PCB3134 Exam 03 Lecture 1 Protein layering 1 Monomer 2 Dimer two monomers 3 Tetramer two coiled dimers 4 Two tetramers packed together 5 Filament 10nm eight packed tetramers twisted into a ropelike filament Intermediate Filament Ex Cytoplasmic keratins vimentin neurofilaments Actin is opposite of keratin in function Ex Nuclear nuclear lamins Lecture 2 Actin has positive and negative charged ends Intermediate Filament building 1 Nucleation Globular G action monomers form around an Filamentous F actin trimer 2 Elongation 3 Steady State Critical concentration the rate of subunit addition loss Barbed end fast growing ATP actin binds Pointed end slow growing ADP actin falls Treadmilling removal of ADP G Actin from pointed end negative and binding of ATP G actin to the barbed end positive Actin Binding Proteins control filament assembly by binding to the ends Nucleators promote specify cellular location of polymerization Ex Cap Z Tropomodulin Ex Formin dimer Arp2 3 nucleator that assembles filaments into branched networks at the front of a moving cell activated by ActA Myosins family of proteins that are actin based motor proteins Ex Myosin I and Mysosin V Two non muscle myosins that bind to membranes Myosin I is small and one headed myosin V is large two headed and takes the largest steps of any known myosin Myosin V is involved in vesicle transport and often collaborates with MT motors MyosinVI only pointed end directed myosin Sweeney Lecture 3 Sarcomere the basic unit of striated muscle contraction linked together lengthwise to form a myofibril several myofibrils a muscle fiber A Thick Filaments myosin contains roughly 300 copies of Myosin II arranged by a combination of head head and head tail interactions to form bipolar filaments Bare Region location of tail tail interactions so that the heads can have a staggered formation B Thin filaments actin Myosin always walks towards the Z disc Syncitium large multi nucleated cell formed by the fusion of several cells early in development Actin Myosin Interaction 1 Myosin head is locked onto an actin filament in a rigor configuration 2 A molecule of ATP binds to the cleft on the back of the head and causes a change in the actin binding site Reducing the affinity of the head for actin 3 Hydrolysis of the ATP causes a displacement of the head about 5nm down the actin filament The ADP and inorganic phosphate remain attached 4 The weak binding of the myosin head to the new site on the actin filament causes the release of the inorganic phosphate 5 The release of the inorganic phosphate triggers the power stroke in which the head regains its original rigor conformation The force generated in the this movement causes the release of the ADP 6 The myosin head has now moved to a new position down the positively charged end of the actin filament Three overlapping cycles 1 Actin bind release cycle 2 The myosin ATPase cycle bind hydrolyze release 3 The myosin head powerstroke recovery cycle Contraction Striated Muscle actin based 1 Transmission of an action potential from a neuron to the plasma membrane of the myofiber a Depolarized T tubule membrane opens its voltage gated Ca 2 channel releasing Ca 2 ions into the cytosol b Ca 2 release channel in sarcoplasmic reticulum is activated and begins to release Ca 2 ions from the lumen of the sarcoplasmic reticulum into the cytosol 2 Addition of the Ca 2 stimulus a Ca 2 binds to troponin C b Tropomyosin moves relative to the actin filament exposing the myosin binding site allows myosin to carrying out contraction cycle Contraction Smooth Muscle modified myosin based 1 Elevated Ca 2 levels activate MLCK myosin light chain kinase 2 MLCK phosphorylates one of the myosin light chains turning on the myosin contraction cycle Lecture 4 Cell Migration Challenges motility is a whole cell process requiring integration of many parts not all cells are the same Cell Crawling 1 Protusion 2 Retraction 3 Attachment Myosin II when genetically manipulated cells are found to still be able to crawl move but cannot successfully divide impaired cytokinesis myosin II forms the cleavage furrow Reconstitution Assay rebuilding a biological process by combining its essential parts Listeria 1 Establishes that all we need for cellular movement is Actin ATP and Arp 2 3 2 Cellular movement is myosin independent 3 Movement is powered by actin polymerization at the rear of the cell pushing the front forward Dendritic Nucleation Hypothesis edge 1 Activation of Arp 2 3 leads to assembly of branched networks at the leading 2 Capping protein prevents growth of non productive filaments 3 Cofilin cuts filaments and promotes network disassembly of the comet tails Actin Polymerization filaments along the membrane due to thermal motion bend away from the membrane enough to create space for the addition of an actin monomer movement occurs when the lengthened filament bends back and pushes against the membrane Lecture 5 Tubulin Dimer The basic building block of microtubules Tubulin heterodimer consists of an alpha and beta tubulin dimers and serves as the microtubule subunit Protofilament connected strain of tubulin heterodimers has positive and negative charged ends similar to an actin filament y TURC gamma tubulin ring complex arranged in a lock washer shape on the negative end of a microtubule nucleates MT polymerization analogous to Arp 2 3 and formin for actin Positive ends extend away from the centrosome and y tubulin on the negative ends towards the centrosome MT s grow from the y tubulin ring complexes of the centrosome Growth and shrinkage of MT s occurs at the free positive end Centrosome two centrioles and other proteins Dynamic instability slow growing and rapid shrinking of MT s co exist catastrophe rescue cycles Lecture 6 Male sperm pulmonary cells female reproductive system only ciliated flagellated cells in the human body Axenome shaft inside the plasma membrane covering cilia and flagella Axonemal dynein consists of heavy chains held together by intermediate and light chains ATPase activity is in the head which powers the stalk to walk along the microtubule toward the negative end Caused Movement If the nexin links are removed by enzyme treatment protease the force produced by the dyneins causes the adjacent outer doublets to slide past each other without restriction causing no bending IFT intraflagellated transport associated with polycystic kidney disease PKD Case Study 2 Because MT grows out from the centrosome their positive