It can also assist with changing the cell’s shape, repositioning t...

10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology VBDI 6010E: Fundamental Cell biology Module 7: The Cytoskeleton 7.1. Introduction The function of the cytoskeleton is to help the cell maintain its structure and organization. It can also assist with changing the cell’s shape, repositioning the internal organelles, and even moving the cell from one place to another. The cytoskeleton is made up of protein filaments within the cytoplasm of the cell. The three main filaments are: actin filaments, microtubules, and intermediate filaments. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 1/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology 7.2. Filaments The two most important filaments that are found in the cell are actin filaments (aka microfilaments) and microtubules. Both of these are made of globular proteins that can assemble and disassemble within the cell (through polymerization and depolymerization respectively). In regards to their size, intermediate filaments fall between the size of actin filaments and microtubules and are made up of fibrous protein subunits. A key characteristic of intermediate filaments is that they are a lot more stable than the other two types of filaments. Filaments are responsible for cell movements such as muscle contraction and the movement of cilia. These are important for the movement of the cell and a multicellular organism overall. 7.3. Muscle uses filaments for contraction There are 3 different kinds of muscle: smooth muscle, skeletal muscle, and cardiac muscle. Both smooth and cardiac muscles control the involuntary contraction of the heart and other organs https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 2/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology around the body, whereas the skeletal muscle controls the voluntary contraction of the muscles when we perform different activities such as running, walking, exercising, etc. The main similarity between these three muscle types is that they all cause movement by using active contraction. This contraction uses different components of the cytoskeleton and different filaments to produce movement. Myofibrils are the contractile elements of the skeletal muscle cell. The skeletal muscle cell is actually called a muscle fiber and is formed by the fusion of many cells together making it a multinucleated cell. This cell is composed of myofibrils, which contract in the presence of ATP. These myofibrils are made up of repeating units of cytoskeletal proteins. These repeating units that make up the myofibrils are called sarcomeres, and they are the smallest contractile unit of the muscle cells. The sarcomere is what gives the muscle a striated appearance. There are different “bands”, “lines”, and “zones” that make up the sarcomere. They are the A bands, I bands, M bands, https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 3/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology and the Z line/Z disc. The Z line/disc is what separates one sarcomere from another. The A band is the middle portion of the sarcomere that contains both thick and thin filaments. The H zone is the middle portion of the sarcomere that contains both the M band (which is directly in the middle), and only thick filaments. The I band is the part of the sarcomere that contains only thin filaments. A nice mnemonic to remember this is: H is a THICK letter, so it contains only THICK filaments. I is a THIN letter, so it contains only THIN filaments. The Z line/disc is at the endZ of the sarcomere. The M band is in the Middle. When the muscle contracts, these filaments slide past each other and the sarcomere gets smaller. The thin actin filaments move closer, while the thick myosin filaments stay in the same space. Upon contraction, the sarcomere looks like it has been “scrunched up”. It is important to note that the I band and the H zone are the ones that will shorten. Everything else will remain the same. This is https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 4/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology known as the sliding filament model, and describes how the thin filaments slide past the thick filaments. You can watch the following video depicting the process of how the filaments slide against each other to cause muscle contraction. https://www.youtube.com/watch?v=BVcgO4p88AA (https://www.youtube.com/watch?v=BVcgO4p88AA) 7.4. Muscle contraction The contraction of muscle is mediated by the contraction of the sarcomere as mentioned previously through the sliding filament model. The contraction of muscle itself is driven by ATP hydrolysis. The filaments that are involved here are actin and myosin. Actin, as we know, is a thin filament that can also be found as a component of the cytoskeleton. Myosin makes up the thick filaments, and contains an ATPase, where the hydrolysis of ATP for muscle contraction takes place. Once the ATP is hydrolyzed by myosin, it “walks” along the actin to make the muscle contract. This process is called the cross-bridge cycle. However ATP hydrolysis is not the only process involved in https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 5/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology However, ATP hydrolysis is not the only process involved in muscle contraction. It also involves the intracellular increase of calcium. The signal for muscle contractions travels through a structure called the T-tubules. Once it travels through here, it reaches the sarcoplasmic reticulum, which holds the calcium within a muscle cell. The sarcoplasmic reticulum then releases calcium. The regulation of muscle contraction in response to increased intracellular calcium levels is regulated by two proteins: troponin and tropomyosin. These two proteins, in the absence of calcium, inhibit the binding of myosin to actin. However, when calcium is present, it binds to troponin, which then moves tropomyosin out of the way to allow myosin to bind to actin and facilitate muscle contraction. 7.5. Ciliary movement Cilia are hair-like extensions off of the cell that are made of bundles of microtubules. Their main function is to move the fluid that is present over the surface of the cell or to move single-celled organisms through a fluid. The core of the cilia is called the axoneme and it contains microtubules that are assembled in a specific structure. Cilia are essentially hollow tubes made up of microtubules that are arranged in a 9+2 pattern at the axoneme. This pattern is characterized by 9 doublets that make up a ring, and then in the middle, there is 1 doublet, showing the 9+2 pattern. This arrangement of microtubules is characteristic of almost all cilia and eukaryotic flagella. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 6/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology The microtubules that make up cilia themselves are made up of tubulin, another cytoskeletal component. Tubulin is made up composed of alpha and beta-tubulin, and its assembly is organized by different structures in the cell that provide a type of base from which the microtubules can grow called the basal body. Within the basal body, the arrangement of the microtubules is 9+0, not 9+2. Besides the basal body acting as an anchor, the axoneme is also further stabilized by other accessory proteins. Another important component of the cilia is the presence of the motor protein dynein on the microtubule doublets in the ring of the 9+2 arrangement. As a motor protein, dynein plays a very important role in the movement of the cilia. Clinical Correlate: Kartagener’s Syndrome https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 7/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology Kartagener’s syndrome is an autosomal recessive disorder that affects the motor protein dynein in the individuals body. Because the dynein is defective, the cilia in the body can no longer move properly. Think of all of the places where the body has cilia and flagella. Cilia are located on the upper respiratory tract, and there are flagella on the sperm. So what kind of symptoms will this patient experience? They will experience recurrent upper respiratory tract infections, chronic sinusitis, and infertility in males (due to the inability of the sperm to travel). Another interesting symptom of Kartagener’s syndrome is situs inversus with dextrocardia. Situs inversus is a term that refers to the inverting of the normal position of the organs in the body. So for example, if your liver is normally on the right side, then in a patient with Kartagener’s syndrome, the liver will be on the left side. This patient will also have their heart on the opposite side (the right side) which is referred to as dextrocardia (dextro — right, cardia — heart). 7.6. Microtubules and actin filaments. Up until now, we have discussed myofibrils and cilia, which are relatively permanent structures. The cell also has other more dynamic methods of moving itself. These are important because they can rapidly respond to external and internal stimuli. These extremely dynamic and motile structures are made up of microtubules and actin filaments, which can assemble and disassemble rapidly. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 8/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology As we know now, microtubules are made up of alpha and beta-tubulin. During the interphase of cell division, microtubules go from the center of the cell and extend throughout the cytoplasm. When mitosis occurs, these microtubules will disassemble and those that make up the mitotic spindle will begin to form. Once mitosis ends, the whole process reverses. The mitotic spindle microtubules are used for the movement of chromosomes around the cell to allow for the metaphase alignment of chromosomes, and also the separation of sister chromatids during division. When we take a look at the organism amoeba, we can see that its feeding tentacles are made up of microtubules. These tentacles can rapidly extend and retract due to the rapid depolymerization and repolymerization of tubulin to form the microtubules. Because microtubules are very dynamic, it wouldn’t make sense for all of them to be polymerized. In order to keep a backup reserve of tubulin, 50% is kept in a cytoplasmic pool so that it can help with rapid polymerization. 7.7. Drugs that interrupt microtubule polymerization and depolymerization https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 9/14 polymerization and depolymerization 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology Many drugs have been discovered that can disturb/prevent the polymerization of microtubules. These drugs have been used as anticancer drugs. One example is colchicine. This compound was originally extracted from saffron by the ancient Egyptians! Colchicine has the ability to bind to a tubulin dimer and prevent its polymerization. Because of this, it can be used to halt cell division and is therefore classified as an anti-mitotic drug. Other examples of drugs from this same category include vincristine and vinblastine. They are useful as anti-cancer cells because they can preferentially kill the rapidly dividing cells found in cancer. Taxol is another anticancer drug but it has a different mechanism of action. Rather than preventing and decreasing the polymerization of tubulin, it actually increases polymerization. Due to a rapid increase in polymerization, the cytoplasmic pool of tubulin is depleted leading to the discontinuation of the polymerization. Without the presence of tubulin, polymerization cannot take place. Additionally, taxol stabilizes the microtubule polymer and prevents depolymerization from occurring, halting the process of mitosis. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 10/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology 7.8. Actin filaments Actin is another dynamic filament that assembles and disassembles rapidly. They are found in almost every kind of cell. For example, actin filaments make up microvilli, which are thin, finger-like projections that stem from the surfaces of cells. They can extend and retract according to the depolymerization and polymerization of actin. Just as there is a cytoplasmic pool of tubulin in the cell to keep the tubulin concentration relatively stable, there is a similar concept for actin. The cytoplasmic actin pool is bound to a protein called profilin which prevents the polymerization of actin and keeps its concentration constant in the cell. The polymerization of actin is used in many cell processes such as locomotion (movement), phagocytosis, and cytokinesis. 7.9. Drugs that interfere with Actin polymerization and depolymerization An example of a drug that prevents actin polymerization are the cytochalasins, a group of metabolites that are released by different species of molds. These molecules can prevent the polymerization of actin by binding specifically to one end of the actin filament and preventing the addition of more actin molecules to that end. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 11/14 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology Phalloidin is another example of a molecule that can affect actin depolymerization. It is released by the death-cap mushroom, aka Amanita phalloides. In contrast to cytochalasins, phalloidin actually prevents the depolymerization of actin. It does so by stabilizing the actin filaments and preventing their depolymerization. Phalloidin is commonly used in the lab as a cytoskeletal stain. 7.10. Actin and Microtubule polymerization The polymerization of both actin and microtubules requires an energy source. For both of these processes, the energy source is different. For actin, the energy source is ATP. Every time an unpolymerized G-actin molecule is added to the polymer, an ATP molecule is hydrolyzed. Actin polymerization is characterized by a lag phase, in which we can’t really see a distinct change in polymerization. This is because a certain amount of actin has to come together and polymerized in a certain geometric conformation known as the nucleus. Once this occurs, more and more actin can be added at a much faster rate in the elongation phase. We mentioned earlier that the concentration of actin is kept relatively stable within a cell. What happens when we increase polymerization but don’t have much actin left in our cytoplasmic pool? At this time, actin will be released from one end of the polymer at the same rate that actin is being added at the other end of the polymer. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 12/14 g 10/16/23, 5:53 PM VBDI 4997E: Pre-Veterinary Histology p y This is known as the critical concentration or the steady-state. Both actin and tubulin polymers have a positive end and a negative end. This tells us that these polymers are polar. These two opposite ends will grow and depolymerize at different rates. The fastgrowing or elongating end is known as the positive end, while the other end doesn’t grow nearly as fast and is known as the depolymerizing or negative end. Tubulin has a similar polymerization mechanism except that its energy source is GTP. 7.11. Intermediate filaments Intermediate filaments are tough and durable proteins that have a diameter that is between that of microtubules and actin. They are usually found in the parts of the cell that are exposed to mechanical stress such as along a process of a neuron, or between adjacent epithelial cells. They are the most stable component of the cytoskeleton and are also the least soluble. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 13/14 10/16/23, 5:53 PM p y VBDI 4997E: Pre-Veterinary Histology Upon electron microscopy, intermediate filaments look like irregular threadlike molecules. They associate with each other side by side to make “ropes” that look similar to a collagen molecule. The way that intermediate filaments “polymerize” is what gives them their strength against mechanical forces. This assembly is most likely irreversible. If intermediate filaments need to be broken down, they are broken down by proteolytic enzymes that destroy the intermediate filaments. Intermediate filaments are actually made up of a bunch of different polypeptides that are all different sizes. We do not really know much about the structure of intermediate filaments, except for the fact that it is a trimer, not a dimer like actin or tubulin. There are many different classes of intermediate filaments, and it is assumed that one cell type usually only has one type of intermediate filament. For example, a neuron has neurofilaments, epithelial cells have keratin filaments, and other cells have vimentin, which can be copolymerized with other proteins. https://uga.view.usg.edu/d2l/le/content/2926415/viewContent/47443581/View 14/14