Unit 3 Cytoskeleton 2023-24 PDF

INTRODUCTION The cytoskeleton is a dynamic network of protein filaments extending throughout the cytoplasm. The cytoskeleton is critical for many essential cell functions : cell division, cell movement, cell growth, differentiation ... The main functions of the cytoskeleton are : § To provide a stru...

INTRODUCTION The cytoskeleton is a dynamic network of protein filaments extending throughout the cytoplasm. The cytoskeleton is critical for many essential cell functions : cell division, cell movement, cell growth, differentiation ... The main functions of the cytoskeleton are : § To provide a structural framework for the cell and support the large volume of cytoplasm (especially in animal cells that have no cell wall). § To serve as a scaffold that determines the position of organelles. § To facilitate the internal transport of organelles or other structures (including chromosomes during cell division). § To allow cell movements (cell migration), cell contraction, or changes in cell shape. CYTOSKELETON COMPONENTS IN EUKARYOTIC CELL The cytoskeleton is composed of proteins that assemble through non-covalent interactions to form long filament structures . Three types of structures can distinguished based on their diameter, protein component and subunit arrangement : Ø MICROTUBULES (22-25 nm) hollow cylinders made of Tubulin Ø INTERMEDIATE FILAMENTS (10 nm) show a rope-like structure and are composed of fibrous proteins (keratin, vimentin ...) Ø ACTIN FILAMENTS OR MICROFILAMENTS (5-9 nm) two stranded helycal polymers of Actin CELLULAR DISTRIBUTION OF THE CYSTOSKELETON Each of the three cytoskeleton components show a different distribution in the cell. a) b) MICROTUBULES a) MICROTUBULES Centrosome c) MICROFILAMENTS INTERMEDIATE FILAMENTS radiate from the microtubule-organizing center (MTOC) or b) MICROFILAMENTS show a scattered distribution in the cytosol, but are especially concentrated just beneath the plasma membrane. c) INTERMEDIATE FILAMENTS extend accross the cytosol forming a framework that attaches to the plasma membrane and provides mechanical strength to the cell. MICROTUBULES Cells contain 2 types of microtubule populations: Ø UNSTABLE MICROTUBULES, short-lived and very dynamic. They form the Mitotic Spindle and mediate changes in cell shape and organelle movements Ø STABLE MICROTUBULES, long- lived and non dynamic Present in Cilia, Flagella and Centrioles Mitotic Spindle Flagellum Centrioles STRUCTURE OF MICROTUBULES A microtubule is a polymer of globular tubulin subunits arranged in a hollow cylindrical tube. 13 Each microtubule subunit is a heterodimer of α - Tubulin and β-Tubulin. The tubulin subunits are aligned end to end forming protofilaments that pack side by side to create the wall of the microtubule. Each microtubule contains 13 protofilaments . Tubulins are very similar in all animal species and together with Histones are the most evolutionary conserved proteins. Diameter: 25 nm Length: variable (1- >100 μm) MICROTUBULE DYNAMICS Microtubules are dynamic structures that go through continuous assembly and disassembly processes and are constantly growing and shrinking . This behavior is known as DYNAMIC INSTABILITY. In addition to their structural polarity, microtubule display GROWTH POLARITY because tubulin dimers are added preferentially at one end, designated the (+) end (fast growing end) , and are lost preferentially from the other end, the (-) end (non-growing or slow growing end). The (+) end is the one where the β-tubulin monomers are exposed. The (-) end is the one where the α-tubulin monomers are exposed. In a cell the (-) ends of all microtubules are found around a microtubule organizing center or MTOC (the centrosome for example) and radiate from there to the cell periphery. MICROTUBULE ASSEMBLY AND DISASSEMBLY 1- αβ-tubulin dimers bind head to tail to form short protofilaments. 2- These proteofilaments associate laterally into more stable curved sheets. Eventually sheets made up of 13 protofilaments wrap around forming a short hollow cylinder. The microtubule then grows by the addition of new tubulin dimers to the ends of the protofilaments. 3- The free tubulin dimers have GTP bound to both tubulin monomers. However, shortly after the tubulin dimer is added to the growing microtubule, GTP in the βtubulin monomer is hydrolized to GDP due to the intrinsic GTPase activity of tubulin. 4- GTP hydrolysis weakens the binding affinity of tubulin dimers for each other leading to disassembly or depolimerization of the GDP-bound dimers. MICROTUBULE ASSEMBLY AND DISASSEMBLY THE DYNAMIC INSTABILITY MODEL § Only microtubules with (+) ends associated with GTP-tubulin are stable and can prime tubulin polymerization. § MTs with bound GDP-tubulin depolymerize more rapidly and can disappear in 1 min. § At high concentrations of non-polymerized GTP-tubulin, the rate of tubulin assembly is faster than the rate of GTP hydrolysis. Therefore the MT grows. § At low concentrations of non-polymerized GTP-tubulin, the rate of tubulin assembly decreases and the GTP hydrolysis rate is higher. An unstable GDP cap is formed, the protofilaments spring apart and tubulin subunits are released. The MT shortens. MICROTUBULE ASSEMBLY AND DISASSEMBLY α-tubulin à non-hydrolyzable GTP β-tubulin à GTP is hydrolyzed to GDP Both ends of the microtubule have the ability to grow, but they will do it at very different rates. Tubulin polimerization rate at the (+) end is 2-3 times higher than at the (-) end. The concentration of free tubulin dimers determines whether a microtubule will grow or shrink. (-) (+) [Tubulin] = Critical Concentration (Cc) The Critical Tubulin Concentration (Cc) is the concentration at which microtubule length is constant. (-) (+) If the tubulin dimer concentration is higher than Cc, polymerization will be favored and the microtubule will grow. [Tubulin] > Cc Polymerization If the tubulin dimer concentration is lower than Cc, depolymerization will be favored and the microtubule will shrink. The concentration of tubulin in the cytoplasm depends on the rate of synthesis and degradation but varies also with polymerization or depolymerization events. (-) [Tubulin] < Cc (+) Depolymerization MICROTUBULE-ASSOCIATED PROTEINS (MAPs) They bind specifically to microtubules and modulate their stability of and their association with other cell structures. There are two types of MAPs : Ø PROTEINS THAT STABILIZE MICROTUBULES : they bind to the negatively charged Cterminal part of Tubulin and stabilize the outer wall of a microtubule. They can increase the growth rate of microtubules or suppress microtubule catastrophe. They are cell-specific. Some are present in neurons : MAP1A, MAP1B, TAU (in axons and dendrites) and MAP2 (only present in dendrites). Others are not present in neurons : MAP4. Ø PROTEINS THAT DESTABILIZE MICROTUBULES : they may sever intact cytosolic microtubules through an ATP-dependent process by breaking-up internal bonds between tubulin dimers (Katanin ) or they may promote disassembly of tubulin dimers at the (+) end (Catastrophin) MICROTUBULE-ASSOCIATED PROTEINS (MAP) The binding of MAPs to Microtubules is inhibited by PHOSPHORYLATION . Therefore, phosphorylation of MAPs favors microtubule disassembly. Hyperphosphorylation can promote disassembly in disorders like alzheimer s disease. + MAPs VESICULAR TRANSPORT THROUGH MICROTUBULES Vesicles can be transported by MOTOR PROTEINS like Kinesin and Dinein moving along microtubules . The movement can be towards the (+) end (anterograde) or towards the (-) end (retrograde) of microtubules. § Kinesins mediate anterograde transport § Dyneins mediate retrograde transport The type of receptors present on the vesicle surface will determine in what direction it will be transported. Vesicle transport is particularly important in neurons where molecules synthesized in the cell body or even mitochondria must be delivered to the axon terminal. Axonal transport occurs in both directions (up and down the axon) , is fast (up to 400 mm/day), and relies on kinesin, dinein and microtubules. http://www.youtube.com/watch?v=kOeJwQ0OXc4&feature=related FUNCTIONS OF UNSTABLE MICROTUBULES 1. MAINTAIN THE CELL SHAPE 2. CELLULAR TRANSPORT. Axonal transport The oriented microtubules in the axon serve as tracks for the directional transport 3. MITOTIC SPINDLE FORMATION, which will distribute chromosomes between daughter cells when the cell enters mitosis. STABLE MICROTUBULES • CILIA AND FLAGELLA • CENTRIOLES CILIA AND FLAGELLA § They are thin and flexible proyections of the cell. § Cilia and Flagella have essentially the same structure but cilia are short (few micrometers) and abundant while flagella are long (e.g. more than 2 mm in an insect sperm) and scarce. § Function: locomotion of free cells (sperm) or movement of fluids along the cell surface (respiratory epithelia). § Structure : 2 main regions • Axoneme: central bundle of microtubules • Basal body: point of attachment to the cell. AXONEME § The axoneme is formed by 9 doublet MT surrounding a central pair of singlet MT (9+2 arrangement). § Each doublet consists of A and B microtubules (A is complete: 13 protofilaments; B is incomplete: 11 protofilaments) with the (+) end located at the distal end of the axoneme. § The outer MT doublets are connected to the central pair by radial spokes and to each other by a protein called Nexin. § Permanently attached to each A tubule of the doublet are inner-arm and outer-arm dyneins, that drive the movement of cilia and flagella. BASAL BODY It is the growing point of cilia and flagella, where the (-) end of axoneme microtubules are oriented. It has the same structure as centrioles : 9 triplets of microtubules tilted towards the central axis. DIFFERENCES BETWEEN CILIA AND FLAGELLA Type of movement : Cilia show a pendular or whiping movement, with an effective stroke followed by recovery stroke to initiate a new movement. They sweep materials across tissues. Flagella show a waving movement that propels cells forward. Length: Cilia are shorter than flagella. Number : Cilia are present in high numbers in each cell while flagella are scarce (1 or 2) FLAGELLA CILIA 106/µm2 CENTRIOLES § Two cylindrical structures located at the center of the centrosome. § Exclusive of animal cells. § In interphase they are perpendicularly oriented and constitute the DIPLOSOME. § Together with the pericentriolar (PC) matrix they form the Centrosome § Each centriole is formed by 9 triplets of microtubules, tilted towards the central axis of the structure. § Each triplet is attached to the adjacent triplet by the NEXIN protein. § Before entering mitosis cells must duplicate their centrioles. Each centriole gives rise to a new centriole so that once mitosis is complete each daughter cell gets a diplosome. § They are involved in the formation of the mitotic spindle but they are not essential (e.g. plants do not have centrioles but form mitotic spindles). C C diplosome B A THE CENTROSOME § The centrosome functions as a Microtubule Organizing Center (MTOC) . § It is made up of pericentriolar material (PCM) and sometimes, but not always, it contains 2 centrioles oriented perpendicular to each other. § The (-) ends of cytosolic microtubules are immersed in the pericentriolar matrix but they do not contact the centrioles. § A gamma-tubulin ring (γTuRc) is found at the (-) end of microtubules , but the (+) ends are free. MICROFILAMENTS Microfilaments are the thinnest filaments in the cytoskeleton (5-9 nm) They are two stranded helical polymers of ACTIN Actin is the most abundant intracellular protein in eukaryotes Microfilaments show structural and growing polarity : (+) end , fast growing, and (-) end, slow growing. § The rate of polymerization and depolymerization is very high. § They are usually shorter and more flexible than microtubules. § § § § Actin F-ADP Actin G-ATP ACTIN § Actin can be found as a free globular monomer (G-ACTIN) or it can form fibrous polymers (F-ACTIN). § Free Actin monomers are bound to ATP, but ATP is hydrolyzed to ADP shortly after it is incorporated into the filament. § Hydrolysis of ATP reduces the binding strength between actin monomers and leads to depolymerization. G-Actin F-Actin POLYMERIZATION OF ACTIN MICROFILAMENTS 1) NUCLEATION : formation of small aggregates of Actin. It is the rate-limiting step in the formation of an actin polymer. 2) ELONGATION : the small initial nucleus elongates by addition of new actin units to both ends. The (+) end grows faster. 3) HYDROLYSIS OF ACTIN-BOUND ATP AND STABILIZATION : a steady state is reached at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation. The filament length does not change. ACTIN FILAMENT TREADMILLING § ATP-actin monomers are added rapidly to the (+) end of the microfilament and the ATP is hydrolyzed to ADP after polymerization. The ADP-actin is less tightly bound and can dissociate from the (-) end. § This cycle is called TREADMILLING. It creates a flow of actin monomers from the (+) end to the (-) end of the filament. § Treadmilling illustrates the dynamic behavior of actin filaments. ACTIN-BINDING PROTEINS The formation and stability of actin filaments in the cytosol is controlled by different actin-binding proteins Ø PROTEINS THAT PROMOTE POLYMERIZATION: PROFILIN Ø PROTEINS THAT PREVENT POLYMERIZATION: THYMOSIN Ø SEVERING PROTEINS : GELSOLIN AND COFILIN Ø CAPPING PROTEINS : CapZ AND TROPOMODULIN REGULATION OF POLYMERIZATION-DEPOLYMERIZATION Actin polymerization is regulated by proteins that bind free actin monomers (Gactin). These proteins either promote or inhibit actin polymerization. PROTEINS THAT PROMOTE POLYMERIZATION: PROFILIN : it forms a complex with G-actin-ATP that contributes to monomer aggregation at the (+) end. It is a nucleotide-exchange factor. PROTEINS THAT INHIBIT POLYMERIZATION: THYMOSIN : it binds to G-actin-ATP, sequesters it and prevents its binding to the (+) end of the filament. SEVERING PROTEINS Another group of proteins control the length of actin filaments by breaking them into shorter fragments . These proteins stabilize a conformational change in the actin subunit to which they bind and create a small gap between neighboring subunits and, eventually, a break . These proteins are GELSOLIN and COFILIN. After breaking a filament , the severing protein remains bound at the (+) end of one of the resulting fragments, where it prevents the addition or exchange of actin subunits (FILAMENT CAPPING). CAPPING PROTEINS They bind to actin filament ends and stabilize them . § CapZ: binds to the (+) ends of actin filaments and prevents addition or loss of actin subunits at that end . § TROPOMODULIN: Caps the (-) ends of actin filaments CapZ An actin filament that is capped at both ends is effectively stabilized, undergoing neither addition nor loss of subunits. Such capped filaments are needed in places where the cytoskeleton organization does not change, as in a muscle sarcomere or in the erythrocyte membrane. ORGANIZATION OF ACTIN FILAMENTS Actin filaments filaments are assembled to form two types of stable structures : • ACTIN BUNDLES • ACTIN NETWORKS ORGANIZATION OF ACTIN FILAMENTS Actin filaments filaments are assembled to form two types of stable structures : • ACTIN BUNDLES • ACTIN NETWORKS Both structures require ACTIN CROSS-LINKING PROTEINS to link one filament to another in different ways. The filament pattern created by this proteins is determined by their size and shape. • Actin-Bundling proteins : FIMBRIN, α-ACTININ • Network-forming proteins : FILAMIN MICROFILAMENT FUNCTIONS 1- DEFINING AND CHANGING THE CELL SHAPE : For example : changes in platelet shape during blood clotting. Resting platelets have a biconcave, disk-like shape Following activation by clotting Finally, platelets spread out agents platelets extend and form LAMELLIPODIA numerous FILOPODIA (thin (sheet-like extensions) cellular projections) These changes in morphology are the result of complex rearrangements in the actin cytoskeleton that is cross-linked to the plasma membrane. MICROFILAMENT FUNCTIONS 2- FACILITATING CELL MOTILITY (CELL CRAWLING) AND MUSCLE CONTRACTION: 1) Extension of the cell membrane and formation of a lamellipodium. 2) Polymerization of actin filaments and further protrusion of the lamellipodium. 3) Formation of focal adhesions in the lamellipodium and adhesion to the substratum. 4) Stable contacts with the underlying surface prevent the membrane from retracting. There is a cytosolic flux forward. 5) The cell “ tail” eventually detaches and retracts into the cell body. Cell contraction as well as muscle contraction involve Actin and Myosin II MICROFILAMENT FUNCTIONS 3- FORMATION OF THE CONTRACTILE RING AND CYTOKINESIS: Cytokinesis is the process that divides a cell in two daughter cells following mitosis. Contractile ring (Actin and Myosin II) Each daughter cell must receive the same amount of cytoplasm and organelles. A CONTRACTILE RING consisting of Actin filaments and Myosin II is assembled at the equator of the dividing cell. Its contraction pulls the plasma membrane progressively inward until it closes off and splits the cell in two. Actin filaments Cleavage furrow Myosin Daughter cells STABLE MICROFILAMENT STRUCTURES Ø MICROVILLI : § Fingerlike extensions of the plasma membrane that are particularly abundant in cells involved in absorption such as the epithelial cells lining the intestine. The purpose of these thin protrusions is to increase the surface area available for absorption. § They do not possess intrinsic movement capacity § They are smaller than cilia (1/10 or 1/20 of the cilia size) § A single cell can have several thousand microvilli. ACTIN FILAMENTS with associated proteins MYOSIN I VILLIN FIMBRIN INTERMEDIATE FILAMENTS § Intermediate size (10 nm) § Present in all animal cells § Highly stable, felixible and resistant polymers of fibrous proteins (there is NO polymerization and depolymerization and no structural polarity. § They do not require ATP or GTP for assembly or disassembly. § They are composed of different proteins depending on the cell type : • Epithelial cells : Keratins • Neurones : neurofilament proteins • Muscle cells : Desmin § Abundant in the cytosol of cells that are subject to mechanical stress FUNCTION: Intermediate Filaments DO NOT contribute to cell motility. Their functional role is to provide mechanical strength to the cell and to dissipate tensile forces to avoid cell or tissue damage. IF also provide support to the nuclear membrane. INTERMEDIATE FILAMENT STRUCTURE All IF proteins have a central α-helical region flanked by two globular N- and C-terminal domains. The helical segments of two monomers interwind around each other to form a coiled-coil dimer with both N- termini on one side and both C- termini on the other side (PARALLEL DIMER). Two parallel dimers associate in an antiparallel orientation to form a staggered TETRAMER . Tetramers assemble end-to end to form PROTOFILAMENTS Protofilaments associate laterally to form PROTOFIBRILS Protofibrils wind around each other to form a ROPELIKE FILAMENT INTERMEDIATE FILAMENTS IN THE NUCLEAR LAMINA The NUCLEAR LAMINA is a fibrous meshwork that lines the inner face of the nuclear envelope. It is composed of Intermediate Filaments (made up of Lamins) and other associated proteins. LAMIN INTERMEDIATE FILAMENTS support and strengthen the nuclear envelope. DYNAMICS OF NUCLEAR INTERMEDIATE FILAMENTS During the initial stages of mitosis the nuclear envelope breaks down and the nuclear lamina intermediate filaments disassemble in a tightly regulated process. At the end of mitosis the nuclear envelope forms again and the nuclear lamina re-assembles. HUMAN DISORDERS ASSOCIATED WITH INTERMEDIATE FILAMENTS q Epidermolisis Bullosa Simplex : Caused by mutations in several Keratin genes. As a result, cells in the epidermis become fragile and easily damaged. In affected individuals skin is less resistant to friction and minor trauma and blisters easily. Normal Keratin Mutant Keratin q Amyotrophic Lateral Sclerosis (Lou Gehrig's syndrome) : It is a Motor Neuron Disease that has been linked to alterations in Neuron Intermediate Filaments and to accumulation and abnormal assembly of neurofilaments q Neurodegeneration q Laminopathies : Progeria, Muscular Dystrophy