Classical cytoskeletons. Lecture 8 & 9: 1. Cytoskeleton functions. 2. Cytoskeleton functions. Intermediate filaments structure and function

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1 Cell biology 2017 Lecture 8 & 9: Classical cytoskeletons Microtubules Text book Alberts et al.: Chapter 16 (Topics covered by the lecture, focus on general principles) Cell Biology interactive media video or animation 1 Actin filaments (Microfilaments) Intermediate filaments 2 1. Cytoskeleton functions Cell containing cytoskeleton Cell without cytoskeleton 2. Cytoskeleton functions Cell containing cytoskeleton Cell without cytoskeleton Establishment of cellular shape and intracellular organization ER Golgi Cell appendages Cell locomotion Resistance against tensile stress 3 Genomic and cellular division 4 rinciple architecture of cytoskeletal filaments Intermediate filaments (8) Actin Filaments (2) Microtubules (13) Intermediate filaments structure and function Apolar filaments amphipathic helical monomers 6 Tetramer of coiledcoil dimers Cytosol: support of cell layers ( tensile stress ) Cell adhesion (desmosome) Subunits: 10 nm A family of coiledcoil proteins 7 nm Actin 25 nm Tubulin heterodimer Desmo: a prefix meaning "adhesion" rotofilament (proto = a prefix meaning the earliest ) 5 Nucleus: supporting the nuclear envelope animation 16.4 Intermediate _filament 1

2 rotein: Location: Tissue specific intermediate filaments Intermediate filaments can be composed of either: Homodimers or heterodimers Intermediate filament superfamily: >60 genes in mammals Epithelia Cytosolic Cytokeratins Neurofilaments Neurons Vimentin, Desmin Cells in connective and muscle tissue Nuclear Lamins 7 Lining of the nuclear membrane Keratin: ECM (Basal lamina) Intermediate filaments in epidermis 8 Ectoderm Mesoderm All cells Different means to achieve mechanical strength Fig. 191 Epithelial cells Basal lamina (dense ECM) Connective tissue (ECM cells) Epithelial tissue: The intermediate filaments of the cells themselves (linked from cell to cell by desmosomes) provides mechanical strength. Hemidesmosomes (integrin binding to laminin, hemi= half)) are only found in the epithelial cells that connect to the basal lamina. Epithelial cells are normally nonmotile. Connective tissue: ECM provides mechanical strength. Residual cells produce ECM components and migrate within ECM, e.g. to sites of tissue damage. Connective tissue cells are motile (actindependent) Cells are resistant to mechanical stress ECM (but not cells) are resistant to mechanical stress 9 Actin filaments in nonmuscle cells 5 nm Structure olar filaments composed of actin Functions Linking the interior to the exterior ( ECM ) Contraction ( ) Spreading & protrusions cell shape Locomotion Contractile ring during cell division Video 01.1keratocyte_dance Video 22.7 neurite_outgrowth 10 Subunit interactions within actin filaments 11 Myosin: a family of () enddirected actin motors olymer with both longitudinal and lateral subunit interactions stability within the polymer but dynamic ends Head Myosin bound to actin filament AT binding dissociates myosin AT AD i AT is rapidly hydrolyzed, which cause a simultaneous conformational change Dynamic ends Filaments may become stable by capping of ends Actin binding proteins may bundle filaments into a stable arrangements, e.g. filaments that exert contraction Magnetic tape: Maria BäckLindgren (T1, vt14) AD i Video 16.9 crawling_actin AD i Following AT hydrolysis, myosin binds an actin subunit Binding to actin causes the release of AD i. This results in a conformational change termed the power stroke 12 2

3 Myosin family members in nonmuscle cells A family of () end directed motor proteins Example of functions: Monomeric myosin Short range transport (not coved by this course) Nonmuscular myosin II Contraction (movement towards the ends of two antiparallel actin filaments) 3 distinct types of muscle cells Blood vessel Relaxed and contracted skeletal muscle Skeletal muscle: fused myoblasts giant multinucleated cells video beating_heart 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 Sarcomere Muscle contraction Contraction Actin Myosin Sarcomere The actin and myosin filaments remain the same length The sarcomere length shortens because the actin and myosin filaments slide relative each other Sarcomere: Greek sarx "flesh", meros "part" animation 16.8 myosin ( error search for a functional video) 15 binds along the actin filament: No contact between actin and myosin filaments Contraction is initiated by an increase of cytosolic Ca 2 Troponin mediated translocation of tropomyosin Ca 2 Regulation of skeletal muscle contraction animation muscle_contraction 16 Actin filaments: a polymer with dynamic ends Significance of the free monomer concentrations Steady state On rate = Off rate No net effect on polymer length Cooperative longitudinal and lateral subunit interactions within the polymer high affinity due to cooperativity Low cooperativity at ends low affinity Capping of ends results in stable filaments Actin binding proteins may bundle filaments into various arrangements, e.g. filaments that exert contraction animation 02.1 noncovalent _bonds 17 Increased monomer pool Net polymerization Decreased monomer pool Net depolymerization 18 3

4 Concept of the critical free actin concentration The actin monomer ( = soluble subunit) concentration ( = [Free]) at steady state is referred to as the critical concentration Nucleation of actin filaments in cells Spontaneous is an unlikely event. This is because the affinity of monomer interactions is low % % olymerized actin = Elongation Spontaneous (in vitro only) Steady state (equilibrium) 0[Free subunits] Nucleation phase Local activation of factor local Elongation phase (polymerization) 0 % Time 19Actin monomer concentration Active factor stabilizing assembly template Control of actin filament Conformation changes of actin 22 No factor No (specific) Inactive factor No (specific) Actin monomers bind AT, AD and AM [AT] >> [AD] Actin hydrolyzes bound AT conformational change Actin bound to AT Global activation of factors Local activation of factors Nucleotide hydrolysis i Actin bound to AD Global Local 21 The intrinsic ATase activity of actin is very low, but increases when actin monomers are part of a filament I. AT fueled actin treadmilling Low affinity (critical conc. e.g. 1 µm) () end () end High affinity (critical conc. e.g. 0.2 µm) Treadmilling occurs when [Monomers] (i.e. [Free subunits]) are between the critical concentrations at the two ends (1 0.2 µm) AD AT During treadmilling the filament length remains constant, while subunits dissociate from the () end and are added at the () end 23 II. AT fueled actin treadmilling AD AD AD Time AD AD AD AD AD AD AD AT AT AT :interaction strength AT AT AT [AT] >> [AD] = Fluorescent actin Subunits "move" towards the () end 24 4

5 Treadmilling requires actin severing 25 Actin filaments are dynamic in nonmuscle cells [Gactin] = [Monomer] = [Free] stabilizes the () end i) Treadmilling is the mechanism behind attachmentdependent cell motility ii) Cell motility and remodelling requires rapid assembly/disassembly of actin filaments olymerization ceases due to low [Gactin] A severing protein ADF/Cofilin binds to ADactin containing filament olymerization at the () end can resume and the filament will treadmill, which will facilitate continuous growth at the () end Stimuli AD AT exchange Time 26 Motility: movement of cells ( mot is the same root that's found in "motor ) Higherorder architecture of actin filaments Actin factors Arp 2/3 Actin filaments (in nonmuscle cells) may associate into bundles or networks via different crosslinking proteins Formin Antiparallel bundles allowing access to myosin II Network actinin actinin Arp 2/3 may also bind preexisting filaments to create branching Tight parallel bundles Fimbrin Fimbrin Formin Arp 2/3 27 Locally acting switches direct cell migration Rho 28 Epidermis Cdc42 1. Basal lamina Actin web (treadmilling) 3. Stratum Corneum 1. Stress fibers (contraction) 2. Dermis 30 Connective tissue cells migrate to an injury Actin bundles (protrusions) Basal layer Rho//Cdc42: Gproteins regulated by specific GEFs & GAs ECM /residual cells 3. ( fibroblasts ) 5

6 Actinmediated cell migration Stress fibers (contraction) Function: Lamellipodia (treadmilling) Actin and bundling by Cdc42 Chemotactic signal Filopodia (protrusions) Chemoattractant (e.g. DGF) Local switches (Rho family members): Rho Cdc42 31 Video 23.9 wound_healing dependent lamellipodia formation 3 GEF Cdc42 Arp 2/3 Fimbrin Actin Tight parallel actin bundles Video 10.1 membrane_fluidity 33 Chemotactic signal (I3K activation see slide 38) Cdc42 GEF GD Cdc42 Internal (migrationdependent) signals ADF/Cofilin Rho GD 2. Stable filaments Formin ADF/Cofilin 1. Actin Stable actin meshwork Actin and branching actinin actinin Myosin II 4. Contraction ADF/Cofilin dependent severing treadmilling Summary of cell motility Actin reorganization drives neutrophil migration Bacteria, releasing peptides containing NformylMethionine 2 3 ECM 4. Detachment at trailing end Video 01.2 crawling_amoeba 3. Antiparallel actin bundles 1 Neutrophils have fmetleuhe receptors Chemoattractant rotrusion 34 Rho dependent stress fiber formation 3. Contraction and translocation ECM attachment at the leading edge (focal adhesions) Video 15.2 chemotaxis Video 16.2neutrophil_chase The activated receptor provides the direction of lamellipodia formation The bacteria is internalized by phagocytosis 36 6

7 .M..M. Reminder: heterotrimeric Gproteins No ligand (default state) GD Ligand binding causes a conformational change GD GD The Gprotein is recruited to the receptor, which acts as a GEF the subunit exchanges GD for dissociation of the subunit and subunits 37 Neutrophil chemotaxis local regulation of fmetleuhe receptor GCR GD I3 Kinase Arp 2/3 zzz GD GEF 3 Filamin Nformylated bacterial protein Actin and branching I3 Kinase 3 GEF Actin meshwork 38 Structure Hollow polar tubes (13 tubulin protofilaments) Function Microtubules Tubulin heterodimer tubulin tubulin rotofilament Microtubule Exert both pushing and pulling forces Structural support and railroad tracks, which establish intracellular organization Cellular appendages (cilia and flagella) Segregation of chromosomes during mitosis 39 ushing and pulling by microtubules during mitosis Interphase (G2) rophase Centrosome (mother & daughter) Telophase/ cytokinesis Video 13.2 biosy_secret_path Video 17.7 mitotic_spindle Anaphase Metaphase rometaphase 40 hosphatidylinositol Astral Overlap Kinetochore MT The centrosome the site for microtubule The centrosome contains ~100 tubulin ring complexes sites (templates) for microtubule assembly Tubulin Ring Complex Minusend Centriole pair 41 Different microtubules arrangements Cell types with proliferative potential Columnar epithelial cells (small intestine) Microvilli (actin protrusions) lusend Inherited templates direct protofilament arrangement TuRC= assembly manual Tubulin heterodimers may assemble into a variety of structures different templates Neurons 42 7

8 hydrolysis at the Esite of the tubulin heterodimer Esite Esite Catalytic loop Esite = Exchangeable site roteins that control microtubule dynamics Stabilization by Microtubule Associated roteins (MAs) Multivalent binding along the polymer () end () end Destabilization by catastrophe promoters eeling of protofilaments Catalytic loop 43 The () end is stabilized by the tubulin ring complex 44 MT dynamics catastrophe 45 MT dynamics rescue 46 cap (Esite exposed) tubulin GDtubulin tubulin GDtubulin () end (delay in hydrolysis) Catastrophe promoting protein : Rescue promoting protein The () end is capped by tubulin () end (nucleated at the centrosome) eeling of protofilament Catastrophe, followed by depolymerization Depolymerization Video 16.1 MT_instability aus Regain of cap (due to reinitiated polymerization) Dynamic instability stochastic switches () end olymerization [GD] << [] GD Catastrophe Rescue () end (nucleated at the centrosom) : Rescue promoting protein : Catastrophe promoting protein Dynamic instability serves to search and capture a variety of structures Depolymerization 47 Cell cycle regulation of microtubule dynamics Interphase (inactive Cdk/M) CdkM Few and long microtubules: Few events Slow dynamics Mitosis (active Cdk/M) CdkM Many and short microtubules: Many events Rapid dynamics Video 16.5 microtubule_dynamics Note visualization by fusion to a fluorescent protein (EB1GF & TubGF) 48 8

9 Capture of kinetochores by microtubules 49 Unidirectional transport on polar polymers 50 1 MTs continuously searches the cellular space... Generation of pulling force 2...and are stabilized by kinetochore attachment 3 Finally, both kinetochores are captured by MTs from opposite centrosomes. Sister chromatids are positioned at the cellular equator by the polar ejection force generated by MTs ( ) Candy Checkout olarity in a queue at the supermarket Motor proteins (unidirectional movement) () end () end Movement of MT dependent motor proteins 51 MT dependent pushing forces during mitosis Dynein Kinesin Kinesin dimer () end () end Kinesin dependent pushing forces via antiparallel MTs are required for: Headoverhead walking (an AT dependent process) rophase A B B B B B A Anaphase (B) Animation 16.7 kinesin 52 Control of division plane in epithelia Correct Incorrect (a wart!) 53 Astral microtubules direct the division plane 1. Dynamic (astral) microtubules are stabilized by tip binding proteins ( ) at specific sites at the cell cortex Membrane anchored dynein ( ) pulls at astral microtubules Basal lamina (ECM) 3. ulling forces specify the correct division plane CellECM contacts (hemidesmosomes: integrins) 9

10 Cell polarization by localized MT stabilization 55 Motor proteindriven transport on microtubules A nonpolarized cell in which MTs search the intracellular space Dynein Kinesin Stabilization of MTs that encounters localized tipbinding proteins ( ) Virus ER 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 Golgi Exocytosis Lysosome Vesicle Axon Endocytosis Mitochondrion Synapse 56 Video 13.2 biosy_secret_path Video 16.6 organelle_movement Cilia Microtubuledependent cell appendages 5 10 m appendages projecting from cell surfaces Capable of movement Moves fluids over cell surfaces (e.g. lung epithelia) Flagella In essence a cilia, but longer ( m) Only one per cell Move the cell in a wavelike fashion Distinct from flagellas on bacteria Microtubule arrangement in cilia and flagella Axoneme Basal body Cilia Flagella Axoneme: the part of a cilia or flagella that bends back and fourth The beating of a cilia ower Stroke (energi input) The beating of cilia is dependent on MT bending forces Axoneme 59 Dynein dependent MT bending in cilia and flagella 2. Anchorage to dynein tail 1. Nexin, holds the MTs together Recovery Stroke (back to default) Basal body Video XX.X cilia/flagella beating??!! 3. Bending of MTs upon dynein movement 60 10