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Transcript
Cell biology 2014 (revised 11/2-14) Lecture 8 & 9: Chapter 16 965-1020 1026-1050 A lot of reading! Focus on general principles and topics highlighted in the lecture synopsis Cell Biology interactive media ”video” or ”animation” 1 Classical cytoskeletons Microtubules Actin filaments (Microfilaments) Intermediate filaments 2 1. Why do we need a cytoskeleton? Cell containing cytoskeleton - Establishment of cellular shape and intracellular organization Cell without cytoskeleton ER Golgi - Resistance against mechanical stress 3 2. Why do we need a cytoskeleton? Cell containing cytoskeleton Cell without cytoskeleton - Cellular appendages - Cell locomotion - Genomic and cellular division 4 Principle architecture of cytoskeletal filaments Intermediate filaments 10 nm Subunits: A family of coiledcoil proteins Actin filaments Microtubules 7 nm 25 nm Actin Tubulin heterodimer 5 Intermediate filaments – structure and function 6 Non-polar filaments + amphipathic a-helical monomers Tetramer of coiled-coil dimers Cytosol: support of cell layers ( tensile stress ) Cell adhesion (desmosome) Nucleus: supporting the nuclear envelope animation 16.4 -Intermediate _filament Tissue specific intermediate filaments Intermediate filaments can be composed of either: Homodimers or heterodimers - Intermediate filament super-family: >60 genes in mammals Cytosolic Nuclear Protein: Cytokeratins Vimentin, Desmin Neurofilaments Lamins Location: Epithelia Cells in connectiveand muscle tissue Neurons Lining of the nuclear membrane of all cells 7 8 Intermediate filaments in epidermis Keratin: 1 + 10 5 + 14 ECM (Basal lamina) PP PP PP PP P P PP P P PP P P P P P P P P Actin filaments – structure and function Structure - Polar filaments composed of actin Functions - Linking the interior to the exterior ( - Contraction ( ) - Spreading & protrusions cell shape - Locomotion - Contractile ring during cell division Video 01.1-keratocyte_dance Video 22.7 –neurite_outgrowth ECM ) 9 Actin filaments are dynamic in migrating cells Rapid assembly and disassembly is central to a variety of functions, such as cell remodeling and locomotion Stimuli Time 10 Subunit interactions within actin filaments A polymer with only longitudinal subunit interactions uniform (and poor) polymer stability + + Protofilament (proto = a prefix meaning the “earliest”) A polymer with both longitudinal and lateral subunit interactions stability within the polymer but dynamic ends Internal stability Dynamic ends 11 Nucleation of actin filaments Spontaneous nucleation is slow because the initial interactions are unstable (low degree of cooperativity) Spontaneous nucleation Spatially regulated nucleation factors local nucleation Nucleation factor 12 Control of actin filament nucleation No availabile nucleation factor Inactive nucleation factor No (specific) nucleation No (specific) nucleation Global activation of nucleation factors Localized activation of nucleation factors Global nucleation Local nucleation 13 Actin nucleation factors Arp 2/3 Formin + end Arp 2/3 may also bind pre-existing filaments to create branching + end - end - end 14 15 Concept of the critical free concentration The monomer ( = soluble subunit) concentration ( = [Free]) at steady state is referred to as the critical concentration 0 Elongation Monomer concentration [Free subunits] % Subunits in filament (% Bound) 100% Steady state Spontaneous nucleation 0% Time (minutes) 60 Dynamics at different free concentrations [Monomer] = [Free subunits] [Monomers] > critical concentration, e.g. 3 nM Net polymerization [Monomers] = critical concentration, e.g. 2 nM No net effect on polymer length [Monomers] < critical concentration, e.g. 1 nM Net depolymerization 16 Nucleotide turnover in cytoskeletal subunits Actin subunits bind ATP Tubulin heterodimers bind GTP The subunit changes its conformation upon nucleotide hydrolysis Subunit bound to a nucleoside triphosphate Nucleotide hydrolysis Subunit bound to a nucleoside diphosphate 17 A nucleoside is the portion of a nucleotide that doesn't include the phosphate groups I. ATP fueled actin treadmilling ”Low” critical concentration ”High” critical concentration (e.g. 2 nM) (e.g. 10 nM) - end + end Treadmilling occurs when [Monomers] (i.e. [Free subunits]) is between the critical concentrations at the two ends (2 10 nM) ADP ATP 18 During treadmilling the filament length remains constant, while subunits are added at the (+) end and dissociate from the (-) end II. ATP fueled actin treadmilling :interaction strength - ADP + ADP ADP ADP ADP ATP ADP P P ADP ATP ADP P P ADP ADP Time - ATP ATP ATP ATP [ADP] << [ATP] + = Fluorescent actin Treadmilling actin subunits "move" towards the (-) end 19 Treadmilling requires actin severing [G-actin] = [Monomer] = [Free] Arp2/3 stabilizes the (-) end - 20 + Polymerization ceases due to low [G-actin] A severing protein – ADF/Cofilin – binds to ADP-actin containing filament Polymerization at the (+) end can resume and the filament will treadmill, which will facilitate continuous growth at the (+) end ADP/ATP exchange Significance ?! slide 10 & 52 Microtubules Structure - Hollow polar tubes of tubulin protofilaments Tubulin heterodimer b-tubulin a-tubulin Protofilament Microtubule Function Exert both pushing and pulling forces Structural support and railroad tracks, which establish intracellular organization Locomotion by cellular appendages (cilia and flagella) Segregation of chromosomes during mitosis 21 Pushing and pulling by microtubules during mitosis Interphase (G2) Prophase Telophase/ cytokinesis 22 Prometaphase Anaphase Metaphase Astral- video 13.2 – biosy_secret_path Video 17.7 – mitotic_spindle OverlapKinetochore MT The centrosome – the site for microtubule nucleation The centrosome contains ~100 g-tubulin ring complexes, which act as nucleation sites for microtubule assembly Centriole pair g-Tubulin Ring Complex Minus-end Plus-end All subunits are encoded for by the genome, but assembly requires an inherited copy as a template 23 Different microtubules arrangements • “Most” cell types • Columnar epithelial cells (small intestine) - - - - -- + + + + ++ • Neurons + + - - -+ +- ++ + 24 GTP hydrolysis at the E-site of the tubulin heterodimer E-site = Exchangeable site E-site GTP b GTP a E-site GTP b Catalytic loop GTP GTP GTP GTP GTP GTP GTP a Catalytic loop 25 Proteins that control microtubule dynamics Stabilization by Microtubule Associated Proteins (MAPs) Multivalent binding along the polymer Destabilization by catastrophe promoters Peeling of proto-filaments at the end 26 MT dynamics – catastrophe GTP cap (E-site exposed) GTP-tubulin GDP-tubulin Catastrophe promoting protein + end (delay in GTP hydrolysis) - end (nucleated at 27 the centrosome) The + end is “capped” by GTP-tubulin Peeling of proto-filament Catastrophe, followed by depolymerization MT dynamics – rescue GTP-tubulin GDP-tubulin : Rescue promoting protein Depolymerization video 16.1- MT_instability Paus Regain of GTP cap through re-initiated polymerization 28 Dynamic instability – stochastic switches 29 [GDP] << [GTP] GTP GDP Catastrophe Rescue - end (nucleated at Depolymerization Polymerization + end the centrosom) : Rescue promoting protein : Catastrophe promoting protein Dynamic instability ”search and capture” of a variety of structures Cell cycle regulation of microtubule dynamics Interphase Mitosis (active Cdk/M) P CdkP M P P CdkP M Few and long microtubules: - Few nucleation events - Slow dynamics Many and short microtubules: - Many nucleation events (5x) - Rapid dynamics (10x) video 16.5- microtubule_dynamics Note- visualization by fusion to a fluorescent protein (EB1-GFP & aTub-GFP) 30 Capture of kinetochores by microtubules 31 1 MTs continuously searches the cellular space... 2 ...and are stabilized at the kinetochores of chromosomes * * * * ** * * * * 3 Finally, both kinetochores are captured by MTs from opposite centrosomes. Generation of pulling force *The sister chromatid pair is positioned at the cellular equator by the polar ejection force generated by MTs (* ) Unidirectional transport on polar polymers Candy Check-out Polarity in a queue at the supermarket Motor proteins (unidirectional movement) - end + end 32 Movement of MT dependent motor proteins Dynein Kinesin - end + end Head-over-head walking (an ATP dependent process) 1. 2. A B animation 16.7- kinesin 3. B 4. B 5. B B A 33 MT dependent pushing forces during mitosis + Kinesin dimer + Kinesin dependent pushing forces via anti-parallel MTs are required for: Prophase Anaphase (B) 34 Control of division plane in epithelia Correct Incorrect 35 Control of division plane by astral microtubules 1. Dynamic (astral) microtubules are stabilized by tip binding proteins ( ) at specific sites at the cell cortex - 1. + 2. Membrane anchored dynein ( ) pulls at astral microtubules 2. Basal lamina (ECM) 3. 3. Pulling forces specify the correct division plane 36 Cell polarization by localized MT stabilization A non-polarized cell in which MTs search the intracellular space Stabilization of MTs that encounters localized tip-binding proteins ( ) Reorientation of the MT- system by membrane anchored dyneins ( ) A polarized cell: stabilized MTs serve as rail tracks that transport membrane vesicles and actin regulatory proteins to MT (+) ends 37 38 MT-dependent trafficking in the cell + Virus Dynein Kinesin ER Golgi Vesicle - + Axon Lysosome Synapse Endocytosis Exocytosis Mitochondrion video 16.6- organelle_movement Cellular appendages built of microtubules Cilia Flagella - 5 -10 mm appendages projecting from cell surfaces - In essence a cilia, but longer (100-200 mm) - Capable of movement - Only one per cell - Moves fluids over the cell surface - Move the cell in a wavelike fashion 39 Arrangement of microtubules in cilia and flagella Flagella Axoneme Cilia Basal body Axoneme: the part of a cilia or flagella that bends back and fourth 40 The beating of a cilia The beating of cilia is dependent on MT bending forces Power Stroke (energi input) Axoneme Basal body Recovery Stroke (back to default) 41 Dynein dependent MT bending in cilia and flagella 1. Nexin, holds the MTs together 2. Anchorage to dynein tail 3. Bending of MTs upon dynein movement 42 Myosin: a family of (+) end-directed actin motors - + Myosin bound to actin filament Head ATP binding dissociates myosin ATP ATP is rapidly hydrolyzed, which cause a simultaneous conformational change ADP +Pi Following ATP hydrolysis, myosin binds an actin subunit ADP +Pi ADP +Pi Video 16.9 –crawling_actin Binding to actin causes the release of ADP + Pi. This results in the conformational change termed the “power stroke” 43 “Non-muscular” myosin family members A large family of related (+) end directed motors. Example of functions: Monomeric myosin Transport (short range) 44 + - + Non-muscular myosin II Contraction (movement towards the +ends of two anti-parallel actin filaments) Cargo - - + - + Muscles – a brief overview • Skeletal muscle, fused myoblast that forms a multinucleated cell (fast but non-persistent) • Cardiac muscle cells (persistent) • Smooth muscle cells i) surrounds hollow organs – intestines and blood vessels ii) Arrector pili muscles attached to hair follicles (slow and very persistent) Lumen video 16.11- beating_heart Lumen 45 Principle of skeletal muscle contraction When stimulated to contract, the heads of the bipolar myosin filament walk along actin in repeated cycles of attachment and detachment contraction of the sarcomere unit Actin Myosin Actin Myosin + + + + Contraction Sarcomere Sarcomere - The actin and myosin filaments remain the same length - The sarcomere length shortens because the actin and myosin filaments slide relative each other animation 16.8- myosin (compare with picture 43) 46 Regulation of skeletal muscle contraction Tropomyosin binds along the actin filament: No contact between actin and myosin filaments Tropomyosin Tropomyosin Tropomyosin Tropomyosin Contraction is initiated by an increase of cytosolic Ca2+: Troponin mediated translocation of tropomyosin Tropomyosin Tropomyosin Tropomyosin Tropomyosin Ca2+ animation 16.10- muscle_contraction 47 Higher-order architecture of actin filaments Actin filaments (in non-muscle cells) may associate into bundles or networks via different cross-linking proteins Anti-parallel bundles allowing access to myosin II - a-actinin a-actinin Sparse 3D network + Tight parallel bundles - + Fimbrin Fimbrin + + - short and thin fibers Long and thick fibers 48 Cell migration requires locally acting GTP switches GTP Rho GTP Rac + - + GTP Cdc42 - + + - - Stress fibers (contraction) Actin web (tread milling) Actin bundles (protrusions) Rho/Rac/Cdc42 are GTP switches (similar to Ras) 49 An integrated view of actin dependent migration Actin structure: Stress fibers (contractile) Lamellipodia Filopodia (pseudopodia) (microspikes) - + - + + - Rho family member: Rho video 23.9- wound_healing Rac Cdc42 Chemoattractant (e.g. PDGF) + - 50 Actin nucleation and bundling by Cdc42 Chemotactic signal GTP Cdc42 Cdc42 GEF GDP Cdc42 Arp 2/3 Actin nucleation video 10.1- membrane_fluidity Fimbrin Tight parallel actin bundles 51 Rac dependent lamellipodia formation GTP Rac P 3P P Rac GEF Chemotactic signal (PI3-K dependent – see slide 56) GDP Rac ADF/Cofilin Actin nucleation and branching ADF/Cofilin dependent severing treadmilling Stable actin meshwork 52 Rho dependent stress fiber formation Internal (localization dependent) signals ADF/Cofilin GTP Rho 2. Stable filaments Formin - end a-actinin 1. Actin nucleation a-actinin Myosin II - end 4. Contraction 3. Anti-parallel actin bundles 53 Summary of cell motility 3. Contraction and translocation 54 1. Protrusion + + - - + + - Chemoattractant - ECM 4. Detachment at trailing end Video 01.2 –crawling_amoeba 2. ECM attachment at the leading edge (focal adhesions) Role of actin for neutrophil migration 1 Neutrophils have fMet-Leu-Phe receptors Bacteria, releasing peptides containing N-formyl-Methionine 2 3 video 15.2 chemotaxis video 16.2-neutrophile_chase The activated receptor provides the direction of pseudopodia formation Neutrophil engulf bacteria through phagocytosis 55 56 Neutrophil chemotaxis fMet-Leu-Phe receptor N-formylated bacterial protein GPCR GDP a b g P 3 P Phosphatidylinositol GTP a + b PI-3 Kinase GTP Rac PI-3 Kinase zzz GDP Rac Rac GEF Filamin Arp 2/3 P 3P P Rac GEF Actin nucleation and branching Stable actin meshwork II. Regulation of hetero-trimeric G-proteins No ligand (default state) P.M. GDP b ga Ligand binding causes a conformational change P.M. GDP b ga b + GTP a GDP GTP The G-protein is recruited to the receptor, which acts as a GEF the a-subunit exchanges GDP for GTP dissociation of an active a-subunit 57