Fluorescence Microscopy of MTs and the Cytoskeleton.

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Fluorescence Microscopy of MTs and the
Cytoskeleton.
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Intracellular Transport Motors
Motor Domains
Kinesins
Dyneins
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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-6 Kinesin-4
KLP-8/KLP-12/KLP-19 Kinesin-5
KLP-14 Kinesin-6
KLP-9 Kinesin-7 Kinesin-8
KLP-10/KLP-13 Kinesin-9 Kinesin-10
Kinesin-11
KLP-5 Kinesin-12 Kinesin-13
KLP-7 Kinesin-14
KLP-3/KLP-15/KLP-16/KLP-17 Orphan
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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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.
(Johnson and Rosenbaum, 1992 Kozminski et al,
1993)
1. Transport of axonemal precursors for assembly
at distal tip. 2. Delivery of ciliary membrane
proteins e.g signaling proteins.
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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 particleGFP fusion proteins ? Anaesthe
tize worms in 10mM Levamisole ? Mount on
slide ? Monitor transport using time-lapse
spinning disc confocal fluorescence
microscopy ? Kymograph analysis
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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
(Snow, Ou et al (2004) Nature Cell Biol., 6 1109)
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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
sliding in muscles.