Introduction to MCB 252 Topic 25 MT MotorsProf David Rivier MCB 252 Spring 2015 MCB 252 Cell Biology Topic 25 Microtubule Motors Reading Lodish 18 4 Kinesin and Dynein Mediated transport Lodish Fig 18 27 Kinesins plus end directed motors Dyneins minus end directed motors MT based motility Axonal transport Newly synthesized proteins must be moved from cell body to tip of axon anterograde Old membranes must be transported back retrograde Lodish Fig 18 5 remember axons can be up to 1 meter long Experimental demonstration of axonal transport pulse chase Different materials move at different speeds fast 0 5 2 5 m sec slow 10 100 nm sec So round trip from spinal cord to foot takes 3 weeks to 3 months Lodish Fig 18 16 Visualizing axonal transport Squid Loligo Prof George Langford Dartmouth Univ dissect giant axon Dr Ron Vale UCSF Movie T25M1 07 DissAxo avi microscopy Alberts Panel 11 3 Bidirectional movement on MTs Lodish Fig 18 17 Purification of anterograde motor Vesicles ATP MTs no movement Above squid cytoplasm extract movement Substitute non hydrolyzable ATP analog no movement vesicles bind tightly to MTs So incubate MTs cell extract ATP analog this should result in tight binding of motor to MTs Collect MTs release bound protein with ATP Identified kinesin In vitro motility assay with purified kinesin 1 Adsorb purified kinesin to glass slide 2 Add rhodamine MTs ATP 3 Visualize T25M2 Movie 07 mov invitmtglid avi Sliding filament assay Dr Ron Vale UCSF Kinesin structure 2 heavy chains 2 light chains Head binds ATP and MTs Central stalk coiled coil Tail binds cargos Plus end directed motors Linker region also called the neck Lodish Fig 18 18 Myosin and Kinesin Heads Kinesin linker region neck Myosin and Kinesin Heads Myosin and Kinesin heads have related structures Kinesin Family Conventional N terminal head Unconventional head elsewhere Model of kinesin directed vesicle transport Lodish Fig 18 19 Kinesin Movement Kinesin 8 nm steps one dimer step Kinesin walks along one protofilament Kinesin Two headed Kinesins each has its own neck Movement in kinesins is generated by reversible binding of the neck to the head Binding of the neck to it s head throws the other head forward The energy of ATP hydrolysis in one head is used to move the other head The two heads interact with each other they influence the nt binding of each other Kinesin Power stroke Neck NOT bound to head Neck BOUND to head ATP binding binding of the neck to the head therefore ATP binding to leading head throws trailing head forward Properties of Kinesin Heads nt bound to head Binding to MT Neck None 0 ATP ADP Pi ADP Tight No Tight Yes Weak on off Yes Weak on off No Interaction with Kinesin ATP cycle Kinesin Movement Movie T25M3 1804 kinesin mov General Roles of Kinesin Family Members Lodish 18 20 Kinesin and dynein mediated transport Kinesins plus end directed motors Dyneins minus end directed motors Lodish Fig 18 27 Dyneins Dyneins were purified based on similar ideas and assays used to study myosins and kinesins Cloning of genes that encode dyneins revealed 1 that they were substantially different than myosins and kinesins and 2 that dyneins have a domain that is part of the AAA ATPase superfamily AAA ATPases Associated with many Activities ATPase Evolution and Motor Head Evolution Kinesin heads distantly related to myosin heads Their ancestral progenitor gene predates the 3 domains of life What are the properties of the AAA ATPase family members 3 Domains of Life Three Domains of Life Eukaryotes Bacteria Archaea What was the progenitor of all three domains LUCA Last Universal Common Ancestor LUCA 3 6 4 1 Billion years ago Age of the Earth 4 5 Billion years old Structure of AAA ATPases A Hexameric Disk 6 AAA ATPase domains form a Hexamer Sometimes 6 domains in one polypeptide Sometimes 6 domains in six polypeptides Remember the 26 S Proteasome AAA ATPase Cap contains an AAA ATPase that unfolds the ubiqutinated protein and threads it into the chamber Remember Ubiquitination and the 26S Proteasome ATPase threads the ubiquitinated protein into central chamber where degradation occurs Threading AAA ATPases Protein Degradation 26 S proteasome Recombination Helicase DNA Replication Threading protein dsDNA ssDNA Arrow direction threaded material moves AAA ATPase moves in the opposite direction Hexameric AAA ATPase DNA Helicase Arrow direction helicase moves DNA threaded in opposite direction Spastin Katanin like AAA ATPase Spastin MT severing Spastin sequence very closely related to Katanin Spastin Katanin Model AAA ATPase Inside of Microtubule Tubulin A Cool Gadget Sliding Clamp Complex Disassembly Assembly Clamp Loader Complex Disassembly ATPase Evolution and Motor Head Evolution Kinesin heads distantly related to myosin heads Their ancestral progenitor gene predates the 3 domains of What are the properties of the AAA ATPase family members Dynein Structure by EM Stem cargo binding Head ATPase and Salk MT binding Classic Cytoplasmic Dynein Different Family Members monomers dimers trimers Dimeric Dynein How does dynein move Compare structures by EM before and after the Power stroke EM of dynein conformations Phosphorus P Phosphate PO4 Pi Vanadium V Vanadate VO4 Vi Dynein ADP Vi trapped in pre power stroke conformation Dynein Apo no nucleotide bound post power stroke conformation Experimental strategy Compare ADP Vi and Apo forms by EM to identify the change that occurs as a result of the power stroke Look at Lots of Dynein Molecules by EM Dynein Powerstroke Average the angles of stalk to stem from many EM micrograph images of ADP Pi form and no nt Apo form Dynein conformation change Change from pre to post power stroke Dynein Power stroke Dynein PowerStroke How Do Dyneins Attach to Vesicles and Organelles Kinesins plus end directed motors Dyneins minus end directed motors Lodish Fig 18 27 Dynein and Dynactin A Tractor Trailer System Plus our old friends Ankyrin and Spectrin Dynein Dynactin and Company Dynactin Lodish Figure Arp1 Actin Related Protein 1 Yet another use of an actin family polymer Modifiers of Dynein Processivity Dynactin Arp1 Modifiers of Dynein Processivity Lissencephaly Modifiers of Dynein Processivity NudE Lis1 Post Power Stroke Stage A Road Map of Vesicle Traffic Still identifying motors and sorting out which family members move which vesicles Lots of Kinesin and Dynein Family Members Still sorting out which motors move which vesicles etc We currently have little understanding of regulation of dyneins