Molecular Cell Biology

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Molecular Cell Biology. Microtubules and their Motors Cooper. Microtubules and their Motors. Intro Vesicle Trafficking Cilia Mitosis. Microtubule Structure. Cross-section Hollow tube 24 nm wide 13-15 protofilaments Helical structure Polar Plus ends generally distal - PowerPoint PPT Presentation

Transcript of Molecular Cell Biology

  • Molecular Cell Biology

    Microtubules and their Motors


  • Microtubules and their MotorsIntroVesicle TraffickingCiliaMitosis

  • Microtubule StructureCross-sectionHollow tube24 nm wide13-15 protofilamentsHelical structurePolarPlus ends generally distalMinus ends generally proximal (at MTOC)Composed of Tubulin / Heterodimer

  • Microtubule Structure & Assembly

  • Microtubule MotorsDefinitionMicrotubule-stimulated ATPaseMotility along MTsSequence of known motorDyneinMoves to Minus End of MtLarge, multi-subunit proteinKinesinMoves to Plus End of MtException - Ncd/Kar3

  • Discovery of KinesinSearch for Motor for Axonal TransportDevelopment of Video-enhanced DIC ImagingMovement Requires ATPAMPPNP Freezes ParticlesMicrotubule Affinity ChromatographyBind in AMPPNP, Release in ATP

  • Kinesin Structure

  • Kinesin Movement and Processivity

  • Kinesin Superfamily Structures

  • Kinesin Superfamily Phylogenetic Tree

  • Cytoplasmic DyneinDiscovered BiochemicallyMinus End Motor for Vesicle TransportRequires Dynactin Complex for FunctionMoves the Mitotic Spindle

  • Dynein and Kinesin Motor Domain Structures

  • Dynein Motor Subunit Architecture

  • Model for Interactions between Dynein, Dynactin Complex, Microtubules, and Cargo

  • Membrane Trafficking - ER and GolgiPositioning ER & GolgiGolgi near MTOCMinus Ends are at MTOCGolgi Position Requires DyneinERTubular network spread about the cellKinesin moves the tubules peripherally

  • Microtubules (Red) and ER (Green)

  • Vesicle Traffic: Trans-Golgi to Plasma MembraneKinesin - KIF13ADiscovered by sequencingPlus-end Directed, fast (0.3 m/s)Binds AP-1 (affinity chromatography) and mannose 6-P receptorInhibit function (express tail as dominant negative) -> less M6PR at cell surface

  • Xenopus MelanophorePigment GranuleMovementVesicle Move Along MicrotubulesVesicles Carry Dynein, Kinesin & Myosin-VRegulation of the motors accounts for the dispersion / aggregationInward Motion(Movie Loops)

  • Xenopus MelanophorePigment GranuleMovementOutward Motion(Movie Loops)Vesicle Move Along MicrotubulesVesicles Carry Dynein, Kinesin & Myosin-VRegulation of the motors accounts for the dispersion / aggregation

  • Cilia in Action

  • Chlamydomonas CiliaSperm Flagellum

  • Cilia on Surface of Epithelial Cells

  • Structure of Axoneme: Cross-section

  • Axonemes are Anchored at theirBase in Basal Bodies

  • Conversion of Sliding to Bendingto Wave FormationSlide on only side of axonemePropagate down the long axis

  • Rotation of Central PairWhole ChlamydomonasCell w/ Two FlagellaAxonemes Isolated from ChlamydomonasDark-Field Microscopy

  • Experimental Approaches to Study Cilia in ChlamydomonasAxoneme 2-D gel - 250 polypeptides!Mutants - Collect & CharacterizeWhat Structures and Polypeptides Missing?

  • Missing Structures in Mutant

  • Missing Polypeptides in Mutant

  • Primary CiliumKidney Tubule EpitheliumDefective in Polycystic Kidney Disease 4th most common cause of kidney failureAutosomal DominantHow does loss of the cilium cause the disease?

  • Mitosis BackgroundNames of Stages: Interphase, prophase, metaphase, anaphase, telophaseInterphase MTs disassemble then reassembly as Spindle MTs

    Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNAEarly AnaphaseLate AnaphaseMetaphasePrometaphaseCytokinesis OnsetLate Cytokinesis

    Onion Root Tipc/o KU Med Ctr

  • Boveri: Centrosome and Centriole

  • CentrosomesAnimals: Centriole Pair in Amorphous CloudEnds of MTs in Cloud.No Relationship to Centrioles. Different from Relationship of Basal Body and Axoneme MTs.Flowering Plants: Lack Centrioles

  • Centrosome Ultrastructure

  • Centriole Fine Structure

  • Mitotic Spindle AssemblyCentrosome duplicates and separatesNuclear envelope breakdown in animalsMTs rearrange via dynamic instability

  • Spindle MTs

  • Mitotic Spindle Rotation in C. elegans EmbryoDynactin RNAiControl

  • Chromosome Congression to Metaphase PlateKinetochores capture MTsChromosome pulled to PoleForce at KinetochoreChromosome pushed away from PoleForces on armsForce at Kinetochore

  • Microtubule / Kinetochore Attachment

  • Metaphase Normal

  • Types of Mt / Kc Attachment

  • Metaphase - Merotelic Chrom

  • Metaphase to Anaphase

  • Metaphase/Anaphase Lagging

  • Anaphase

  • Anaphase A: Chromosome to PoleCentromere splits and Chromosomes MoveGFP-labeledCentromeres

  • Models for Chromosomes Moving to the PoleTreadmilling?Depolymerization at PoleDepolymerization at KinetochoreHow remain bound while end shrinks?Motors at Kinetochore or Pole

  • Pac-Man and Poleward Flux Models for Anaphase A

  • Poleward Tubulin Flux in Anaphase AMovement to Pole...

    Blue: Photobleach Mark, 0.7 m/min

    Yellow: Edge of Chromosome, 1.2 m/min

  • Kinetochore as a slip-clutch mechanismHigh tension:Switch to polymerization to prevent detachmentLow tension: Depolymerization generates force and movement

  • Anaphase B

    Pole - Pole Separation


    Not used.From Rogers PNAS 97 paper...Melanophores offer a promising model system to address the issue of motor regulation (6). The sole physiological role of these cells is the simultaneous transport of hundreds of membrane-bound pigmented organelles, termed melanosomes, either to amass at the center of the cell or to disperse throughout the cytoplasm. The net effect of this transport is to give lower vertebrates, such as fish and amphibians, the ability to change color. The animals appear darker when the cells have dispersed pigment and lighter when the cells have aggregated pigment. Melanophores transport melanosomes along a highly developed radially organized microtubule cytoskeleton. As with most cell types, the microtubules are oriented with their minus ends associated with a perinuclear microtubule-organizing center and their plus ends extending out to the cell periphery (7). Melanophores regulate the direction of pigment transport by modulating the intracellular second messenger, cAMP (8, 9). Upon stimulation of the cells with the appropriate hormonal stimulus, adenylate cyclase activity is up-regulated, thereby increasing cytosolic cAMP levels. Increased cAMP activates pigment dispersion, most likely through the activation of cAMP-dependent protein kinase (PKA; refs. 9 and 10). Conversely, a decrease of cAMP levels within the cell permits an as yet unidentified phosphatase to trigger pigment aggregation (11, 12). It is unknown whether the motors responsible for pigment transport are phosphorylated directly by PKA or are downstream in a multistep pathway.

    MelanophoresPigmented cells in vertebrate dermis are being studied by several laboratories as a model for intracellular organelle transport. Fish and amphibians possess specialized cells, called melanophores, which contain hundreds of melanin-filled pigment granules, termed melanosomes. The sole function of these cells is pigment aggregation in the center of the cell or dispersion throughout the cytoplasm. This alternative transport of pigment allows the animal to effect color changes important for camouflage and social interactions. Melanophores transport their pigment in response to extracellular cues: neurotransmitters in the case of fish and hormonal stimuli in the case of frogs. In both cases, melanosome dispersion is induced by elevation of intracellular cAMP levels, while aggregation is triggered by depression of cAMP. The regulatory mechanisms downstream of these second-messengers are poorly understood.

    Early studies of pigment transport in melanophores established a role for microtubules. Reconstitution of melanosome motility along microtubules in vitro demonstrated the presence of two distinct motor activities of opposite polarity. Using a dominant negative approach, our laboratory subsequently identified the microtubule plus-end directed motor responsible for pigment dispersion as the heterotrimeric Kinesin-2 (formerly kinesin-II or KRP85/95) (Tuma et al., 1998). Cytoplasmic dynein has also been shown to carry melanosomes to the cell center during aggregation (Nilsson and Wallin, 1997).

    It was recently demonstrated that pigment in fish melanophores is transported along actin filaments in vivo and that filamentous actin and melanosomes are closely associated (Rodionov. et al., 1998). Concurrently, our laboratory reconstituted melanosome motility along actin filaments in vitro and tentatively identified the motor attached to melanosomes as myosin V (Rogers and Gelfand, 1998). The model that has evolved from these observations is that melanosomes are transported across long distances via microtubules in both types of cells, while actin-based transport is probably utilized to achieve uniform distribution of pigment throughout the cell. Work with melanophores points to cooperation between microtubule- and actin-based transport for several types of organelles.

    Mammalian melanocytes also produce melanosomes but, unlike melanophores, pigment in these cells is transported to the cell periphery for subsequent exocytosis to surrounding epithelial cells. Analysis of dilute, a naturally occurring mouse mutant exhibiting coat-color defects, showed that the unconventional myosin, myosin V, is essential for melanosome movement. Recently, video microscopic analysis revealed that a microtubule-based component is involved in pigment transport in melanocytes (Wu et al., 1998),