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29/11/2011 The cytoskeletal system, motor proteins 20.11.2011. Cyto + SKELETON • The cellular "scaffold" or "skeleton" within the cytoplasm of the eukaryotes (prokaryotic cytoskeleton!? (1991)). • It is composed of proteins. • Functions: − maintains cell shape (stability) − protect the cell − enables cellular motion (using structures such as flagella, cilia and lamellipodia) − intracellular transport (the movement of vesicles and organelles) − endocytosis and exocytosis − cell division 1 29/11/2011 The cytoskeleton is made up of 3 components. microtubules cytoskeleton intermediate filaments microfilaments http://www.pdn.cam.ac.uk/staff/roper/kr_images.html http://www.microscopyu.com/articles/fluorescence/filtercubes/yfp/yfphyq/stains/yfpcy2keratinptk2cells.html http://www.lajollaneuroscience.org/sr/homepage/cell/scientific_art_gallery/pasquale.htm Cytoskeletal filament arrangements • filament • bundles (parallel array of filaments – e.g. microvillus) • network (loosley packed, crisscross filaments) – 2D planar (close to plasma and nuclear membranes) – 3D (within the cell) Cross-linking proteins !!! (connect and stabilize) 2 29/11/2011 Microfilaments / Actin ~ 7-10 nm in diameter most concentrated just beneath the cell membrane Functions: • force production (contractile and protrusive forces) • cell movement (20µm/sec.) • wound healing • defend against infection • maintaining cellular shape • participation in some cell-to-cell or cell-to-matrix junctions (signal transduction) • important role in cytokinesis • muscular contraction (myosin) Microfilaments - actin F-actin (polymer – filamentous) ATP 37nm adenosine triphosphate G-actin (monomer - globular) G-aktin (42.3kDa) ~ 375 amino-acid 6.7 x 4 x 3.7 nm 7-10nm Double stranded, right handed helix 3 29/11/2011 Actin – nucleotide hidrolysis - + - + D D D D D D D T D T D D D D D D D T T + The ratio of the actin monomers inside the actin filaments (%) Actin polymerisation nucleation („lag” phase) elongation (increasing the size) „steady-state” (dynamic equlibrium) 100 Critical concentration (non-polymerising actin fraction) + - Formation of oligomers 0 (RATE LIMITING STEP) time 4 29/11/2011 „Tread-milling” shrinking end - + growing end - + + - - + - + Movement of the filament Intermediate filaments 8 to 12 nanometers in diameter Ropelike filaments more stable than actin filaments (easy to bend hard to break) Functions: •maintenance of cell-shape by bearing tension. •organize (STABILISE) the internal three-dimensional structure of the cell (ceratin). •supporting and anchoring the position of the organelles in the cytosol (vimentin) •structural components of the nuclear lamina (lamins) and sarcomeres (desmin) •They involved in some cell-cell and cell-matrix junctions. Types: • vimentins (the common structural support of many cells) • keratin, found in skin cells, hair and nails • neurofilaments of neural cells (NF-L, NF-M) • Lamin (structural support to the nuclear envelope) 5 29/11/2011 Intermediate filaments Polymerisation: • Monomer • Dimer • Tetramer • two tetramers….filamentum No polarity (no + or - end)! Microtubules Hollow cylinders (tubes) of about 25 nm in diameter (lumen = approximately 15nm in diameter) Contains 13 protofilaments (polymers of alpha and beta tubulin). They have a very dynamic behaviour, binding GTP for polymerization. They are commonly organized by the centrosome (MTOC:Microtubule Organizing Center). Functions: • intracellular transport (associated with dyneins and kinesins). • the mitotic spindle (cell division). • connection with IC organelles (ER, mitochondrion) 6 29/11/2011 Microtubules - tubulin β-tubulin GTP v. GDP 25nm dimer + GTP „cap” GDP protofilament GTP α-tubulin - growth shrinkage GTP: Guanosine-triphosphate Actin-binding proteins Functions: • Sequestering monomers (profilin – thymosin) actin cc. in the non-muscle cells ~50-200µM ~ 50% monomer • facilitate polymerisation (Arp2/3; formin) • inhibit polymerisation („capping protein”) • cutting (fragmentation, „digestion”) the polymers (gelsolin) • Crosslinkig (spectrin, fimbrin, α-actinin) 7 29/11/2011 Thymosin - profilin Actin-thymosin complex works against the filament formation - + free monomers - + Actin-profilin complex facilitate the filament formation ARP2/3 70o Stick to the side of a filament Start the polymerisation (growing) of new filaments 8 29/11/2011 Microtubule binding proteins • Sequestering („stathmin”) • Stabilizing („MAP:Microtubule Associated Proteins”) Intermedier filament binding proteins • crosslinking (plectin) 9 29/11/2011 Motor proteins Motor proteins • They can bind to specific filaments • They hydrolyze ATP (chemical energy) • They can move along filaments (kinetic energy) 10 29/11/2011 Types of motor proteins 1. Actin-based: myosins Conventional (miozin II – 1864: Wilhelm Kühne) and nonconventional myosins Myosin families: myosin I-XVIII 2. Microtubule based motors a. Dynein (1965: Gibbons and Rowe) Flagellar and cytoplasmic dyneins. Mw~500kDa They move towards the minus end of MT b. Kinesin (1985: Ron Vale) Cytoskeletal kinesins Neurons, cargo transport along the axons Kinesin family: conventional kinesins + isoforms. Mw~110 kDa They move towards the minus end of MT 3. Nucleic acid based DNA and RNA polymerases They move along a DNA and produce force Motor proteins • “Walk” or slide along cytoskeletal fibers – Myosin on microfilaments – Kinesin and dynein on microtubules • Use energy from ATP hydrolysis • Cytoskeletal fibers: – Serve as tracks to carry organelles or vesicles – Slide past each other 11 29/11/2011 Common properties N 1. Structure C N-terminal globular head: motor domain, nucleotide binding and hydrolysis specific binding sites for the corresponding filaments C-terminal: structural and functional role (e.g. myosins) 2. Mechanical properties, function In principle: cyclic function and work Motor -> binding to a filament -> force -> dissociation -> relaxation 1 cycle requires 1 ATP hydrolysis They can either move or produce force The end! 12