Cytoskeleton and experiments

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Cytoskeleton and experiments
2013-12-04 19:36:35

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  1. microtubules structure
    long, hollow unbranched tubes composed of tubulin; outer diameter of 25 nm and thickness of 4 nm
  2. microtubules (component of)
    mitotic spindle, core of cilia and flagella, etc. 
  3. Microfilaments structure
    solid, thinner structures, often organized into a branching network and composed of actin
  4. intermediate filaments structure
    • Structure: tough, ropelike fibers composed of a variety of related proteins; strong, flexible
  5. Functions of the cytoskeleton (5)
    • ·         structural support that determines the shape of the cell and resist forces that tend to deform it
    • ·         framework that positions the various organelles within the interior of the cell
    • ·         tracks that direct the movement of materials and organelles within cells
    • ·         force-generator that movse cells from place to place
    • essential component of cell’s division machinery (ex: mitotic spindle
  6. Live-cell fluorescence imaging 
    The EM produced only static images and didn’t provide insight into the dynamic structure and function of the various components
  7. Live-cell fluorescence imaging 
    -          Fluorescently labeled proteins are synthesized by fusing GTP to a protein OR the protein subunits are fluorescently labeled in vitro by covalently linking to a small fluorescent dyeà microinjected into a living cell where they polymerizeà followed over time
  8. Live-cell fluorescence imaging 
    -what does it look like?
    -          if there are only small amounts, they look like speckles that serve as fixed markers to follow dynamic changes in length or orientation (fluorescence speckle microscopy)
  9. Live-cell fluorescence imaging 
    • -          to reveal location of proteins present in low concentrations, use antibodies and inject in living cell
    • this will also reveal the function since the antibody destroys the ability of the protein to function 
  10. Single- Molecule Assays
    - what led to it and what does it make possible?
    high-resolution video microscopy led to this development; it makes it possible to detect the activity of an individual protein molecule acting as a molecular motor in “real time”
  11. Single- Molecule Assays
    - how?
    • -          in earlier assays, microtubules were attached to a glass coverslip and beads containing attached motor proteins were placed directly onto the microtubules via laser beans, which are shone through the objective lens of a microscope, producing a weak attractive force near the point of force (called optical tweezers)à followed by a video camera (when ATP is present)
    • o   can also trap a signle bead and determine minute forces generated by a single motor protein as it tries to move the bead against the force exerted by the optical trap
  12. Single- Molecule Assays
    - problem?
    -          Problem? measuring movement of beads, not microtubules; also cannot resolve the molecules themselves
  13. Single- Molecule Assays
    - ways of getting aroubd problem?
    combining fluorescence microscopy ad specialized imaging techniques; clarity of images is made by use of TIRF (total internal reflection microscopy) so that light rays from other fields of view don’t obscure the info; use of AFM to follow motor protein and measure properties of cytoskeletal elements
  14. Single- Molecule Assays
    - benefits?
    Can visualize the movement of individual kinesins inside living cells by labeling and watching interactions between kinesins; led to nanotechnology
  15. Fluorescence recovery after photobleaching (FRAP)
    - how?
    inject cell with tubulin coupled to a fluorescent dye or induced to express GFP-tubulin; laser used to bleach out region and follow specimen over time, measuring recovery of signal into bleached zone, either by dynamics of microtubules turning over in the zone, growth of new microtubules into bleached zone, or movement of microtubules through the bleached zone