CBIO 3400 Week 8 Study Questions 1 Explain the correlation between actin myosin and ATP In the absence of a nucleotide myosin is Actin has 2 subdomains connected by a cleft that binds ATP bound strongly to actin rigor state Myosin is an actin dependent motor that hydrolyzes ATP and converts chemical energy into movement along actin binding of ATP causes an opening of the filaments Myosin has 3 parts actin binding cleft and dissociation from head motor domain binds actin and ATP actin neck lever arm undergoes movements during force generation ATP hydrolysis causing a conformational tail may contain dimerization and cargo such as lipid change moving the head to a new binding domains position closer to the plus end This is Small conformational changes in the motor domain caused a pre powerstroke position by binding and hydrolysis of ATP are transferred into large movements of the neck lever arm In the absence of actin myosin rebinds actin this triggers myosin hydrolyzes ATP very slowly the presence of actin release of Pi myosin undergoes a second conformational change power stimulates binding to actin and the hydrolysis of ATP i e stroke rotation of the lever arm during which force is generated the force moves the actin filament myosin is an actin dependent ATPase 2 What is the function of Arp2 3 How does it work ADP is released and myosin returns to the rigor state Arp2 3 nucleates actin and forms branches You can expect to find Arp2 3 near the leading edge of a membrane much like actin Arp2 3 is found at all branch points in actin filaments ActA stimulates Arp2 3 because Arp2 3 alone has a relatively weak ability to stimulate actin assembly ActA binds to Arp2 3 and enhances its ability to nucleate actin assembly i e make new actin subunits Thus actin will polymerize preferentially near the surface of the pathogen Lodish et al 1 3 Describe the mechanism of how troponin and tropomyosin affect the muscle contraction Tropomyosin is an elongated protein associated with 7 actin monomers It binds Ca2 and undergoes a conformational change that allows tropomyosin to move along actin Troponin is a globular complex with 40 nm spacing and in contact with both tropomyoisin and actin i e 1 troponin for each tropomyosin The mechanism associated with muscle contraction is as follows 1 Troponin TN C subunit binds Ca2 2 The conformational change in troponin is transmitted to tropomyosin 3 Tropomyosin displacement allows myosin to bind actin 4 Ca2 is released from ER in response to plasma membrane depolarization caused by an Troponin and Tropomyosin impulse from a nerve cell sarcoplasmic reticulum Karp 1999 Myosin cid 3 VI cid 3 role cid 3 in cid 3 sterocilia Role cid 3 of cid 3 Myosin cid 3 VI cid 3 in cid 3 stereocilia 4 What is the cause of hearing loss in Snell s Waltzer mouse Why Tropomyosin elongated protein associated with 7 actin monomers tropomyosin and actin 1 troponin for each tropomyosin Troponin a globular complex 40 nm spacing in contact with both Myosin VI mutations are responsible for hearing loss and balance defects in the Snell s Waltzer mouse Inner ear hair cells have stereocilia microvilli The motion of stereocilia in response to sound opens mechanosensitive channels and this depolarizes the plasma membrane leading to activation of exocytosis and release of neurotransmitters at the basal end Neurotransmitters then diffuse to a nerve terminal Within cells myosin VI is enriched at the base of the stereocilia In the SW mouse stereocilia fuse soon after birth and myosin VI concentrated at the base of stereocilia may be pulling the membrane down toward the pointed end of the actin filament near the base of stereocilia The fused stereocilia prevent the appropriate movement of the microvilli in the ear upon the arrival of sound and thus cause hearing loss Myosin cid 882 VI cid 3 mutations cid 3 are cid 3 responsible cid 3 for cid 3 hearing cid 3 loss cid 3 and cid 3 balance cid 3 defects cid 3 in cid 3 the cid 3 Inner cid 3 ear cid 3 hair cid 3 cells cid 3 have cid 3 stereocilia microvilli cid 3 The cid 3 motion cid 3 of cid 3 stereocilia in cid 3 response cid 3 to cid 3 sound cid 3 opens cid 3 mechano cid 882 sensitive cid 3 channels cid 3 and cid 3 this cid 3 depolarizes cid 3 the cid 3 plasma cid 3 membrane cid 3 and cid 3 leads cid 3 to cid 3 activation cid 3 of cid 3 exocytosis cid 3 and cid 3 release cid 3 of cid 3 neurotransmitters cid 3 at cid 3 the cid 3 basal cid 3 end cid 3 cid 3 Neurotransmitters cid 3 diffuse cid 3 to cid 3 a cid 3 nerve cid 3 Within cid 3 cells cid 3 myosin cid 3 VI cid 3 is cid 3 enriched cid 3 at cid 3 the cid 3 In cid 3 the cid 3 SW cid 3 mouse cid 3 stereocilia fuse cid 3 soon cid 3 Myosin cid 3 VI cid 3 may cid 3 be cid 3 pulling cid 3 the cid 3 membrane cid 3 down cid 3 toward cid 3 the cid 3 pointed cid 3 end cid 3 of cid 3 actin cid 3 Normal sterocilia Science Photo Library Fused stereocilia in the myosin VI Mutant mouse Hertzano et al PLOS Genetics 2008 Rodriguez and Cheney Trends Cell Biol 2000 10 307 Myosin cid 3 VI cid 3 is cid 3 concentrated cid 3 at cid 3 the cid 3 base cid 3 of cid 3 stereocilia cid 3 http blocks fhcrc org myosin Myo6 ht ml 2 5 What role does nucleation play in actin microtubule assembly Nucleation is completed by ActA and Arp2 3 ActA binds to Arp2 3 and enhances its ability to nucleate actin assembly i e make new actin subunits Nucleation refers to actin assembly or basically the polymerization of actin 6 What is treadmilling in cytoskeleton assembly When does it occur DATA Treadmilling occurs at actin concentrations above Cc for the end and below Cc for the end This is a very slow process in vitro It is essentially a process where subunits are lost from one end usually the end and added to the other end usually the end 7 Explain the dynamic instability of microtubules Pantaloni et al Science 292 2001 1502 ADP ATP Pi Actin Intrinsic Treadmilling Treadmilling of actin filaments CBIO3400 Treadmilling occurs at actin concentrations above Cc for the plus end but below Cc for the minus end between 0 12 M and 0 6 M actin ATP Rate of treadmilling in vitro 2 cid 80 m hr very slow The dynamic instability of microtubules is their alternation between phases of growth and shrinkage Individual microtubules behave independently Dynamic instability is most frequent in the steady state when the depolymerization of dimers is near the Cc GTP hydrolysis induces a conformational change inside the tubulin dimer that increases the curvature of