The problem of distanceChanges in synapses are thought to underlie learning and memory. How do build new synapses and destroy others when the major site of protein synthesis is in the cell body?Assuming these changes are specific to select synapses,How can the cellular machinery of a specific synapse be altered if transport from the soma takes exceeding long?Synapses (both pre and postsynaptic elements) are often very distant from the cell body. How do you get components to these sites?Microtubules,are,tracks,for,transports2P. Cuddon 2005 Nikon small worldWhat is being transported? how can you figure this out?P-TrkAAPPproximal distal unligatedUltrastructural analysisproximalUltrastructural analysisproximaldistalMultiple different rates of transportRoy 2014 and Ochs S. 1972- Tubulin-neurofilaments- Actin & assoicatedproteins- many cytosolic proteins- membrane cargo- RNPsOptical methods to visualize transport• Confocal and TIRF microscopy combined with fluorescent tagged cargoDistanceTimeOptical methods to visualize transport• Confocal and TIRF microscopy combined with fluorescent tagged cargoDistanceTimeWhat transports components?• Two types of motors for long distance – Kinesins (many different motors)• Mostly + end directed– Cytoplasmic Dynein (one multisubunit)• (-) end directed• Myosins for short distance transportKinesins: motor, stalk and cargo bindingKinesin-1! 8-nm steps from one tubulin dimer to the adjacent one in a direction parallel to the protofilaments! ~ 1 micron per second! 100 ATP molecules per second, each step corresponds to one cycle of the ATPase reaction! Motor activation is coupled with motor binding to organelle! Processive motility: taking steps without dissociatingHow does one deliver many distinct components to processes?• Diverse motors that transport a diverse set of cargoOne Dynein with many regulatory proteins.• Only one retrograde motor• Many associated proteins that regulate dynein functions including cargo binding• Retrograde transport clearly regulated by multiple distinct associated proteins!Multi-subunit protein complex !Responsible for nearly all minus-end directed microtubule-based transport (organelles, proteins, RNAs) two identical heavy chains of about 530 kDa each two 74 kDa intermediate chains four 53-59 kDa intermediate chains several light chains!Processive motor (dimerization required for processivity)DYNEIN-+ Heavy chain:!member of the AAA+ family of ATPases!ATP hydrolysis provides the energy for movement!Compared to kinesins and myosins, the molecular mechanism of dynein-based microtubule transport is much less well understood!Different cargo linked by binding to different light chains and other accessory subunits (dynactin)Dynein: the retrograde motorCargo specificity of dyneinNGF retrograde transport-Many neurons in mammalian brain are dependent on trophic factors for survival-Nerve Growth Factor (NGF) required for survival of sensory neurons-NGF is secreted by the peripheral targets of sensory neurons (salivary glands in original isolation of NGF)NGF-TrkA receptor complexes are endocytosed and are transported back to soma in signaling endosomes by dynein.ANTEROGRADE!Establishing neuronal polarity!Deliver growth and guidance molecules to the growth cone!Deliver synaptic components!Deliver cytoskeletal construction along the axon!Neurotrophin transport!Energy supply - mitochondrial transport!Transport of mRNARETROGRADE!Growth factor signaling!Injury signaling!Neurotoxin / viral transport!Energy supply - mitochondrial transportRoles of axonal transport17Slow axonal transportWang and Brown 2000, 2002-used FRAP techniques to visualize rare events-stop and go mechanism-neurofilaments transported primarily as assembled polymers-Transport dependent on Kinesin 1neurofilaments and MTs18Slow axonal transportcytosolic proteins-used photoactivation techniques- some proteins transported by fast transport and slow transport-Transport dependent on Kinesin 1Scott et al. 201119Myosin I: the effect of load on motor activitySingle molecule imaging with optical tweezersMyo1b and Myo1c behave very differently under load but not without load.Myo1b probably functions as anchor, not motor20Regulation of motor proteinsverhey and hammond 200921Activation of motorsPhosphorylation, or de-phosphorylation is also a common mechanism to stimulate cargo bindng and motor activityMotors usually auto-inhibited by inter-actions between tail and motor to disrupt either MT binding, or movementBinding of adaptors or cargo relieves inhibitionverhey and hammond 200922unloading of cargokif17 transports NMDA receptorsAlly et a.; 2008; Guillaud et al. 2008a Lin2,7,10 (Mint1) complex acts as an adaptor between NR2B and Kif17calcium entry at spines activates CaMKIIkif17 transports NMDA receptorsthis drives selective release of cargo in processes near active spines23Regulation of transport- mitochondria link to motors via milton (TRAK), which acts as an adaptor between the mitochondria rho protein Miro and both Kinesin and Dynein.- mitochondria in axons consist of stationary (~60%), anterograde (20%) and retrograde (20%) moving pools - ^^ Ca arrests mitochondria by decoupling the motor through miro’s Ca binding EF hand domains.-Anchoring is driven by both myosinV interactions with cytoskeleton and syntaphilin’s interaction with MTs.24Subcellular localization of cargoHow are cargo targeted to the correct destination?25Polarity of Neuronal MTsStone et al. 2008 ; Goodwin et al. 2012Axon initial segmentSor3ng,of,axonal,and,dendri3c,cargo27AIS actin filter selectively permits certain motor/cargo combinations into axon. Dynein through mixed direction runs trafficks cargo in the proximal dendritesKinesin take over transport in distal dendrites where MT orientation is biasedMyosin may also return mis-sorted cargo from axon back through AIS Lewis et al 2010, Song et al. 2009, Kapitein et al. 2010Consequences,of,disrup3ng,transportKinesin,motor,lesions,Charcot,Marie-Tooth,2A,(Kif5A)Hereditary,Spas3c,paraplesia,(,Kif1A,,Kif5)28Dynein,motor,lesions,Spinal,muscular,atrophy,,(dynein,heavy,chain)Amyotrophic,lateral,sclerosis,(p150,glued)Extensive,evidence,of,transport,defects,other,diseasese.g. Alzheimer’s, Huntington’s and Fragile X-Syndromeexamples: Millecamps And Julien; 2013Review,Transport29Neuronal cells are highly polar and have dedicated cellular mechanisms whichmediate transport of cellular constituents from the soma to axons