Molecular Cell Biology
Microtubules and their Motors
Cooper
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
End
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), as in melanophores. These findings underscore the importance of interplay between actin- and microtubule-based organelle motility.
Contributed by V. Gelfand and S. RogersSpinning-Disk Confocal Images of Mitotic PtK1 Cell Mitosishttp://www.kumc.edu/instruction/medicine/anatomy/histoweb/cytology/cell05.htm
Want to Add Dam1 Complex Here...Sorger Kc Complexes...from my journal club talk.
Three models based on different contributions of kinetochore motility and kinetochore microtubule flux to metaphase kinetochore tension and anaphase A. For each model, only a single kinetochore microtubule (KMT) is shown with the polar minus end () at the left and the plus end (+) attached to the kinetochore on the right. Arrows indicate sites of polymerization or depolymerization. Metaphase includes sequential times, t1 and t2; anaphase onset occurs at t2 and continues for sequential times t2, t3, and t4. At metaphase, the centromeric linkage between sister kinetochores and polar ejection forces on the arms support tension at kinetochores (large blue arrow). Tension is lost at anaphase onset when sisters separate and the polar ejection forces on the arms are inactivated (Funabiki and Murray, 2000). See text for details.
Maddox P et al. J Cell Biol 2003;162:377-382Figure 3. Flux contributes to the anaphase A segregation rate of chromosomes. (A) Measurement of flux and segregation rates in an anaphase spindle. Pre, prebleached spindle; microtubules, green; chromatin, red. Numbers are elapsed time (seconds) after photobleaching. The blue arrow marks the progression of the photobleach mark toward the pole as a result of flux. (The dashed yellow line marks the end of a pole and, coincidentally, the center of a centrosome.) The yellow arrow marks the leading edge of a selected chromosome as it moves poleward. Scale bar, 2 m. http://www.molbiolcell.org/cgi/content/full/18/8/3094/F3Maiato, H. et al. J Cell Sci 2004;117:5461-5477. From Maddox... http://jcb.rupress.org/content/162/3/377.full
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