Cytoskeletal Motor Proteins Myosins Kinesins and Dyneins Last Class ABPs MAPs Today Motor Proteins special ABPs MAPs Ac n based motor proteins Myosins Myosin motor proteins move along ac n laments by coupling the energy from ATP hydrolysis to conforma onal changes They are considered mechanochemical enzymes because they convert chemical to mechanical energy The Myosin superfamily consists of at least 15 classes I XV based on sequence homology in the head motor domains Most Myosins are end directed motors Figure 16 57 Molecular Biology of the Cell Garland Science 2008 Myosins are composed of heavy light chains The heavy chains have head neck and tail domains The head motor domain contains the ac n binding and the ATPase ac vi es The neck domain has a regulatory func on and is the site of aJachment for the regulatory and essen al light chains or calmodulin The tail domains di er between Myosins and determine speci c proper es of each one what it will bind to whether it will be a monomer or dimer and whether it will polymerize into thick laments Myosin II was rst described as the motor protein in muscle that powers contrac on Other Myosin II isoforms are also found in non muscle cells It consists of two heavy chains 200kD each with an essen al and a regulatory light chain each 20kD 6 proteins total The tails are long helices that mediate dimeriza on by forming a coiled coil structure The tails also mediate the polymeriza on of Myosin II into bipolar thick laments Figure 16 54a Molecular Biology of the Cell Garland Science 2008 Ac n lament sliding assay The motor func on of Myosins can be directly demonstrated by video microscopy of lament sliding assays In these experiments myosin molecules are stuck to glass such that some bind by their tails with their head domains free If uorescent ac n laments are added labeled with phalloidin the laments s ck to the Myosin on the glass If ATP is added the laments slide along the glass The velocity of movement di ers among dis nct Myosins Which are the ends Which are the ends Is the or end leading Figure 16 56 Molecular Biology of the Cell Garland Science 2008 ATP hydrolysis is coupled to Myosin mo lity and Myosin ATPase ac vity is ac n ac vated In the absence of ac n the rate of hydrolysis is 4 ATP hour whereas in the presence of ac n it is 20 ATP second This ensures that Myosin is ac ve and using ATP only when ac n is around Myosin Ac n Cross bridge Cycle Rigor state at the start of the cycle the ATP binding site is empty and Myosin is ghtly bound to ac n In muscle this state corresponds to rigor mor s when muscle is depleted of ATP a er death the muscle becomes s because all of the Myosin is bound to ac n 0 ATP binding when ATP binds to Myosin the ATP binding cle closes which opens the ac n binding cle and weakens the interac on with ac n 1 ATP hydrolysis a er detaching from the ac n lament ATP is hydrolyzed to ADP and Pi This causes a conforma onal change that moves the head to a new posi on before rebinding the lament 2 3 Pi release As the phosphate is released the Myosin head undergoes a second conforma onal change called the power stroke that restores Myosin to the rigor conforma on This exerts a force on the ac n lament causing it to move rela ve to the Myosin 4 ADP release a er ADP release the Myosin remains in the rigor state ATP exchange releases the head from ac n 5 The exact nature of each of these steps is s ll debated 0 D Drubin Another look at the Cross bridge Cycle Figure 16 61 Molecular Biology of the Cell Garland Science 2008 The Myosin II power stroke hJp www youtube com watch v j8F5GGPACkQ Mammalian cells express mul ple Myosins Myosin I proteins have shorter tail domains and do not assemble into laments they are singleheaded The tails of di erent Myosin I proteins are dis nct Some have a second ac n binding site and mediate movement of laments past one another Some have membrane binding sites that aJach to and move vesicles or organelles Myosin V proteins are dimers like Myosin II but are thought to be involved in vesicle transport like Myosin I Myosins III IV and VI XV have conserved head domains with variable tail regions Their func ons are not well characterized Myosin VI is the only known Myosin to move towards the end of F ac n 8 14 3 Unconven onal Myosin V can walk along F ac n Does Myosin V walk hand over hand or like an inchworm The step size of a Myosin V motor walking along a single ac n lament is 72nm If the cargo was labeled instead of the neck how far would it move per step Muta ons in Myosin genes can cause disease More than 45 muta ons in the MYH9 gene encoding non muscle Myosin IIA have been found to cause diseases called MYH9 related disorders They are characterized by bleeding problems hearing loss kidney disease and clouding of the front surface of the eye cataracts Muta ons at Arg702 in the protein result in many problems including a severely reduced amount of platelets thrombocytopenia early onset renal disease and hearing loss in infancy Myosin VI abla on has been associated with deafness in mice Myosin VII muta ons have been associated with deafness and blindness in humans Usher Syndrome 1B autosomal dominant non syndromic deafness Microtubule associated motor proteins Kinesins and Dyneins Advances in video microscopy in the 1970s and 1980s facilitated the observa on of organelles and par cles moving along single MTs This phenomenon could also be observed when cell cytoplasm from the axons of giant neurons in squid was added to MTs in the presence of ATP These in vitro mo lity assays were used to isolate two classes of MT motor proteins Kinesins and Dyneins Kinesins have conserved motor domains Kinesins are MT ac vated mechanochemical ATPases They comprise a large superfamily of related proteins that fall into 3 major classes Each of these classes shares a high degree of sequence similarity in the motor domain which contains the ATPase and MT binding ac vity The 3 classes di er in the loca on of the motor domain in the primary sequence of the protein Kin N Kinesins have the motor domain at the N terminus of the protein Kin N Kinesins including conven onal Kinesin move towards the MT ends Kin C Kinesins have a C terminal motor domain Kin C Kinesins move towards the ends these Kinesins are rare Kin I Kinesins have an internal motor domain centrally located in the protein sequence Kin I Kinesins do not move along MTs but bind MT ends and promote proto lament peeling Figure 16 58a Molecular Biology of