Cytoskeleton PDF

Prof Wanda Lattanzi Dept Life Science and Public Health Section of Biology Room 352bis 1st Floor Istituti Biologici [email protected]  Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbook...

Prof Wanda Lattanzi Dept Life Science and Public Health Section of Biology Room 352bis 1st Floor Istituti Biologici [email protected]  Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbooks’ reference for pictures) and are therefore protected by the current regulations governing the Protection of Copyright. All rights are reserved. The reproduction and/or diffusion, even partial, by any analogical and/or digital means, without the consent of the rights holder is FORBIDDEN. Any unauthorized use of the above mentioned "Contents" is under the full and exclusive responsibility of the users who will be responsible for it, according to the laws and regulations in force. It is allowed the use of the material for private and study use, however not for profit and without commercial purposes. The cytoskeleton In all eukaryotic cells 3D network formed by: Microtubules Intermediate filaments Microfilaments Interconnected structures joint by noncovalent bonds, extremely dynamic (rapid assembly and disassembly). Functional facts 1. Mechanical and structural support 2. Organelles’ positioning 3. Movements and transport of intracellular cargoes 4. Cellular motility and contractility 5. Cellular division https://www.youtube.com/watch?v=tO-W8mvBa78 MICROTUBULES Microtubules In all eukaryotic cells Support network throughout the cytoplasm Mitotic spindle during cell division Central axis of cilia and flagella Tracks for the displacement of macromolecules and subcellular structures Microtubules : functions Support network spread throughout all the cytoplasm of interphase cells Resistance to compression and bending guide movements of cargoes inside the cell Mitotic spindle in dividing cells Axial shaft (axoneme) in cilia and flagella Microtubules: overview Extremely dynamic structures that can rapidly grow (via polymerization) or shrink (via depolymerization) in size, depending on how many tubulin molecules they contain. Microtubules: structure Tubular hollow non-branched structures Polimers of / tubulin Microtubules: structure Hollow cylinder Ext diameter: 25nm Wall thickness: 4nm Walls are formed by 13 protofilaments Each filament: linear chain of globular protein subunits tubulin / heterodimers → polarity Microtubule dynamics Microtubules are polarized: Plus end (β tub) Minus end (α tub) Tubulin subunits are added on the + end and removed from the - end (required GTP hydrolysis) of each protofilament The entire wall is built through a centripetal renewal «Treadmilling» Microtubule dynamic instability The ends of a microtubule grow at different speeds Stretch and shorten according to a GTP-dependent dynamic equilibrium At the + end the speed of adding dimers is greater than that of removal: net elongation At the - end the rate of adding dimers is slower than that of removal: net shortening Microtubule dynamic instability: the structural cap model GTP cap In a growing microtubule: 1. the + end has tubulin-GTP dimers that polymerize the protofilaments 2. as the protofilaments close the microtubule completing the loop, GTP is hydrolyzed 3. Tubulin-GDP dimers have a kinked conformation that causes tension at the - end 4. This tension is released when protofilaments open externally in the absence of new GTP dimers ("catastrophic shortening") Microtubules’ associated proteins (MAPs) Heterogeneous group of proteins Two ends: 1 anchored end to the microtubule wall 1 protruding outside the wall Form transverse bridges among microtubules Take part in microtubule assembly Contribute to multiple functions Microtubules’ associated proteins (MAPs) Binding is regulated through Ser-Thr phosphorylation by microtubule-affinity- regulating-kinase (MARK) MAP Phosphorylation leads to their detachment from microtubules causing microtubule instability and disruption Drewes, G. (2012). Microtubule Affinity Regulating Kinases (MARK). In: Choi, S. (eds) Encyclopedia of Signaling Molecules. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-0461-4_161 Tang, E., Cheng, C.Y. (2018). MAP/Microtubule Affinity-Regulating Kinase. In: Choi, S. (eds) Encyclopedia of Signaling Molecules. Springer, Cham. https://doi.org/10.1007/978-3-319-67199-4_101717 Microtubuli: struttura Proteine associate ai microtubuli (MAP) Proteina Tau nei neuroni è iper- fosforilata → grovigli neurofibrillari che non legano i microtubuli → degenerazione dei neuroni Tau protein is a neuronal MAP found hyperphosphorylated in AD brain → neurofibrillay tangles failing to bind to microtubules → neuron degeneration in Alzheimer’s disease. Microtubule motor proteins: kinesins and dyneins Convert chemical energy (ATP hydrolysis) into mechanical energy (conformational changes in the protein structure) Transport cargoes along the trails formed by microtubules: macromolecules (ribonucleoprotein complexes), vesicles, mitochondria, lysosomes, chromosomes, cytoskeletal fractions. Move along a unidirectional path based on microtubule polarity Microtubules motor proteins Kinesins Superfamily of related proteins (KRPs, kinesin‐related proteins: 14 different families (kinesin‐1 to kinesin‐14). Tetramers of heavy and light chains that form: two globular heads that bind microtubule and hydrolyze ATP a neck, a rodlike stalk a tail, fan‐shaped, that binds cargo to be hauled. Microtubules motor proteins Kinesins Move along single protofilaments towards the plus end “hand‐over‐hand” mechanism (at least one of the heads is attached to the microtubule at all times). highly processive (well adapted for independent, long‐distance transport of small parcels of cargo) The two heads behave in a coordinated manner: when one head binds to the microtubule, the resulting conformational changes in the adjacent neck region of the motor protein cause the other head to move forward toward the next binding site on the protofilament https://www.youtube.com/watch?v=gbycQf1TbM0 Microtubules: motor proteins Dyneins huge heteropolymeric protein two identical heavy chains + different intermediate and light chains Each dynein heavy chain consists of a large globular head with an elongated projection (stalk), force‐generating engine. Each stalk contains the all‐important microtubule‐binding site situated at its tip. The longer projection, known as the stem (or tail), binds the intermediate and light chains Microtubules: motor proteins Dyneins Move towards the minus end Force‐generating agent in positioning the spindle and moving chromosomes during mitosis Role in positioning the centrosome and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm https://www.youtube.com/watch?v=IvJrDsRuWxQ  Microtubule-organizing centers (MTOC) The minus ends of microtubules are anchored in structures called microtubule organizing centers (MTOCs), which represents the specialized sites in the cytoplasms where microtubule assembly starts. ✓Centrosomes (usually adjacent to the nucleus) ✓Basal bodies (apical MTOCs, below the plasma membrane) Microtubule-organizing centers (MTOC) Centrosomes Main site of microtubule initiation in animal cells, found at the center of the microtubular network They control the number and polarity of microtubules, the number of protofilaments in their walls, and the time and location of their assembly Centrosomes 2 barrel‐ shaped centrioles, perpendicular to each other cylindrical structures 0.2 μm in diameter, 0,5 μm in length 9 parallel fibrils, each formed by 3 microtubules: A (complete), B, C A tubules are connected to a central hub with nine spokes (cartwheel) Amorphous electron‐dense pericentriolar material (PCM) Tubulin  Pericentrin Basal bodies Site of microtubule nucleation at the base of cilia and flagella Same structure as centriole Microtubule biogenesis Nucleation Interaction among γ and β tubulin Assembly of a starting nucleus Stabilization of bonds among subunits Elongation Rapid and progressive polimerization with addition of new subunits γ-tubulin ring complex (γ-TuRC)  Vibratile cilia Cilia and flagella Structures elongating from the cell surface, having a citoskeletal shaft (axoneme) Motile CILIA: with their movement the displace the cell or sustances/particulates around the cell, along a perpendicular direction. FLAGELLA 1-2 per cell, they move as a wave Flagella pushing the cell forward along a parallel direction. Cilia and flagella AXONEME Series of parallel microtubules running longitudinally (+ end at the apex,– end at the base) 9 + 2 microtubule organization 9 peripheral doublets of A+B microtubules Joint by interdoublet nexin bridges 1 central pair of microtubules Wrapped by a sheath Joint to peripheral doublets by radial spokes Cilia and flagella A-B microtubules elongate from the basal body to form the doublets of the axoneme Cilia and flagella Intraflagellar transport (IFT) Motor proteins (kinensins and dyneins) mediate the bidirectional transport along the axonme of cilia and flagella Cilia and flagella Cilar and flagellar motility Motor proteins (kinensins and dyneins) mediate the conformational changes needed to modify dynamically the angle of inclination by making each microtubules slide on one another. Primary cilia Non motile Cellular antennae, able to capture mechanical stimuli and transduce them inside the cell as a molecular cascade mechanotransduction. The primary cilium A microtubule-based, non-motile organelle that extends as a solitary unit from the basal body of most cell types in the human body, receiving and processing molecular and mechanical signals. 1898 – Karl Zimmerman discovered the nonmotile cilium 1968 - Sergei Sorokin named it primary cilium The primary cilium structural facts Axoneme derived from the centrosomal mother centriole, lacking the central doublet of microtubules (= non-motile). Ciliary membrane continuous with the plasma membrane, with unique composition The primary cilium Intraflagellar transport structural facts (IFT): continuous movement of particles, from the ciliary base to The primary cilium is a highly polarized the tip (anterograde IFT) and back (retrograde IFT) structure: Basal body is a modified (mother) centriole, from which the axonme extension starts Transition fibres (TF) mediate docking of the basal body to the plasma membrane or vesicles during early stages of ciliogenesis Transition zone (TZ) between the basal body and the cilium, contains specialized gating structures that control the entrance and exit of ciliary proteins. Anvarian et al. Nat Rev Nephrol. In press doi: 10.1038/s41581-019-0116-9 The primary cilium structural facts Ciliary pocket (CiPo): depression of the plasma membrane in which the cilium is rooted. Membrane trafficking-specialized domain, establishes Frequently primary cilia do not project beyond the cell surface closed links with the actin-based cytoskeleton and is enriched in clathrin-coated pits The primary cilium structural facts Ciliary assembly and maintenance rely on the vesicular transport of proteins and lipids from the TGN that are incorporated in the periciliary membrane of the CiPo. Some proteins may enter by lateral transport of trans-membrane diffusion. IFT mediates the docking and fusion of vesicles on the mother centriole to form the TZ → axoneme extension Anvarian et al. Nat Rev Nephrol. In press doi: 10.1038/s41581-019-0116-9  The primary cilium structural facts The cilioplasm is a highly compartmentalized intracellular environment with a finely tuned directional flux of molecules The base of the cilium provides a membrane diffusion barrier to prevent the lateral diffusion of membrane proteins between plasma and cilia membranes. Ciliary trafficking is regulated by the BBSome, a multi-protein complex  The primary cilium functions The primary cilium is a multifunctional antenna involved in apicobasal and planar cell polarity (PCP) maintenance sensing both mechanical (fluid flow, pressure, touch, vibration) and chemical changes in the extracellular environment conveying signaling information to the cell to regulate: cellular proliferation, migration, interaction with the ECM, ecc. Multiple interconnected signalling pathways: Sonic Hedgehog (SHH) Trasforming Growth Factor β (TGFβ) Pedersen et al, Trends Biochem Sci. 2016;41(9):784-797 Wnt The primary cilium functions Primary cilium expression is tightly regulated during the cell cycle: During mitosis is dismantled and centrioles are duplicated to form the spindle poles Transiently resorb the primary cilium before the S phase Usually lost during G0 as quiescent cells cease to sense and signal environmental clues → STRUCTURAL CHECKPOINT FOR CELL CYCLE PROGRESSION The primary cilium functions The cilium senses changes in physical forces acting in the extracellular environment and transduce them inside the cell → SIGNALING HUB FOR MECHANOTRANSDUCTION kidneys Role of policystins in mechanotransduction bone The primary cilium functions Crucial for the establishment and maintenance of cell polarity (in both apicobasal and planar directions), guiding cell divisions towards the appropriate path during key morphogenetic events → STEER FOR CELLULAR POLARIZATION Nigro et al, Cells 2015 The primary cilium Role in development kif3Bwt kif3B-/- The correct ciliary signalling is needed for left-right asymmetry in vertebrates Role in body patterning Nonaka et al, Cell 1998 The primary cilium Role in development During brain development, the integrity of ciliary signalling is needed for: Radial glia progenitor migration and cortical scaffolding Orientation of neuroblasts’ mitoses Neuronal migration Correct cortical patterning The primary cilium in human diseases “CILIOPATHIES”: defects in ciliary and basal body proteins Cilia are present in most vertebrate tissues → pleiotropy in ciliary disorders Clinical expressivity depends on the spatiotemporal context of the mutated protein Variable complex phenotypes with overlapping features: Retinal degeneration Situs inversus Mental retardation Polydactily NTD Liver, kidney, pancreas cysts Genetic heterogeneity: mutations in > 40 genes known to date; mostly AR The primary cilium in craniofacial malformation Intermediate filaments Fibrous non-branched non-polarized filaments 10-12 nm diameter Spread throughout the entire cytoplasm Confer mechanical resistance Less sensitive to chemical agents than other types of cytoskeletal elements and more difficult to solubilize Intermediate filaments composition >70 different proteins form subunits Classified into 5 categories: I-IV cytoplasmatic V nuclear All IF polypeptides share a central rod‐shaped α‐helical domain of similar length and homologous amino acid sequence  Intermediate filaments composition All IF polypeptides share a central rod‐shaped α‐helical domain of similar length and homologous amino acid sequence Gae et al., 2019, Structure27, 1547–1560  Intermediate filaments structure Basic unit: tetramer composed by 2 homodimers (each dimer contains 2 polypeptides that spontaneously interact, as their α‐helical Coiled-coil rods wrap around each other, are aligned parallel to one dimer Protofibril tetramer another) COILED-COIL DIMER Homodimers are then paired longitudinally, misaligned and antiparallel PROTOFIBRIL TETRAMER 8 tetramers associate laterally to form a thicker filament ~60nm in length PROTOFIBRIL UNIT 60nm filaments associate in series INTERMEDIATE FILAMENT No chemical energy required for the assembly Intermediate filament Protofibril unit  Intermediate filaments function IF radiate throughout the cell and form a cagelike network. Connect to the plasma membrane via specialized sites of adhesion such as desmosomes and hemidesmosomes Connect to microtubules and microfilaments by members of the plakin family of proteins (e.g. plectin) Connect to the nuclear envelope (lamins) the IF network serves as a scaffold for organizing and maintaining cellular architecture and for absorbing mechanical stresses applied by the extracellular environment. Intermediate filaments composition Type I-II IF proteins: Keratins Each consisting of about 15 different proteins expressed in epithelial cells. Each type of epithelial cell synthesizes at least one type I (acidic) and one type II (neutral/basic) keratin, which copolymerize to form filaments. Some keratins (hard keratins) are used for production of hair, nails, and horns. Other keratins (soft keratins) are abundant in the cytoplasm of epithelial cells, with different keratins being expressed in various differentiated cell types. Epidermolysis bullosa Rare disease [8.2 cases per million live births] characterized by blistering of the skin and mucous membranes that occur with minor trauma or friction. Different forms with varying severity and inheritance Hereditary disease due to mutations in one of 16 different genes There is no cure; treatments for wound care, pain control, infection control, nutritional support; prevention and treatment of complications Intermediate filaments composition Type III IF proteins Vimentin: expressed in fibroblasts, smooth muscle cells, and white blood cells. Desmin: expressed in muscle cells, where it connects the Z discs of individual contractile elements Glial fibrillary acidic protein (GFAP): in glial cells Peripherin: expressed in neurons of the peripheral nervous system. Intermediate filaments composition Type IV IF proteins: neurofilament proteins Three neurofilament (NF) proteins (light, NF-L,medium, NF-M, and heavy, NF-H) expressed in neurons; particularly abundant in the axons of motor neurons α-internexin: expressed at an earlier stage of neuron development Nestin: expressed even earlier during the development of neurons, in stem cells of the central nervous system. Intermediate filaments composition Type IV IF proteins: neurofilament proteins Head domains have a microtubule polymerization inhibitory domain that regulates the number of MT Rod domains have important roles in the polymerization of NF subunits, and serve as a binding site for the myosin to modulate levels and local topography of specific vesicular organelles (ER, endosomes, synaptic vesicles) within the axoplasm Intermediate filaments composition Type IV IF proteins: neurofilament proteins Maintain symmetrical shape of neurons Required for axon radial growth Docking and organization of different axoplasmic constituents Intermediate filaments composition Type V IF proteins: lamins Expressed in the euchariotic nucleus They assemble to form an orthogonal meshwork underlying the nuclear membrane: the nuclear lamina  Microfilaments 8nm in diameter Subunits of globular actin (G-actin) Polimerize into a flexible branched filament: F-actin FUNCTIONS Cellular motility Intracellular movements Phagocytosis Cytokinesis Microfilaments The G-actin monomer is bound to ATP ATP-actin hydrolyzes ATP to polimerize F-actin are polimers of ADP-actin G-actin subunits are oriented towards the same direction → F-actin is polarized Microfilaments F-actin is polarized: “barbed” (plus) end “pointed” (minus) end S1 proteolytic subunit of myosin tightly attach to F-actin: “S1-decoration”  Microfilaments’ assembly-disassembly The events that occur during actin assembly/disassembly in vitro depend on the concentration of actin monomers The critical concentration of the barbed end is much lower than the pointed end → the barbed end can continue elongating at lower ATP‐actin concentrations than the pointed end can 1. As long as the concentration of ATP‐actin monomers remains high, subunits will continue to be added at both ends 2. As the monomers are consumed the concentration of free ATP‐actin continues to drop until a point is reached where net addition of monomers continues at the barbed end but stops at the pointed end 3. As filament elongation continues, the free monomer concentration drops further: monomers are added exclusively to the barbed end Once a steady‐state concentration of monomers is reached, subunits are added to the + end at the same rate they are released from the - end. As a result, subunits treadmill through the filament Myosins: the microfilaments’ motor protein Motor proteins associated to microfilaments Humans contain about 40 different myosins from at least 12 classes, each presumed to have its own specialized function(s). Two broad groups 1. Conventional (type II) myosins: first identified in muscle tissue 2. Unconventional myosins (type I and types III– XVIII), classified based on amino acid sequence, some expressed widely among eukaryotes, whereas others are restricted to species. Myosins: the microfilaments’ motor protein Myosin features a typically saymmetric structure Heteroexamer: 6 polypeptidic chains (1 pair of heavy chains + 2 pairs of light chains) The heads bind F-actin Head domains of various myosins are similar, the tail domains are highly divergent. Myosins: the microfilaments’ motor protein Myosin subunits form a bipolar filament Tails towards the center Heads protruding towards opposite ends → attract two F-actin filaments towards each other Basic mechanism involved in muscle contraction Muscle contraction Dystrophin and DAPC Dystrophin is a rod-shaped cytoplasmic protein that interact with other proteins (α-dystrobrevin, syncoilin, synemin, sarcoglycan, dystroglycan, and sarcospan) to form the dystrophin-associated protein complex (DAPC) DAPC connects the cytoskeleton of a muscle fiber to the surrounding ECM through the cell membrane. The N-term of dystrophin binds to F-actin and the C-term to the DAPC at the sarcolemma.  Dystrophin and muscle dystrophy The DMD gene, encoding the dystrophin protein, is the longest in the human genome. Mutations in this gene cause Muscular Dystrophies (XLR): Duchenne and Becker Reduced or missing Dystrophin causes the DAPC to destabilize leading to progressive fibre damage and membrane leakage DMD patients are usually wheelchair-bound by 12 years of age and die of respiratory failure in their late teens or early twenties. Many boys have an abnormal electrocardiogram by the age of 18, indicating that diaphragm and cardiac muscles are affected too. Suggested readings https://www.ncbi.nlm.nih.gov/books/NBK9834/ Suggested readings https://jcs.biologists.org/content/125/17/3923 Suggested readings https://jcs.biologists.org/content/125/14/3257