MOLECULAR BASIS OF AXONAL TRANSPORT

Project: Research project

Description

The general aim of this research project is to develop a more complete
understanding of the molecular interactions which determine how
organelles are transported along microtubules in axons. Our proposed
investigations are particularly concerned with how kinesin and dynein two
soluble, force-generating ATPases that promote movement in opposite
directions along microtubules, interact with specific populations of
organelles, programmed to move either toward or away from the cell body.
In recent experiments, the movement of purified organelles depended on
the presence of an axoplasmic cytosol fraction containing many proteins
in addition to kinesin and dynein. The specific aim of this proposal is
to identify, more precisely, the particular cytosolic factors required
for organelle movement. We will isolate, by AMP-PNP induced microtubule
affinity, followed by salt or nucleotide induced release, a subset of
cytosolic proteins form axoplasm, sufficient to promote organelle
movement. Our proposed experiments will determine whether purified dynein
and kinesin from axoplasm can alone drive organelle movement, or whether
soluble "accessory factors" are also required, as suggested by previous
studies. We will also explore the possibility, suggested by recent
experiments, that both dynein and kinesin are required for retrograde
organelle movement. We will do so in part by recombining the purified
components and testing their ability to promote directed movement of
organelles; in addition, electron microscopic studies of organelles will
localize kinesin and dynein, to determine whether they are bound together
on the organelle surface. Finally, nm-scale motion analysis, which
previously indicated that kinesin-coated beads track along single
protofilaments, while dynein-coated beads "wander" over the microtubule
surface, will be applied to organelle movement to test if these same
features of kinesin and dynein driven movement are apparent. Tracking of
organelle movement at the nm level will provide an assay for molecular
interactions between particular proteins in the reconstituted system.
StatusFinished
Effective start/end date8/1/8912/31/97

Funding

  • National Institutes of Health
  • National Institutes of Health: $291,231.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $26,985.00

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Transport Vesicles
kinesin
Microtubules
Organelles
Kinesin
organelles
Dyneins
microtubules
Optical Tweezers
Proteins
Detergents
Tail
Microtubule Proteins
Decapodiformes
Molecular Imaging
Molecular Models
Avidin
Eukaryotic Cells
Glutathione Transferase
Sepharose

ASJC

  • Medicine(all)
  • Neuroscience(all)