Rao lecture 68

Microtubules 1 lecture 68 CMB

Start comparing the microfilaments and microtubules. Pay attention to comparison for exam purposes.
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functions that need microfilaments
Most extent- microfilaments do not explain transport of vesicles.

Different structure, subunits, etc.
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Learning objectives
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Structure of microtubules
Larger and stiffer than microfilaments.
Nerve axons are very long and filled with microtubules. Stiffer than microfilaments.
Microtubules fill cytoplasm between nucleus and plasma membrane. Start from center of cell and radiate into periphery.
2 populations:
1. stable structural component (cilia, flagellum. RBC, axons)- like actin in muscle and actin cortical network
disruption- catastrophic consequences
2. unstable undergo constant polymerization and depolymerization. Example: cell undergoing mitosis.
Structure microtubules- building block is tubulin heterodimer (microfilament monomer) alpha and beta, mostly. Gamma is in centrosomes. Similarity to microfilaments- subunits bind nucleotide, but it is GTP instead of ATP. And 2 molecules bind, one to each monomer. GTP is trapped in interface in alpha and cannot be hydrolysed. Not exchangeable. Bound to beta is exchangeable and can be hydrolysed.
Green- binding site for taxol, a plant product.
Tubulin is highly conserved.
Be able to compare microfilament and microtubule subunits on exam.
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Tubulin dimers bind. Lateral and longitudinal reactions are important. Long chain is protofilament. Lateral interactions form curved and tube-like structures.They are staggered a bit. Dimers in similar orientation. One end of tube always lined by alpha- minus end. One end lined by beta-+ end. Structural polarity similar to microfilaments. Difference is helix vs tubular structure. Most microtubules are singlets of 13 protofilaments. Rarely less or more. C. elegans neural may have 11-15 protofilaments. Higher animals always 13. Doublets seen in cilium and flagellum. 13(A)+10(B).
Triplet present in centrosomes- 13+10+10 protofilaments.
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MTOC- microtubule organizing center- also called centrioles. Left picture- green is stain for microtubules. Yellow stained for pericentrin in centrosome. Localized close to nucleus. Microtubules radiate from centrosome. Right- closer look under EM. See cross-section one, longitudinal section of other. 10 triplet microtubules form one centriole. Dense area is pericentriole matrix of proteins like gamma tubulin- not polarized, but may play role in nucleation. Pericentrin and other proteins localized in matrix. Microtubules radiate from centrosome. Minus end toward centrosome, plus toward periphery.
Do microtubules elongate toward periphery, or are they formed in other parts of cell?
You could microinject labelled tubulin, cool cell to dissolve microtubules, warm to 37 deg C, and look at microtubules. Time course suggests they start nucleating and elongating from the centrosome.
Central body close to nucleus. In neurons, there are extensions like axons and dendtrites. Microtubules are organized with the minus end toward cell body in axons. Dendrites- mixed organization. Some minus or plus end toward end. Reason not clear. Evidence suggests in axon, microtubules formed in cell body from centrosome. In dendrite they are probably formed without a centrosome. How? We do not know.
When centrosomes divide into 2 centrioles- bottom right.
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Similarities microtubules and microfilaments- undergo polymerization in nucleation, elongation, steady state phases.
Difference- microtubules are temperature sensitive. Cool- mass drops due to depolymerization. Warm- see rapid increase in mass due to repolymerization.Cannot use cold buffer looking at microtubules to wash cells. Critical concentration- concentration at which they start polymerizing.
Critical concentration in vitro is .03 micromolar. Cell concentration is 10-30 micromolar. Favors polmerization. Critical concentration at + and- end are different. Depends on GTP or GDP. Assembly favored at plus end. Treadmilling occurs.
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phases on slide. Protofilaments, curved sheets, tubule.
Elongation added in dimer form. Stable as dimer.
Depolymerization at low concentration- see end differences in pictures. Splayed appearance occurs during depolymerization.Lateral interactions fall apart first.
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Flagellum can act as nucleus. More elongation at plus end.
Unlike microfilaments, depolymerization and polymerization at plus end predominantly occur.
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disruptive drugs:
Ancient colchicine- from plant extract. Prevents polymerization at low concentration and induces depolymerization at higher conc. Can block mitosis.
Can prevent cell division used as treatment agent in diseases. A chemical analog is reversible and can be used to synchronize cells in a culture. All cells in M phase after a day, wash off colcemid, watch cells divide.
Taxol is product from bark of Yew tree. Anticancer. Binds beta tubulin to prevent depolymerization. Stabilizes microtubule. Cell division cannot continue.
Vinblastine- similar to taxol.
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Unlike microfilaments, dynamic instability is seen in microtubules. Length of inducible microtubules drastically decreases (catastrophe phase), then rescue. Repeats in same solution and conditions. Change in length is called dynamic instability. Cycle goes several times. All microtubules do not change at same time. Some elongate, some shorten. Live cell- images over time used to compare microtubules. Each tubule behaves independently.
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GTP cap hypothesis- microtubule during polymerization may have GTP hydrolysed after a while. Not required for polymerization. Depending on rate of polymerization, may see GTP cap. If rate polymerization is >GTP hydrolysis, see GTP cap. With GTP-cap, more stable. GDP cap- unstable. One hypothesis.
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Other explanation is that there are proteins that bind to change stability of microfilaments. Not understood. MAPs bind and colocalize with microtubules.
See slide for rest.
Mutant forms of tau are seen in diease.Transfect fibroblasts with tau- get some elongation of processes.
MAPK phosphorylate MAPs- cannot bind microtubules. Regulate microtubules.
MAP study is open area.
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Transport vesicles and organelles.
Microfilaments only transport vesicles at periphery. Microtubules form tracks for transport of vesicles from center of cell. Intermediate filaments in axon stabilize microtubules. Vesicles align on microtubules. Speed of transport is determined by motor protein, not directly by cargo.Axonal transport does not require an intact cell. Just piece of axon and ATP.
Experiment on right- pulse chase experiment. Cell bodies localized in ganglion. Different proteins move at different rates using microtubule motor system.
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directionality depends on motor protein- plus or minus end directed.
Classic experiment to discover classic motor protein Kinesin.
Isolated pure microtubules.
AMPPNP is analog of ATP that cannot be hydrolysed. Used to purify kinesin 1.