Rao Microtubules 2

Going back to microtubule motor proteins page of Microtubule Lecture 1.
We stopped there last week.A classic experiment demonstrates the eistence of microtubule motor protein.
Add axoplasm- get binding and transport, suggesting something in axoplasm is required . Kinesin 1 was the first motor protein isolated using this system.
Kinesins have 2 or 3 heavy chains. A few have 4. Bipolar means heavy chains on both sides.
Globular domain is where Microtubules binding site is on heavy chain.Also has ATPase activity to induce conformational change for motor function. Neck domain similar to myosins. Long stalk- Coiled coil alpha helix. Light chains attach to tail instead of neck region as in myosins.
Specific function determined by light chain associated with tail domain.
N,M C type depends on what part of heavy chain forms head domain.
+ or – end directed- walk from plus to minus or minus to plus. Very specific for part kinesin.
Cytosolic or mitotic- mitotic part in formation spindle and segregation of chromosomes in mitochondria.
Directionality depends on neck domain.
If you create a chimeric protein(switch neck domain with one for a protein with different directionality), the neck domain determines directionality.
K-dependent vesicle movement:
How can you examine experimentally?
Vesicle migration assay?
Attach microtubules to surface. Vesicle attaches to kinesin and you visualize transport of vesicles by high resolution phase contrast. Can generate video.
Can also show other way around in microtubule sliding assay. Kinesin on slide, microtubules, no fluorescent probe required, just phase contrast.Microtubule moves.
How does kinesin walk along microtubules?
2 theories:
Hand-over hand walking model: 2 head domains bind only to beta tubulin. Beta is present alternatively. Other head domain after conformational change turns and binds to next b tubulin.
Inchworm model- one domain binds, next takes small step and binds to same beta tubulin, then moves forward. Each head domain binds to every b tubulin.
Hand over hand would be faster. How to demonstrate which is true? Paper in Science. Took kinesin and mutated amino acids. Glu and Thr to Cys to bind dye. Followed dye to see how fast it moved. Only one head domain had the dye. Alpha and beta tubulin span 8 nm. If you follow dye in hand-over-hand model, see step distance 16 nm. Inchworm, see 8 nm steps. Hand over hand is most likely model.
Hydrolyses one ATP for 16 nm movement.
Other microtubule motor proteins are called dyneins.
Minus end directed. Move from plus to minus end. Retrograde transport from nerve ending to cell body.Cytosolic dyneins- vesicle transport and segregation of chromosomes. Axonemal dyneins in cilia and flagella. 2 or 3 heavy chains with intermediate and light chains. Dyneins cannot transport vesicles by themselves, because their light chains cannot bind vesicles. Need dynactin which consists of Glued and Dynamtin. Glued has microtubule binding domain, but no ATPase activity. That comes from heavy chain. Glued helps it to stay attached.Microtubules does not reach all the way to cell membrane. Cortical network of actin microfilaments is at border. Vesicle travels along microtubules to periphery. Transfers to microfilaments to go along microfilament to plasma membrane.

Lecture 2
Stable microtubules provide a track.
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Stable in cilium and flagellum. Cross section of flagellum under EM on left. Bundle microtubules. Singlets, doublets. Right- diagrammatic representation. Flagellum has MTOC at base with 9 triplet microtubules. Minus end toward cell, plus end at tip of flagellum.
3 types crosslinking proteins: One bridges singlets in center like rungs of ladder. Nexin binds the “a” tubule of one doublet to the “b” tubule of adjacent doublet.
3rd- spokehead radiates from center to bind “a” tubule of doublet. Motor proteins bind a tubule. 2 rows are dyneins. Tail domains are attached to a tubule with head toward b of next doublet. 2 rows of dyneins.
Beating of flagellum or cilium is characterized by series of bends. Originate at base and propel toward distal end. How? Dynein movement important. Nexins prevent Microtubules from sliding all the way. Generates bend. Digest nexin- no crosslink. Microtubules slide instead of bending.
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Role in mitosis
Unstable microtubules- constant dynamic changes in length. Take part in mitosis.
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Mitotic apparatus- bundles of microtubules. Functions listed on slide.
“To understand” section is repeated list of objectives.
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EM of mitotic apparatus. Football shape is mitotic apparatus. Asters are poles with centrosomes at center.
3 types microtubules in structure: Centrosomes, aster microtubules radiate toward periphery. Purple is called polar microtubules. Minus end at centrosome overlap with similar polar microtubules from other end. Kinetochore microtubules have their origin at centrosome with + end at chromatids.
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Alignment during prophase
Kinesins involved. They are microtubular motor proteins. Bind to one at head, other at tail, to align polar microtubules. Pushing forces push polar microtubules apart with motor protein BimC. 2 head groups at each end bind and walk. Pulling force on asters- dynein attached to plasma membrane. Walk toward minus end and pulls centrosome to periphery.
Spindle disrupted when dynein neutralized in photo at right.
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Chromatid has centromeric DNA which binds to proteins in kinetochore. Inner layer and outer layer of protein . Inner binds to centromeric DNA. Outer binds to + end microtubules.
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Binding to microtubules.
+ end shrinks and elongates- gets in contact with chromatids, hits kinetochore. Oscillates until it hits it. A little like fly fishing (the person throws out a fishing line with a hook on the end and reels it in over and over until he hooks a fish). Metaphase:
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Alignment at equator
Several forces are involved. Motors generate forces- walking along. They generate forces to align the microtubules.
Also polar microtubules form contact with chromatids at ends, but not stable contact. Helps position chromatids. Chromokinesins form a degree of contact for positioning.
Treadmilling may also help.
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Anaphase
Shorten microtubules by depolarize ar +end- pulls apart. Motor prots also pull apart. Polar microtubules may polymerize so motor based forces can extend.
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Intermediate filaments
Slide FYI. Mostly structural. No information is available on any dynamic role for intermediate filaments in the cell. Very hard to depolarize in vitro. Tissue specific.
He will not be here during review session. Will email list of objectives for learning. 2 questions park, 2 pfeffer,2 kriwacki,2 ST. Kriwacki may review at end second lecture. Exam is 1/30.