AMCB Past Paper PDF 2022/2023

Year 3 - Advanced Cell Biology Topic AMCB2 Microtubule motors Mark Dodding Kinesin [email protected] School of Biochemistry 2022/2023 1  Index o...

Year 3 - Advanced Cell Biology Topic AMCB2 Microtubule motors Mark Dodding Kinesin [email protected] School of Biochemistry 2022/2023 1  Index of AMCB2 Topic – Microtubule motors Lecture 1 Introduction to microtubule motors proteins Case study 1: Structure and mechanism of kinesin-1 regulation and cargo recognition Lecture 2 Case study 2: Structure and mechanism of cytoplasmic dynein regulation and cargo recognition  Key concepts and learning outcomes An understanding of the different types of cytoskeletal motors inside the cell Overview of current understanding of structure and mechanism of kinesin-1 – a major microtubule plus-end directed motor. Overview of the Cytoplasmic-Dynein 1 transport machinery – the main microtubule minus end directed motor complex. A basic understanding of some the key techniques used to study motor protein biology  Background reading Cross and Dodding (2019) Motor-cargo adaptors at the organelle cytoskeleton interface. Current Opinion in Cell Biology https://doi.org/10.1016/j.ceb.2019.02.010 Most relevant for live lecture, but also background useful for recorded lecture. Reck-Petersen et al (2018) The cytoplasmic dynein transport machinery and its many cargoes. Nature Reviews Molecular Cell Biology 19, 382 https://doi.org/10.1038/s41580-018-0004-3 Most relevant for the recorded lecture I will cite various other papers on slides – do read if you are interested and would like to know more but it is not essential reading Molecular Biology of the Cell has useful background on motors and microtubules Cellular content is organized by cytoskeletal motors cells Virus we need mese to organise attaches to dynein The illustrations are by David S. Goodsell, Scripps Research Institute.  Cytoskeletal Motors Kinesins – almost exclusively Actin Microtubules plus-end directed microtubule - based transport (except ncd, a MINUS end directed kinesin) Cargo binding / regulation Dyneins – Exclusively minus- heads end directed. Axonemal - Ciliary beating, flagellar motility) Cytoplasmic - Microtubule- based organelle transport and Motor heads cell division Myosins – actin-based transport, contractility Myosin Kinesin Dynein almost exclusively plus- - ATP end directed Carter, JCS, 2013 endomat's domain far hydrolysing large (except myosin VI, MINUS-end directed). DOI: 10.1242/jcs.120725 family 145 types in humans)  The Kinesin Superfamily (KIFs) – many Kif’s for +ve end transport domains far dig &junctionsa ho membranes · has plt domain Common, well some enables conserved ATPase - specialisation motor domains KIF5B = “Conventional kinesin” ↳ coil coil domains I allows Functional KIF16B homooemerisation specialization through addition of protein domains. evolution sarred by generally nomenclature but messy , https://doi.org/10.1038/nrm2774  Composition Kinesin-1of kinesin-1 chairs involved in light moshy cargo selection Expressed in most cell-types and important role in KLC1 KLC2 KLC3 KLC4 axonal transport 312 Heterotetramer composed of two light and two heavy Kif5A chains (3 HC genes (KIF5A, KIF5B and KIF5C) 4 KLC genes (KLC1 to 4). Kif5B combo , Kif5C Diseases of kinesin-1 composition Paralogue dysregulation Gene Expression and Dysregulation Disease TPR diseases Kif5A Neuronal Point mutations Exon skipping ALS, HSP KHC Kif5B Ubiquitous Broad Point mutations Kif5C MCD Ubiquitous Splice variant E KLC1 expression level AD Ubiquitous Upregulation KLC2 SPOAN KLC KLC3 Restricted Broad Premature truncation KLC4 HSP Classic EM evidence that kinesin-1 undergoes large scale conformational change ATPase Cargo motor binding domains tail > auto ↳ inhibited Rotary shadowing EM Amos 1987; Hisanga et al; 1989; Hirokawa et al; 1989; Scholey et al 1989 Molecular Visions, Harvard  New electron microscopy data from Bristol provides a higher resolution view of kinesin-1 conformations staining BUT the crying protein. 2D plus aug Weijman et al. 2022 Science Advances (Dodding Lab)  Folding is thought to enable regulation of enzymatic activity enablamaric activity Coldingto Tail peptide has an important IAK motif inhibits ATPase still to rying each get -- resolanon. shuddes - of Motor domains are cross-linked and locked in an ADP bound state Autoinhibition governed by conformational change is a fundamental principle of motor protein regulation Kaan et al. Science 2011  How is kinesin-1 activated? – by cargo binding via adaptor proteins => Lysosomes Lysosomes are a kinesin-1 cargo ( ofhow cargo binds. n in A small GTPase called Arl8 binds to the surface of lysosomes 10xx I Arl8 recruits a kinesin adaptor called SKIP which binds t TPR chain domain of light SKIP binds directly to kinesin-1 (to its light chain TPR domains).  A close-up view of the motor-cargo adaptor interface ? Tonig The kinesin light chain TPR domains recognises a ‘W-acidic’ motif in SKIP (A.K.A. a WD motif) WD / W-acidic motif Pernigo et al. Science 2013 (Dodding Lab) How does cargo adaptor binding trigger conformational changes associated with motor activation? ? How does binding of a short peptide motif effect a large-scale change in ? organisation of the complex? ? Are other factors required? Short answer – we don’t know (yet) – but there have been some important recent insights.  ↑ Adaptor proteins co-operate with microtubule associated proteins to drive activation Nesprin-4 is a cargo adaptor recruits and activates kinesin-1 at the nuclear envelope a has a kinesin-1 binding W-acidic motif ↳ almost identical Helps to drive cell polarity sequence Nucleus is the ‘cargo’ – nesprin-4 is the cargo adaptor ↳ motif is new nucleus Binding is dependent on a WD-motif in nesprin-4 10.1073/pnas.0808602106  Adaptor proteins co-operate with microtubule associated proteins to drive activation binds to minotables&d interacts MAP 7 wrespring Nesprin-4 promotes limited kinesin-1 activity in a W-acidic motif dependent manner Huenseince ↓ onal- diagenement ! evig Kymograph analysis of a single-molecule motility assay Nesprin-4 activity is enhanced by the Ae e microtubule associated protein, MAP7 active if moms we , ball along misole - MP. to more A general framework is now established, but with many unanswered questions  Summary Introduction to the kinesin family Focussed on kinesin-1sub family - case a case study in cargo recognition and regulation (and a focus of our own work) Composition and architecture of the complex and its regulation by conformation change Introduced the idea of a ‘cargo-adaptor’ protein that triggers activation and links motor to its cargo Shown how this is likely regulated by other (microtubule associated) proteins (more development around single molecule motility and kymographs in recorded lecture)  Year 3 - Advanced Cell Biology Topic AMCB2 Microtubule motors Mark Dodding Cytoplasmic dynein [email protected] School of Biochemistry 2022/2023 19  Index of AMCB2 Topic – Microtubule motors Lecture 1 Introduction to microtubule motors proteins Case study 1: Structure and mechanism of kinesin-1 regulation and cargo recognition Lecture 2 Case study 2: Structure and mechanism of cytoplasmic dynein regulation and cargo recognition  Key concepts and learning outcomes An understanding of the different types of cytoskeletal motors inside the cell Overview of current understanding of structure and mechanism of kinesin-1 – a major microtubule plus-end directed motor. Overview of the Cytoplasmic-Dynein 1 transport machinery – the main microtubule minus end directed motor. An understanding of the key techniques used to study motor protein biology  Background reading Cross and Dodding (2019) Motor-cargo adaptors at the organelle cytoskeleton interface. Current Opinion in Cell Biology https://doi.org/10.1016/j.ceb.2019.02.010 Most relevant for live lecture, but also background useful for recorded lecture. Reck-Petersen et al (2018) The cytoplasmic dynein transport machinery and its many cargoes. Nature Reviews Molecular Cell Biology 19, 382 https://doi.org/10.1038/s41580-018-0004-3 Most relevant for the recorded lecture I will cite various other papers on slides – do read if you are interested and would like to know more but not essential reading Molecular Biology of the Cell has useful background on motors and microtubules  Point to note: The Dynein motor family is diverse DOI: 10.1007/978-3-642-16712-6_765  Only one Cytoplasmic Dynein-1 for all –ve end transport specific is adapter prote for each carga Functional specialization via additional subunits and accessory proteins. https://doi.org/10.1016/S0092-8674(03)00111-9 how is it so versatile ?  Three key components in dynein mediated transport 1. 3. 1. The dynein complex contains motor domains, microtubule binding domains and binding sites Juydrolysis for accessory proteins 2. The dynactin complex – a critical -mirotubule dynein co-factor 2. bindingto 3. The ‘activating’ cargo adaptors – enable dynein to couple to many Laynaction different cargoes Lots of achine-line in cre components Critical role for coiled-coil domains ↳ led from wide range of proteins long +extended CCds.  Structural organization of the dynein motor Cartoon of the dimeric dynein motor complex & +termxial Structure of a single tail domain Note: the stalks are where the binding to microtubules occurs https://doi.org/10.1038/s41580-018-0004-3  Dynactin is the essential co-factor to Dynein-1 wide of range other intergacce for a Multiple binding interfaces to engage with regulatorycomponen Dynein-1, kinesin, possibly microtubules and other accessory factors. Increasing processivity Lau et al, 2021 https://doi.org/10.15252/embj.201488792 In vitro reconstitution of the dynein-1 motor using baculovirus – MultiBAC system All genes synthesized. Co-expressed from a single baculovirus Ensures subunit stoichiometry is more likely to reflect in vivo situation Purified to homogeneity from insect cells. MultiBAC system. Schalger et al, 2014 https://doi.org/10.15252/embj.201488792  Similarity between recombinant and endogenous Dynein-1 inhibited > fly and EM analysis of dynein structures – purified from tissue or recombinant Schalger et al, 2014 https://doi.org/10.15252/embj.201488792  Cryo-EM structure of full length Cytoplasmic Dynein-1 = side in Zhang and Foster et al, 2017 DOI: 10.1016/j.cell.2017.05.025  Single molecule experiments show components required for motility Kymographs used to show motility – note axes of time and distance. Processive movement only seen with dynein + dynactin + BicD2 Schalger et al, 2014 https://doi.org/10.15252/embj.201488792  Single molecule assays used to track individual transport events Microtubule + Dynein 1 (motor) + Dynactin (co-factor) + Hook3 (activating adaptor) Kendrick et al, 2019 https://doi.org/10.1083/jcb.201812170  Single molecule experiments help track co-transport events - I microtubule o multiple dyneinecules Kymographs used to show motility of single molecules. Association of dynein and dynactin unstable in the absence of BicD2. Processive movement only seen with dynein + dynactin + BicD2 McKenney et al, 2014 DOI: 10.1126/science.1254198  Cryo-EM reveals structure of the dynein transport machinery logeme stabilise come Mitochondria > - + a n Endosomes X2D - - some Viruses Cargo Adaptors only wiss Motor head 2 2x Structural studies show how Cargo adaptors Stalks I cargo adaptors thread through the assembled dynein-dynactin complex. Chaaban and Carter, 2022 https://doi.org/10.1038/s41586-022-05186-y drivesmoremovement  New models are beginning to emerge on how kinesins and dyneins work together 1. Hook adaptor for endosomes is shown in green 2. Hook can bind to a kinesin-3 family member called KIF1C (so not kinesin-1 discussed in live lecture) 3. KIF1C binding opens up hook to allow it to bind to dynein-dynactin One cargo (in this case an endosome) can recruit both kinesin and dynein to engage in ‘bidirectional transport’ See later membrane contact sites lecture for more biological context for this mechanism https://doi.org/10.1101/2023.10.26.564242  Summary Dynein functional specificity comes from subunits and cargo adaptors engaging with different cargo Dynein is autoinhibited in the absence of cargo binding Dynactin is an essential co-factor but not sufficient for processive motility, need for activating cargo adaptors In vitro studies allow functional analysis of Dynein-1 Cryo-EM studies reveal new features of the Dynein-1 motor Techniques discussed can be used to study other cytoskeletal motors