L5 Cytoskeleton and cell.pdf

MD110/CMM110 Cytoskeleton and cell motility Kittikhun Kerdsomboon [email protected] CICM 1  Introduction: cell morphology facilitates cell function Our bodies contain over 200 different types of cells. Most of those cells...

MD110/CMM110 Cytoskeleton and cell motility Kittikhun Kerdsomboon [email protected] CICM 1  Introduction: cell morphology facilitates cell function Our bodies contain over 200 different types of cells. Most of those cells have a unique shape, size and structure to facilitate their biological functions. For example, neurons must relay signals between cells and often over long distances. To accomplish this, neurons extend long processes called axons and dendrites that send and receive signals, respectively, from other cells. Dendrites and axons can be hundreds of times the length of the cell body. To maintain these structures, neurons need to provide structural support and deliver biochemical components along the length of axons and dendrites. Enterocytes are cells that line the inner lining of the small intestine. Their primary function is to absorb nutrients from the lumen of the intestine and release them to the blood stream. To increase the efficiency of the uptake of nutrients, enterocytes form many small projections of the cell membrane, called microvilli, into the lumen of the intestine. This increases the overall surface area of the enterocyte. 2 Overriding question: What is the structural scaffold that holds all of the cellular organelles in place? WIRE TUBE ROPE 3 Outline Cytoskeleton Microtubules Actin filaments Myosin filaments Intermediate filaments Cell motility 4 MICROTUBULE Protofilaments are head-to-tail arrays of tubulin dimers arranged in parallel. Microtubules have polarity (plus and minus ends), which determines direction of movement. Microtubules are made of the globular protein tubulin. Tubulin dimers consist of α-tubulin and β-tubulin, which are encoded by related genes. γ-tubulin in the centrosome helps in initiating microtubule assembly. 5 MICROTUBULE - Microtubules are hollow tubes made of globular tubulin subunits. - Main functions + - maintenance of cell shape - cell motility (cilia or flagella) - chromosome movements in cell division - organelle movement - 6 MICROTUBULE 7 MICROTUBULE If GTP is hydrolyzed more rapidly than new subunits are added, GDP-bound tubulin at the plus end of the microtubule leads to disassembly and shrinkage. 8 MICROTUBULE Microtubules usually grow out from an organizing center. - microtubules extend from organizing centers such as a centrosome, the two poles of a mitotic spindle and the basal body of a cilium. 9 10 MICROTUBULE ➤ Drugs that affect microtubules: antimitotic drugs -treatment of human cancer 11 TAXOL (PACLITAXEL) A Pacific yew tree, the natural source of taxol. Taxol (B) Immunofluorescence micrograph showing the microtubule organization in a liver epithelial cell before the addition of taxol. (C) Microtubule organization in the same type of cell after taxol treatment. Note the thick circumferential bundles of microtubules around the periphery of the cell. Taxus brevifolia 12 MICROTUBULE ➤ Both kinesins and dyneins move along microtubules using their globular heads. - + 13 MICROTUBULE https://youtu.be/UHUXRyCY7LM 14  Actin filaments support and modify cell morphology. The actin cytoskeleton plays a critical role in cell morphology. It provides structural and mechanical support to plasma membrane, stabilize interactions between cells and between cells and the ECM. It also allows cells to change their morphology and to move. 15  Actin filaments Nucleation is the first step of actin polymerization—dimers and trimers are formed, then monomers are added to either end. Actin polymerization is reversible; the filaments can be broken down when necessary. 16 Treadmilling and the Role of ATP in Actin Filament Polymerization: Actin bound to ATP associates with the rapidly growing barbed end. ATP is then hydrolyzed to ADP. ADP-actin dissociates more rapidly from filaments https://youtu.be/VVgXDW_8O4U than ATP-actin. Results in treadmilling, where ATP-actin is added. at the barbed end while ADP-actin dissociates from the pointed end. 17 18 Stabilization of actin filaments 19 Filament severing by cofilin 20  Microvilli supported by parallel arrays of long actin filaments. To generate structures like microvilli, cells use villin and fimbrin which has a short gap between its two actin-binding domains. Villin and fimbrin create a tightly packed, parallel array of actin filaments that to generate dense, parallel arrays of actin filaments that provide increased mechanical strength. 21  Myosins generate changes in cell shape and transport vesicles and organelles. Actin filaments not only provide stability for static structures such as microvilli but also allow cells to change shape. Actin filaments can be used by myosin filaments to generate tension on the cell membrane and cause cells to contract. Examples are contraction of all muscle cells, contraction of cells during wound healing, and cytokinesis. 22  Bipolar myosin filaments generate force of contraction. The myosin that forms filaments, called muscle myosin, is similar to myosin that transport organelles except that it lacks a domain for binding organelles. In addition, muscle myosin has a longer coiled coil domain that allows it to polymerize into filaments. One important feature of myosin filaments is that they are bipolar as the motors one side of the filaments want to move in a direction opposite to the motors on the other end of the filament. 23  Calcium triggers contraction of actin and myosin filaments. The bipolar arrangement of myosin filaments allows them to pull on two different actin filaments. If these actin filaments are attached to different regions of the cell membrane, the myosin filament will cause the cell to contract at these two regions. Contraction by myosin filaments is regulated by calcium. When calcium levels are low, the myosin is inactive. When calcium concentration increases, the myosins become active and start pulling on the filaments, causing cell to contract. When calcium levels fall, the myosins lose activity, releasing the actin filaments and relaxing the cell. 24 Calcium triggers contraction of actin and myosin filaments. https://youtu.be/99R-XCGme8Q 25 26 Cytokinesis 27 28  Intermediate filaments integrate cells into a mechanical network. Intermediate filaments extend from the nucleus to the cell membrane. At the cell membrane they interact with proteins that bind proteins in the cell membranes of neighbor cells. The proteins in the neighbor cells are also linked to intermediate filaments. In this way, intermediate filaments are integrated into large network that spans a group of cells, increasing the mechanical strength of the cells and tissue and protecting it against external stress. For this reason intermediate filaments are found predominantly in cells that face significant mechanical stress, such as skin. 29  Intermediate filaments provide tensile strength. The unique properties of intermediate filaments is that they respond differently to low and high forces. At low forces intermediate filaments will stretch but as the force increases, intermediate filaments become less flexible and start resisting the force. This property makes intermediate filaments more mechanically robust than either microtubules or actin filaments. 30 Mechanical properties of actin, tubulin and intermediate filament polymers Microtubules: easily deformed and rupture Actin filaments are more rigid and also rupture easily Intermediate filaments: easily deformed and don’t rupture—maintain cell integrity 31  Intermediate filaments assemble through coiled coil interactions. Monomer Parallel dimer Antiparallel tetramer Protofilaments Intermediate filaments The extensive lateral interactions are in part what gives intermediate filaments their tremendous strength.  Intermediate filament are a family of proteins with tissue-specific expression. Intermediate filaments comprise large family of proteins. Keratin is the largest class with ~50 members and found prominently in skin and hair. Neurofilaments are found in axons and lamins localize to the inner nuclear membrane. 33 Intermediate Filaments 34 35  Mutations in intermediate filaments cause blistering diseases. Mutations in keratin genes cause several human genetic diseases. For example, when defective keratins are expressed in the basal cell layer of the epidermis, they produce a disorder called epidermolysis bullosa simplex, in which the skin blisters in response to even very slight mechanical stress, which ruptures the basal cells. 36 37 Cell motility 38  Neutrophils chase bacteria through tissues or across a substratum. https://youtu.be/hacbn_xcZdU Neutrophils track and chase a bacterium. Neutrophils move at ~ 0.1 to 0.2 μm/s. 39  Metastatic cancer cells crawl through tissues and along a substratum. https://youtu.be/LOIR7PPztXY Metastatic cells use the same crawling ability to escape from a localized cancer and enter the lymphatic or circulatory system and spread to other organs. 40  Cells can follow the concentration gradient of a single chemical. https://youtu.be/RUyMvFn58fo When cells move, they usual follow some external chemical that guides the direction of their movement. 41  Cell motility involves three steps: pushing, attaching, pulling. Cell motility involves several steps. To move across a surface, cells generate force that pushes forward a broad section of the plasma membrane. This is called the leading edge and extends a part of the cell in certain direction. Attachments between the cell and the substratum give the cell something to push against and stabilizes the leading edge. The cell pulls on attachments at back end of cell to detach them from the substratum. This allows the cell to move its backend forward toward the leading edge. 42  Polymerization of new actin filaments, but not from existing filaments, has a lag phase. To initiate motility, cells must regulate when and where they polymerize actin. They do this use actin nucleating factors. Normally, there is a delay in the polymerization of actin called the lag phase. However, in the presence of preformed filaments or nucleating factors, there is no delay in actin polymerization. By controlling the activity and location of nucleating factors, the cell can specify where actin polymerization occurs in the cell. 43  ARP2/3 resembles actin dimers and nucleates filament formation to overcome lag phase. For cell motility, the most important nucleating factor is the ARP2/3 complex. The ARP2/3 complex is set of proteins, two of which resemble actin. When one actin monomer associates with ARP2/3, it forms a stable platform for filament growth. 44  Actin filaments grow from their plus ends. Like microtubules, actin filaments are polarized. The plus end of actin filaments is the fast growing end as monomers are readily incorporated into this end. Minus ends add monomer very slowly and often shrink. Minus ends are often stabilized by nucleating factors 45  Capping proteins control the length of actin filaments. Cell motility depends upon forming short filaments because long filaments are less rigid and wouldn’t support the cell membrane. Cells control length of actin filaments with capping proteins. Capping proteins bind plus ends and prevent further addition of monomer to stop growth. In the presence of high concentration of capping protein, cells form many short actin filaments. 46  Cofilin severs actin filaments leading to depolymerization. To support the continuous pushing forward of the cell membrane, the cell needs a continuous supply of actin monomers to polymerize new filaments. To maintain a supply of actin monomer, the cell recycles old filaments that are no longer required to support cell motility. Cells recycle old filaments by severing them and allowing them to depolymerize. Cofilin binds to the sides of actin filaments and induces a twist in the filament. The twist causes the filament to sever, exposing a minus end from which the filament depolymerizes. To differentiate old from new filaments, cofilin only binds to filaments with actin that is bound to ADP. When actin is incorporated into filaments, it is bound to ATP. Thus, new, growing filaments will contain mostly actin-ATP. Over time, actin hydrolyzes ATP to ADP. When most of the filament is actin-ADP, cofilin can bind and sever the filament. 47  Arp2/3 and capping protein generate short, branched filaments. Cells use a combination of ARP2/3 and capping protein to generate short, branched filaments. ARP2/3 nucleates the polymerization of new filaments by binding to the side of existing filaments. The new filaments grow at ~ 70° angle to the existing filament. This creates a network of branched filaments. The high concentration of capping protein ensures that the filaments remain short. The continuous activation of ARP2/3 generates an expanding network of branched filaments that pushes forward the cell membrane. Toward the center of the cell cofilin severs old filaments to generate a constant supply of actin monomer for new filament growth. 48  External signals control location of actin polymerization via activation of ARP2/3. https://youtu.be/d_uWtjyNb-Y Cells move in the direction of an external signal and must be able to integrate the location of the external signal with the proteins that regulate actin polymerization. A central component in this linkage is WASp (Wiskott-Aldrich Syndrome protein). WASp is activated by receptors in the cell membrane that bind external signaling molecules. Active WASp associates with ARP2/3 and activates it, triggering the polymerization of actin filaments and cell motility. 49  Bacterial molecules activate signaling pathway in neutrophils to initiate actin polymerization. Neutrophils contain receptors that bind to bacterial peptides (peptides with formyl groups). When these receptors bind bacterial peptides, they activate ARP2/3 triggering the growth of a branched network of actin filaments. This pushes the cell membrane towards the source of the bacterial peptides. 50 Take-home message… Actin and myosin filaments allow cell to form different morphologies. Intermediate filaments provide robust mechanical resistance. Coordinated actin polymerization pushes forward the cell membrane to drive cell motility. 51 References Alberts B. et al. 2008. Molecular Biology of the Cell. 5th edition. Garland Science, Taylor & Francis Group, New York. Bruce et.al. 2014 Essential Cell Biology, Garland Science. 4th edition. Geoffrey M. Cooper - The Cell: A Molecular Approach- Oxford University Press (2019) 52