2) Long-distance transport in cells is driven by kinesin and dynein motors that

Question

2) Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. All kinesins share conserved structural and functional motifs including a microtubule and ATP binding terminal motor domain, a coiled-coil, stalk domain and a C terminal tail domain that mediates interactions with various cargoes. In the course of his research Dr. Seneviratne discovers an unusual new kinesin which he names KIF666 which has GTPase motor activity. He has no idea what the cargo of KIF666 is. Based on previous studies, he hypothesizes that KIF666 function is regulated by auto-inhibition and that this auto-inhibition is a result of KIF666 folding into a compact conformation. A schematic of his hypothesis is presented below. He proposes that cargo binding is needed for KIF666 to revert to an open active conformation and GTP hydrolysis to GDP is essential for the open confirmation to revert to the inactive closed confirmation Cargo Cargo Cargo GOP Cargo Pi-energy GDP GDP GTP GTP GDP GDP Closed inactive conformation with GDP bound to motor domain Cargo binds to the tail domain causing the conformation to open Cargo falls off GTPi Kinesin reverts back to the GTP bound closed inactive conformation cytosolic GTP binds hydrolyzed to GOP by the motor domain

Explanation / Answer

To unravel the mechanism of axonal transport, cell biologists sought to identify the protein or proteins in neuronal cytosolic extracts that can propel synaptic vesicles along microtubules assembled in vitro from purified tubulin subunits and stabilized by the drug taxol. When synaptic vesicles were added with ATP to these microtubules, the vesicles neither bound to the microtubules nor moved along them. However, the addition of squid nerve axoplasm (free of tubulin) caused the vesicles to bind to the microtubules and to move along them, indicating that a soluble protein in the nerve cytosol is required for translocation.
When researchers incubated vesicles, nerve cytosol and microtubules in the presence of AMPPNP, a nonhydrolyzable analog of ATP, the vesicles bound tightly to the microtubules but did not move. However, the vesicles did move when ATP was added.

These results suggested that a motor protein in the cytosol binds to microtubules in the presence of ATP or AMPPNP, but movement requires hydrolysis of the terminal phosphoanhydride bond of ATP.
To purify the soluble motor protein, scientists used AMPPNP to promote its tight binding to microtubules, which were used as an affinity matrix. A mixture of microtubules, brain extract, and AMPPNP was incubated; the microtubules with any bound proteins then were collected by centrifugation. Treatment of the microtubule-rich material in the pellet with ATP released one predominant protein back into solution; this protein was named kinesin.

Kinesin isolated from squid axoplasm is a dimer of two heavy chains, each complexed to a light chain, with a total molecular weight of 380,000. The molecule is organized into three domains, a pair of large globular head domains connected by a long central stalk to a pair of small globular tail domains, which contain the light chains. Each domain carries out a particular function: the head domain, which binds microtubules and ATP, is responsible for the motor activity of kinesin, and the tail domain is responsible for binding to membrane vesicles. In light of the transport function of kinesin, a bound membrane vesicle is often referred to as kinesin’s “cargo.”

Within cells, membrane-bounded vesicles and proteins are frequently transported many micrometers along well-defined routes in the cytosol and delivered to particular addresses. Diffusion alone cannot account for the rate, directionality, and destinations of such transport processes. Early video light microscopy studies showed that these long-distance movements follow straight paths in the cytosol, frequently along cytosolic fibers, suggesting that transport involves some kind of tracks.

Subsequent experiments, using nerve cells and fish-scale pigment cells, first demonstrated that microtubules function as tracks in the intracellular transport of membrane-bounded vesicles and organelles, and that movement is propelled by microtubule motor proteins.

Nerve impulses are transmitted from a neuron by release of neurotransmitters from the terminal of the axon, the very long process that extends from the cell body. The neuron must constantly supply new materials — proteins and membranes — to the terminal to replenish those lost by exocytosis at the junction (synapse) with another cell. Where do these new materials originate? Ribosomes are present only in the cell body and dendrites of nerve cells, so no protein synthesis can occur in the axons and synaptic terminals. Therefore, proteins and membranes must be synthesized in the cell body and then transported down the axon, which can be up to several meters in length, to the synaptic regions. This process of axonal transport is now known to occur on microtubules. As noted earlier, the microtubules in axons are all oriented with their (+) ends toward the terminal, which is critical to axonal transport.
The rate at which proteins are transported along axons, and their identity, can be determined by a pulse-chase experiment. Such experiments commonly are conducted on neurons in the mammalian sciatic nerve because their cell bodies are conveniently located in the dorsal root ganglion near the spinal cord and their nerve axons are very long. Studies like these have shown that axonal transport occurs in both directions. Anterograde transport proceeds from the cell body to the synaptic junctions and is associated with axonal growth and the renewal of synaptic vesicles. In the opposite, retrograde, direction other substances move along the axon rapidly toward the cell body. These substances, which consist mainly of “old” membrane from the synaptic terminals, are destined to be degraded in lysosomes in the cell body.

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