Question

1. Using your knowledge of cell & molecular biology techniques, design an experiment to investigate a novel hypothesis for the process of vesicle transport. The hypothesis proposes that a network of defined microfilament tracts in the cytoplasm exist (like railroad tracks!) and that are not visible under a microscope and that do not stain with the regular histochemical dyes that are available. These tracts are of distinctly different types and are made up of proteins – each type of unique tract specifically interacts with specific and unique type of target vesicles and guides them from ER to their final destination (eg., lysosomes, membrane, secretory, etc.).

a. Provide a specific strategy (design) of an experimental plan to investigate this hypothesis with appropriate rationale.

b. Experimental Plan – provide specific details – separate them using sub-topics if a multiple step plan is used. Describe: Cell & Molecular Approach Reagents used Instrumentation used

c. Observations & Results Expected

d. Make – pros and cons – if a approach is challenging – explain why and provide a discussion.

e. Conclusion

Answer 1

Transport vesicles play a central role in the traffic of molecules between different membrane- enclosed compartments of the secretory of pathway. Vesicles are similarly involved in the transport of materials taken up at the cell surface.Vesicular transport is thus a major cellular activity,responsible for molecular traffic between a variety of specific membrane -enclosed compartments. The selectivity of such transport is therefore key to maintaining the functional organization of the cell.

Progress toward elucidating the molecular mechanisms of vesicular transport has been made by three distinct experimental approaches:

1) isolation of yest mutants that are defective in protein transport and sorting

2) reconstitution of vesicular transport in cell - free systems

3) biochemical analysis of synaptic vesicles,which are responsible for the regulated secretion of neurotransmitters by neurons.

Each of these experimental system has distinst advantages for understanding particular aspects of the transport process.Most important is the fact that results from all three of these avenues of investigation have converged, indicating that similar molecular mechanisms regulate secretion in cells as different as yeasts and mammalian neurons.As in other areas of cell biology, yeasts have proved to be advantageous in studying the secretory pathway because they readily amenable to genetic analysis.

By pioneering the isolation of yeast mutants defective in vesicular transport. These include mutants that are defective at various stages of protein secretion,mutants that are unable to transport protein to the vaculoe.The experimental design exploited a mutant mammalian cell line that lacked the enzyme required to transfer N- acetylglucosamine residues to the N-linked oligosaccharide at an early stageof its modification in the Golgi apparatus.