BioE 201 Spring 2013 Sarcomere structure length tension relationship Concepts and terms to understand for Exam 3 Sarcomeres are the contractile units of muscle Made up of thick and think filaments Contraction occurs when actin filaments slide past myosin myosin heads walk along actin thin myosin thick actin at about 5nm per step During each step myosin head binds and hydrolyzes one molecule of ATP About 300 myosin heads per actin and each head can attach and detach about 5 times per second so actin myosin slide past each other at about 15 um sec 3 cytoskeletal filaments Microfilaments o Made up of actin protein o They can carry out cellular movements such as gliding contraction and cytokinesis o Between 6 8 nm in diameter thin flexible and are found in bundles or cross linked making them stronger Microtubules sheets o Diameter is 25 nm cylindrical hollow tubes composed of alpha and beta tubulin o Used to determine cell shape provides tracks for vesicle motor proteins to move in the cell make up mitotic spindles in mitosis and cilia and flagella are made out of microtubules o Monomer is alpha and beta dimers alpha and beta sheets sheets turn into cylindrical shape Intermediate filaments o Diameter 10 15 nm o Provide mechanical strength for cells and tissue Actin polymerization kinetics critical concentration regulation in cell Alpha helix with rate of growth is faster at the barbed end plus then at the pointed end minus ATP ADP when actin monomer binds to filaments When actin hydrolyzes it reduces the strength of binding between monomers and promotes depolarization which is why they are able to dissemble in one location and assemble in another quickly locomotion function Actin polymerizes in 3 steps nucleation elongation steady state o Nucleation need 3 GTP actin to form nucleus o Elongation grows from both ends o Steady state once critical concentration is reached same number of actin molecules come off as actin comes on D pol dt free G actin kon koff Critical concentration free G actin kon koff When free G actin kon koff steady state free G actin kon koff growing free G actin kon koff shrinking BioE 201 Spring 2013 Regulation o High free actin monomer concentration high potential for polymerization and actin binding proteins initiate polymerization for other processes o Actin filaments can be capped Stabilizes filament from shortening and prevents filament from growing o Actin filaments can be severed Exposes free end for polymerization depolarization Myosin structure lever arm model ATPase cycle of myosin Myosin head has active binding site and actin binding site 1 Myosin weakly binds to actin 2 ATP binds to myosin head and causes a conformational change and myosin head 3 Then ATP is hydrolyzed and Phosphate is released 4 When ADP is released causes a power stroke and pulls the rest of the head along the moves 5 nm way The head is the lever arm picture of myosin In vitro motility assays Bead assay actin tracks are secured to glass Myosin heads are attached to beads will move towards the positive end Gliding Assay opposite of bead assay myosin heads are secured to glass filaments and actin filaments will move with minus end leading because myosin is moving towards positive end pushing the actin the other way Optical tweezers Beads are always attracted to region of highest light intensity Actin is secured to the beads and bead is trapped in the brightest light Force is measured by the changes in light intensity Force KX K trap stiffness 1 pN nm Myosin step size experiment X distance Myosin tweezer experiment measures the displacement of myosin head per one step Each even on graph is one power stroke Lever Arm hypothesis Add or remove light chain binding sit to alter length of lever arm Measure gliding velocity compared to lever arm length longer light chain faster velocity Small conformational changes driven by ATP hydrolysis moves the lever arm o Chemical energy pN nm step size nm force pN Microtubule structure and roles in cells Long stiff and hollow tubes of proteins that can rapidly assemble and dissemble in different locations Minus end is anchored to centrosome Heterodimer tubulin subunits alpha and beta protofilament is made up of alpha minus end GTP beta plus end GDP 10 15 protofilaments make up tubulin with hollow inside Growing end looks straight but shrinking end is frayed Kinesins walk toward positive end and dyneins walk towards negative end Kinesins use the hand over hand method to walk BioE 201 Spring 2013 Microtubule dynamic instability Instability comes when tubulin molecules add to the end of microtubule faster than GTP can be hydrolyzed Assembly catastrophy dissembly rescue When there is high GTP in the cell GTP will stay bound to beta tubulin and form a GTP cap Random chemical processes changes GTP to GDP quicker then the next tubulin is added This shifts the balance in favor of dissembly and once depolarization has started it continues at catstrophic rate Clila flagella structure and function Cilia Hairlike structures about 25 um in diameter One cilia contains a core of stable o Cilia moves fluid over the surface of a cell ex Mucus creates a current for microtubules arranged in a bundle moving eggs in oviducts Flagella Much longer than cilia o used to move whole cells through liquids ex Sperm dyneines and microtubules are the motor and filaments Kinesin tweezer experiments step size force kinesins motor proteins with two heads and use the hand over hand method to move Takes 8 nm steps and moves about 1 um sec toward the positive end o about 45 kinesin genes in human genome Optical trap holds beads connected to microtubule in place while microscope measures how much light diffracts to determine the step size of kinesin and measures force by displacement BioE 201 Spring 2013 Chemomechanical efficiency of motor proteins Chemical energy ATP mechanical work o ATP ADP Pi o Delta G 12 kcal mol 100 pN nm o Max Force 6 8 pN Step size 8 nm Work force distance 64pN nm Efficiency mechanical work chemical energy 64 Actin polymerization at leading edge ARP2 3 function Listeria Actin Polymerization activates Arp 2 3 complex to initiate new filaments 3 Elongation on old filaments 4 Growing filaments push membrane forward 5 Capping proteins terminate elongation 6 ATP hydrolyzes when actin binds to other actins 7 Cofilin severs depolymerizing actin ADP Listeria Fundamentals of photolithography and microcontact printing 1 ATP bound to actin binds to profilin 2 When signal