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studying intracellular motility for slow transport mechanisms
- Radiolabeling: Insert radioactive labeled aa, and watch movement of labeled protein
- Photobleaching: Insert fluorescent labeled tubulin, then uniformly fluoresce axon, photobleach zone and zone remains stationary
- Fluorescent Microscopy: Labeled neurofilaments, can observe them through a bleached zone
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cytoplasmic streaming and intracellular localization
- in plants: dependent on actin cytoskeleton
- acellular slime mold: cytoplasmic streaming driven by cortical actomyosin contractions
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Budding yeast
transport of vesicles along actin filament cables from mother to bud
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Passive vs active mechanism
- passive: molecules/structures less than 50nm. Mostly individual molecules & complexes, ie ribosomes
- Active: molecules/structures/ORGANELLES Greater than 50nm. All organelles, many RNAs, viruses. Depend on microtubulues/microfilaments for movement
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MicroTUBULE motors activities
- antero fast axonal 1-2um/s
- retro fast axonal
- Chromosome movement
- ER SLIDING
- SLOW axonal
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Microtubule polymerization activity
ER Elongation 0.1um/s
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MicroFILAMENT motors activities
- Cytoplasmic streaming (Nitella) 60um/s
- Cytoplasmic streaming (Physarum-slime mold) 500um/s
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MicroFILAMENT polymerization activity
Actin-propelled comet (Listeria) 0.5um/s
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DIC mycroscopy
allows for analysis of unstained living material
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Glial cell and RNA
mRNA granules move through branches to disperse, driven by kinesins and dyneins
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General methods of studying Intracellular motility
- Modern microscopy methods: DIC, fluorescence, confocal
- Video recording
- Attaching fluorescent molecules to purified proteins
- bleaching or photoactivating fluorescent tags
- Chimeric gene creation for in vivo synthesis of tagged proteins/organelles
- Developed genetic model systems
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