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

Fluorescence Microscopy of MTs and the Cytoskeleton.

Intracellular Transport Motors:

Kinesins

Dyneins

Motor Domains

Motility Assays for Motor Proteins.

TIRF Microscopy and Optical Trap Assays of Kinesin-1.

ATP hydrolysis is coupled to an ordered series of conformational changes that allows kinesin-1 to “walk” along a MT protofilament.

Microtubule Motors Drive Intracellular Transport.

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

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.

Kinesin-2 is required for Ciliogenesis on Embryos.(Morris and Scholey, 1997, J. Cell Biol., 138, 1009).

Ciliogenesis Uses Kinesin-2

Cilia: cylindrical microtubule-based axonemes surrounded by a membrane.

Intraflagellar Transport (IFT) delivers ciliary precursors to build cilium.

Motile Cilia on a Paramecium

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)

IFT-particles are Protein Complexes.

(Cole et al, 1998, JCB, 141, 993)

Basic Mechanism of IFT.

Ciliary Axonemes Have Two Distinct Domains.

(Snow, Ou et al, 2004, Nature Cell Biol, 6, 1109: Evans et al, 2006, JCB, 172, 663)

1. OSM-3 and kinesin-II redundantly build middle segment.2. OSM-3 kinesin specifically builds distal segment.

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

(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

Kinesin motors drive Sequential pathways of axoneme and ciliary membrane assembly.

(Snow, Ou et al (2004); Pederson and Barr (2005); Qin et al (2005)).

IFT and cilium-based Signaling.

(Scholey and Anderson, 2006, Cell, 125; 439-442)

Ciliopathies: Mechanosensory Cilia and Polycystic Kidney Disease.

(Praetorius and Spring, 2005; Yoder, 2004)

Muscle sarcomeres consist of overlapping actin and myosin filaments.

A sliding filament mechanism drives muscle contraction.

The myosin-2 motor drives actin-myosin filament slidingin muscles.