Question

1. You identify a protein that may play a role in microtubule-based transport. Propose a series of experiments that would aid you in confirming your assessment that this protein has a role in microtubule-based transport.

Please also explain why.

Answer 1

Microtubules are responsible for a variety of cell movements including the intracellular transport and positioning of membrane vesicles and organelles and the separation of chromosomes at Mitosis.

Here, we can take the protein which are the members of two large families of motor proteins, the kinesin and the dyneins.

This proteins move along microtubules in opposite directions, kinesin towards the plus end and dynein towards the minus end.

To Unravel the mechanism of axonal transport cell, the protein or proteins in neuronal cytosolic extracts should be identified.

When synaptic vesicles were added with ATP to these microtubules, the neither bound to the microtubules not moved along them. But the addition of squid nerve axoplasm caused the vesicles to bind to the microtubules and to move along them.

This indicates that a soluble protein in the nerves cytosol is required for translocation.

A mixture of microtubules, brain extract and AMPPNP(used for purification of soluble mortor protein) was incubated;The microtubules with any bound proteins were collected by centrifugation.

Treatment of the microtubule-rich material in the pellets with ATP released one predominant protein back into solution, this protein was named as KINESIN.

Kinesin isolated from squid axoplasm is a diamer of two heavy chains, each complexed to a light chain with the total molecular weight of 380,000.

Systolic kinesin are involved in vesicle and organelle transport; they include the classic axonal kinesin, implicated in transport of lysosomes and other membranous organelle.