CYTOSKELETON, MOTORPROTEINS.

Transcription

1 CYTOSKELETON, MOTORPROTEINS. SCIENCE PHOTO LIBRARY Tamás Huber CYTOSKELETON: Dynamic scaffold in eukaryotic cells Three main filament systems: A. Intermediate filaments B. Microtubules C. Microfilaments 20 µm cytokeratin (PANC1) alpha-tubulin (HeLa) Source: Abcam plc, 332 Science Park, Cambridge CB4 0WN, United Kingdom actin (endothelial cells) Source: Donald E. Ingber, The Architecture of Life Scientific American, Jan Role: 1. cell movement and shape, 2. cell division, 3. Intracellular transport 1

2 Discovery of actin (Brunó F. Straub, 1941) Polymerization (Process of the assembly of subunits) Polymer quantity growth steady state Phases of polymerization: 1. Lag phase: nucleation 2. Elongation 3. Dynamic equilibrium. lag time 2

3 True equilibrium: Polymerization equilibria Tread-milling : (e.g.: actin) Dynamic instability: steady, slow growth followed by catastrophic depolymerization (e.g.: microtubules) Microfilaments (actin) nucleotide 4 2 Subunit: Globular (G-) actin : MW: 42,3 kda, 375 amino acids 1 molecule bound adenine nucleotide (ATP or ADP) Binds: Ca 2+, Mg subdomains 3

4 Actin polymer 37 nm Filamentous (F-) actin: ~7 nm thick, length can be more than 10 µm in vitro, 1-2 µm in vivo Semiflexible polymer chain (persistence length ~10 µm) Right-handed double helix Structural polarization: based on the barbed-wire appearance of actomyosin. "barbed end", and "pointed end" ( barbed = + end: rapid polymerization, pointed = - end: slow polymerization) ATP-cap: stabilize the actin polymer and promotes growth Polymerisation assays Absorption Time (s) 4

5 Tubulin heterodimer: Microtubular system MW: ~50 kda, in neural tissue 10-20% of total cellular proteins - and -tubulin -> heterodimer 1 molecule bound guanosine nucleotide (GTP or GDP); exchangeable( ), and nonexchangeable ( ) Polymer: microtubule ~25 nm thick tubes, contains 13 protofilaments Right-handed short-pitch helix Left-handed long-pitch helix Stiff polymer chain (persistence length: few mm!) Structural polarity: + end: rapid polymerization, - end: slow polymerization GTP-cap Motorproteins: dynein, kinesin, dynamin 5

6 Cell division Source: Wadsworth Center's Featured Image - Archives Intermediate filaments 8 to 12 nanometers in diameter Fibrous monomer (not globular, as actin or tubulin) - amino-terminal head - central rod ( -helix, heptad repeat) - carboxy-terminal tail - the tissue-specific monomers differ in their tail region The subunit of filaments: coiled-coil dimer Ribbon diagram of vimentin 6

7 Polymerization of intermediate filaments Fully polymerized in the cell (not dynamic equilibrium) Hydrophobic interaction between central rods ( -helix) -> colied-coil dimer 2 dimers -> tetramer (antiparallel arrangement, structural apolarity) protofilament Longitudinal arrangement of tetramers -> protofilament filament 8 protofilaments -> filament Cytoskeleton observation with biomimetic assay I. Bugyi et al., FEBS Letters 582 (2008)

8 Cytoskeleton observation with biomimetic assay II. Movie made by Julie Theriot & Dan Portnoy Movie provided by Dr. Beáta Bugyi Salmonella typhimurium invading a fibroblast cell Theriot Lab, Department of Biochemistry, Beckman Center, Stanford University Medical School, in Palo Alto, CA. 8

9 Cytoskeleton associated proteins Large group of proteins with diverse functions, which bind to specific cytoskeletal filaments Groups: A. According to filamentous system 1. Actin-associated (e.g., myosin) 2. MT-associated (e.g., tau) 3. IF-associated (e.g., plektin) B. According to the geometry of binding 1. End-binding ( capping,e.g., gelsolin) 2. Side-binding (e.g., tropomyosin) C. According to function 1. Cross-linking a. Gel-forming (e.g., filamin, spectrin) b. Bundle-forming (e.g., -actinin, fimbrin, villin) 2. Polymerization regulator a. Depolymerizing ( severing, e.g., gelsolin) b. Stabilizing (e.g., profilin) 3. Motorproteins Motor proteins 1. They can bind to specific filament types 2. They can travel along filaments 3. They hydrolyze ATP 9

10 1. Actin-based: myosin protein family. Conventional (myosin II) and non-conventional myosins. Myosin families: myosin I-XVIII 2. Microtubule based motors a. Dynein Flagellar and cytoplasmic dyneins. MW~500kDa They move towards the minus end of MT b. Kinesin Cytoskeletal kinesins Neurons, cargo transport along the axons Kinesin family: conventional kinesins + isoforms. MW~110 kda They move towards the plus end of MT 3. Nucleic acid based motors DNA and RNA polymerases They move along a DNA and produce force Types of motor proteins 4. Rotational motors e.g.: F1F0 ATP-synthase, bacterial flagellar motor/ The working cycle of motor proteins ATP cycle power stroke = working distance attached t on attachment detachment detached t off back stroke r Duty ratio: on on off on total In vitro sliding velocity: v on Attached time: on v Cycle time: total 1 V =working distance (or step size); V=ATPase activity; v=in vitro sliding velocity 10

11 Duty ratio V r v Processive motor: r->1 e.g.: kinesin, DNA-, RNA-polimerase the motor is attached to the track in most of the working cycle Nonprocessive motor: r->0 e.g.: conventional myosin A motor protein can produce force in the pn range. Myosin superfamily 11

12 Myosin, a non-processive motor protein Three-bead assay Bead displacement Time Knight et al, The End! 12