Movement at the Molecular Level Diffusion: <r 2 > = 6 D t (D≈6 π μ a) Typical numbers: Image removed due to 10 nm protein in water D= 10 -10 m 2 /s ….in cells D= 10 -12 m 2 /s (D= 10 -14 m 2 /s lipids) copyright considerations. [<r 2 >] 1/2 =1 μm, t ~0.2 sec in cells [<r 2 >] 1/2 =10 μm, t ~20 sec in cells Slow and isotropic. How to generate fast vectorial motion ? Axonal transport of organelles in giant squids 2/28/03 BEH 410-10.537J 1 Directed (Vectorial) Molecular Movement Polymerization: Living polymerization of actin/microtubules Springs: Conformational changes of molecules Motor Proteins: nucleotide (ATP) hydrolysis: chemical energy -> work Pumps: Hydrolysis of ATP Create concentration gradients 2 Actin Comets Propelling Listeria Listeria monocytogenes moving in PtK2 cells These pathogenic bacteria grow directly in the host cell cytoplasm. The phase-dense streaks behind the bacteria are the actin-rich comet tails. Actin-based motility is also used in cellular motility; this cell is using it's cytoskeleton to crawl toward the lower right-hand corner. Speeded up 150X over real time. --Julie Theriot & Dan Portnoy 3 Actin is Transiently Tethered to the Bacteria Images removed due to copyright considerations. See Cameron, L.A., T. M. Svitkina, D. Vignjevic, J. A. Theriot, and G. G. Borisy. "Dendritic organization of actin comet tails." Curr Biol. 2001 Jan 23;11(2):130-5. Noireaux et al. (2000): it takes about 10 picoN to separate the actin from the comet… 4 Images removed due to copyright considerations. See Kuo, S. C and J. L. McGrath. "Steps and fluctuations of Listeria monocytogenes during actin-based motility??." Nature. 2000 Oct 26;407(6807):1026-9. Steps of 5.4nm 5 Elastic Brownian Ratchets and Tethered Filaments Images removed due to copyright considerations. See Mogilner, A. and G. Oster. "Force generation by actin polymerization II: the elastic ratchet and tethered filaments??." Biophys J. 2003 Mar;84(3):1591-605. Brownian: Actin filament tips fluctuate Some filaments are tethered 6 Actin Ruffles in Motile Cells 7 Supramolecular Springs Energy stored in chemical bonds which act as “latches’ Regulated by Spasmin: Calcium binding protein Images removed due to copyright considerations. See Mahadevan, L. and P. Matsudaira. "Motility powered by supramolecular springs and ratchets." Science. 2000 Apr 7;288(5463):95-100. 8 Horseshoe Crab Sperm Uncoiling of an actin spring (unlike the echinoderm sperm - no polymerization!) Images removed due to copyright considerations. See Mahadevan, L. and P. Matsudaira. "Motility powered by supramolecular springs and ratchets." Science. 2000 Apr 7;288(5463):95-100. 9 Molecular Motors ? Molecules that convert chemical energy into mechanical force ? Motors are specialized for specific tasks: cell division cell movement organelle transport synthesis of ATP ? Most move unidirectionally along polymer filaments ? Coupled mechanical and chemical cycles (fuel) 10 Motor Types Motor Track Functions Myosin F-actin Cell crawling Myosin II F-actin Muscle contraction Cell division Phagocytosis Kinesin microtubule Organelle transport Mitosis & meiosis Dynein microtubule Flagella & cilia Polymerases, ds and ssDNA Replication, Repair Helicases Recombination 11 Muscle Anatomy Muscle Types: Skeletal: fast Cardiac: fast Smooth: slow 12 Sliding filament assay Myosin Heads Walk Along Actin Filaments Polar Biopolymer Molecules : The Tracks Motor Proteins Walk Along Myosin walks along Actin G-Actin (globular) F-Actin (microfilaments) - end Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-2. + end Diameter: 6-8 nm Persistence length: 16 μm Young’s modulus: 1.3-2.5 x 10 9 Pa 14 Myosin: the actin motor protein All myosins have head, neck, and tail domains with distinct functions Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-20. Viewable online at the PubMed Bookshelf: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books 15 Figure 18-20 Myosin Types Conventional Type II muscle contraction cytokinesis cell adhesion, migration Unconventional Types I, III-… type I: cell migration We will concentrate on type II, but other types display similar mechanisms… 16 Skeletal muscle contains a regular array of actin and myosin II: the sarcomere Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-27. Viewable online (Fig. 18-27b only) at the PubMed Bookshelf: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books. Figure 18-27 17 Skeletal muscle contains a regular array of actin and myosin Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-27c. Figure 18-27 18 Capping proteins stabilize the ends of actin thin filaments in the sarcomere Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-28. Viewable online at the PubMed Bookshelf: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.5204. Figure 18-28 19 Thick and thin filaments slide past one another during contraction During contraction the myosin head initially binds tightly the thin filament (actin) to form a cross-bridge Once in contact the head rapidly bends towards Image removed due to the center of the sarcomere during the power stroke copyright considerations. See [Lodish 4th ed.] Figure 18-29. The thin filament is then displaced towards the Viewable online at the PubMed Bookshelf: center of the sarcomere by about 10 nm http://www.ncbi.nlm.nih.gov/ books/bv.fcgi?rid=mcb.figgrp.5208. The head releases from the thin filament, reverts back to the initial conformation and the cycle repeats The heads are only in contact with the filament about 5% of the time Continuous movement because several heads are marching along the filament 20 Muscle Contraction 21 Conformational changes in the myosin head couple ATP hydrolysis to movement Figure 18-25 22 Sliding Filament Model Huxley & Huxley 1954 Image removed due to copyright considerations. See [Lodish 4th ed.] Figure 18-29. Viewable online at the PubMed Bookshelf: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.5208. 23 The Myosin ‘Power Stroke’ pre-stroke Small conformational change In head is amplified by swinging movement of the neck. Light chains increase rigidity of the neck. post- stroke Images removed due to copyright considerations. See Figures 4 and 6 in Geeves and Holmes. "Structural mechanism of muscle contraction." Annu Rev Biochem. 1999;68:687-728. 24 ATP: Cellular Fuel Image removed due to copyright considerations. Chemical bond diagram of ATP. ATP Hydrolysis: ATP + H 2 0 - > ADP + P i K eq = 4.9 x 10 5 Depends on conditions Strongly favored ?Large activation barrier w/o a catalyst= stable fuel ?Free energy change at cellular conditions: -25 kT 25 Mechanochemical Coupling : Myosin II Motor and no actin: low activity ~ 0.1 s -1 M = motor MD + P T = ATP M MT MDP D = ADP Rate limiting step P = phosphate A = actin Motor and with actin: increased activity ~ 25 s -1 MT MDP AMDP AMD AM AMT MT Key ideas: ? release of P (chemical) is catalyzed by binding to actin (mechical) ? w/o ATP myosin bonds strongly to actin ? release of myosin (mechanical) is catalyzed by ATP binding (chemical) 26 The actin-myosin ATPase cycle Allostery: release of P after MDP binds to actin creates a highly strained AMD state that relaxes via a change Diffusion to/ from of configuration and may generate a power-stroke. filament Myosin S1 Image removed due to copyright considerations. ATP hydrolysis induces a recovery stroke. ATP binds to Pi acts as spring “latch” hydrolysis filament l = 10nm Image removed Image removed due to copyright Cycle ~ 50ms due to copyright ADP-Pi ADP-bound, considerations. considerations. released bound Image removed P i pre-stroke Power-stroke P i ATP release from filament ADP Image removed due to copyright considerations. due to copyright considerations. Actin filament “Rigor” state No ATP available – remains attached 27 Images adapted from Vale and Milligan, Science 288, 88 (2000). Mechanochemical Coupling : Myosin Power-Stroke 28 Tropomyosin and troponin regulate contraction in skeletal muscle Ca2+ influences the position of TP & TN on the actin filament. Image removed due to copyright considerations. Binding sites: closed open 29 Increasing the Working Stroke Distance in Myosin Image removed due to copyright considerations. Diagram with caption Figure 4. Processive J. Howard 1997 30 Assays to Study Motor Proteins in vitro Image removed due to copyright considerations. 31 Polar Biopolymer Molecules : Microtubules The Tracks Motor Proteins Walk Along Image removed due to copyright considerations. Diameter: 24 nm Persistence length: 60,000 μm Young’s modulus: 1.9 x 10 9 Pa 32 The Role of Kinesin & Dyneins Image removed due to copyright considerations. MTOC: Microtubule-organizing center Bidirectional transport of organelles 33 Structure of Motor Proteins Myosin Kinesin Image removed due to copyright considerations. See [Lodish]. The head contains ATP and filament binding sites. 34 Mechanochemical Coupling :Conventional Kinesin Key Points: -with ATP kinesin is bound tightly to microtubules -unbinding of kinesin from microtubule requires hydrolysis of ATP - in the absence of microtubule kinesin dissociates P quickly -release of ADP from kinesin is catalyzed by binding to microtubule - 2 heads coordinate movement 35 Hand-over-Hand Model for Conventional Kinesin Image removed due to copyright considerations. http://www.current-opinion.com/jcel/mov1.mov 36 Release of trailing head (K1) is contingent on the binding of the leading head (K2)… Image removed due to …binding of ATP to K1 catalyzes attachment of copyright considerations. K2 to microtubule… …this catalyzes release of ADP from K2… …which catalyzes detachment of K1 from the microtubule… …release of P from K1… 37 Schief & Howard 2001 Motor Proteins: Power Strokes Image removed due to copyright considerations. 38 Common Themes ? Filaments are polar and motor binding is stereospecific. ? This leads to movement in one direction (+ or -). ? Stall forces ~ few (6-10) pN ? Cyclic motors ? Nucleotides roles: 1. regulates attachment/detachment. 2. drives working/recovery strokes. 3. chemical steps are contingent on the completion of mechanical steps. 39 Differences Myosin II Conv. Kinesin non-processive processive (~100 steps of more) 5-15 nm step sizes slips walks towards + end of filament found in large assemblies ‘rower’ 8 nm no slipping walks towards -end of filament works well alone or low #’s porter 40 Motor Speeds (assemblies) Motor type speed in vivo (nm/s) in vitro(nm/s) in vitro ATPase (s-1) Myosin II (skeletal muscle) 6000 8000 20 Myosin II (smooth muscle) 200 250 1.2 Myosin V (vesicle transport) 200 350 5 Conv. Kinesin (axonal transp) 1800 840 44 Nkin (sec. Vesicle transp.) 800 1800 78 BimC/Eg5 (Mitosis/meisos) 18 60 2 Dyneins (cytoplasmic) -1100 -1250 2 Speed in vivo = cell/extracts, motion of motor relative to filament w/o a load. Positive values Indicate movement toward positive end of filament. Speed in vitro = purified motors at high ATP concentrations. ATPase = max rate of hydrolysis per head per sec, measured at high ATP, filament concentrations. 41 Kinesin is attached during it’s rate limiting step while myosin is detached during it’s rate limiting step- ATP hydrolysis. Kinesin on off Myosin on off While one myosin is dissociated 42 from the actin the others can continue pushing (rowing)! = 8 nm ~ 8 nm ~ 5 nm = 36 nm in skeletal muscle Mechanical Models for Motor Proteins Myosin Kinesin 43 Common Themes for Molecular Motors ? ATP hydrolysis drives the motors. ? The motors walk along polar tracks (polymers). ? Type of motor fits the function: processive/non. 44