The Moving Parts: Microtubules and MT- based Motors. MULTIGENE FAMILIES: C. elegans MTs: 9...
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Transcript of The Moving Parts: Microtubules and MT- based Motors. MULTIGENE FAMILIES: C. elegans MTs: 9...
![Page 1: The Moving Parts: Microtubules and MT- based Motors. MULTIGENE FAMILIES: C. elegans MTs: 9 α–tubulins 6 β-tubulins 1 γ-tubulin C. elegans MT-motors: -21.](https://reader035.fdocuments.us/reader035/viewer/2022062304/56649f3d5503460f94c5cc38/html5/thumbnails/1.jpg)
The Moving Parts: Microtubules and MT- based Motors.
MULTIGENE FAMILIES:
C. elegans MTs:9 α–tubulins6 β-tubulins 1 γ-tubulin
C. elegans MT-motors:-21 kinesin heavy chains-2 dynein heavy chains
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Fluorescence Microscopy of MTs and the Cytoskeleton.
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Intracellular Transport Motors:
Kinesins
Dyneins
Motor Domains
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Motility Assays for Motor Proteins.
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TIRF Microscopy and Optical Trap Assays of Kinesin-1.
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ATP hydrolysis is coupled to an ordered series of conformational changes that allows kinesin-1 to “walk” along a MT protofilament.
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Microtubule Motors Drive Intracellular Transport.
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Microtubule Motors in C. elegans.
Kinesins (21 kinesin genes). Kinesin-1 KHC-1
Kinesin-2 KLP-11, OSM-3, KLP20 (plus KAP-1)
Kinesin-3 KLP-1/KLP-4/KLP-6Kinesin-4 KLP-8/KLP-12/KLP-19Kinesin-5 KLP-14Kinesin-6 KLP-9Kinesin-7 Kinesin-8 KLP-10/KLP-13Kinesin-9 Kinesin-10 Kinesin-11 KLP-5Kinesin-12 Kinesin-13 KLP-7Kinesin-14 KLP-3/KLP-15/KLP-16/KLP-17Orphan KLP-18
Dyneins (2 dynein heavy chain genes).Cytoplasmic dynein DHC-1
IFT-dynein CHE-3
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Purification of Heterotrimeric Kinesin-2 complex from Sea Urchin Embryos.
Cole et al, 1993, Nature, 366; 268. Wedaman et al, 1996, J. Cell Biol., 132; 371.
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Kinesin-2 is required for Ciliogenesis on Embryos.(Morris and Scholey, 1997, J. Cell Biol., 138, 1009).
Ciliogenesis Uses Kinesin-2
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Cilia: cylindrical microtubule-based axonemes surrounded by a membrane.
Intraflagellar Transport (IFT) delivers ciliary precursors to build cilium.
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Motile Cilia on a Paramecium
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Discovery of IFT in Chlamydomonas Flagella.
1. Transport of axonemal precursors for assembly at distal tip.2. Delivery of ciliary membrane proteins e.g signaling proteins.
(Johnson and Rosenbaum, 1992;Kozminski et al, 1993)
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IFT-particles are Protein Complexes.
(Cole et al, 1998, JCB, 141, 993)
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Basic Mechanism of IFT.
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Ciliary Axonemes Have Two Distinct Domains.
(Snow, Ou et al, 2004, Nature Cell Biol, 6, 1109: Evans et al, 2006, JCB, 172, 663)
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1. OSM-3 and kinesin-II redundantly build middle segment.2. OSM-3 kinesin specifically builds distal segment.
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In vivo transport assay.(Orozco et al, 1999, Nature, 398, 674)
Heritable transgenic lines expressing IFT motor/IFT particle::GFP fusion proteins
Anaesthetize worms in 10mM Levamisole
Mount on slide
Monitor transport using time-lapse spinning disc confocal fluorescence microscopy
Kymograph analysis
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(Snow, Ou et al (2004) Nature Cell Biol., 6; 1109)
Different Rates of IFT along Middle and Distal Segments of Wild-types.
Protein Vmiddle (µm/sec) Vdistal (µm/sec)
IFT-particle 0.7 1.3
OSM-3-Kinesin 0.7 1.3
Kinesin-II 0.7 None
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Kinesin motors drive Sequential pathways of axoneme and ciliary membrane assembly.
(Snow, Ou et al (2004); Pederson and Barr (2005); Qin et al (2005)).
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IFT and cilium-based Signaling.
(Scholey and Anderson, 2006, Cell, 125; 439-442)
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Ciliopathies: Mechanosensory Cilia and Polycystic Kidney Disease.
(Praetorius and Spring, 2005; Yoder, 2004)
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Muscle sarcomeres consist of overlapping actin and myosin filaments.
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A sliding filament mechanism drives muscle contraction.
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The myosin-2 motor drives actin-myosin filament slidingin muscles.