Neurons are elongate, highly polarized cells consisting of a
cell body (which contains the nucleus, ER and many organelles),
branching projections of the cell body called dendrites,
and a very long extension called an axon. Typically, specialized
regions called synapses are found at the extremities of the dendrites
and axon, and patch-clamp measurements of membrane potentials indicate
action potentials are usually propagated along the neuron from the
dendritic synapses to the axonal synapse. In addition, the axon
exhibits rapid transport of vesicles along its entire length.
To investigate the transport of vesicles in the axon, Kaether,
C. et al. (2000, Mol. Biol. Cell. 11:1213-1224.
PMID10749925) grew hippocampal neurons in culture and examined
axonal outgrowths by phase contrast and fluorescence microscopy.
Their fluorescent images are displayed in the figures and video
below. To follow the movement of axonal vesicles, the gene for a
ubiquitous IMP (amyloid precursor protein, APP) was tagged with
the gene for yellow fluorescent protein (YFP), and the chimeric
gene was expressed in the cultured neurons.
The movement of vesicles containing APP-YFP was followed by fluorescence
microscopy, and the upper figure below, which is a negative
image of the one beneath it, highlights two types of tubular vesicle:
those moving from left to right (outlined in red) towards the axon
tip and those moving from right to left (outlined in green) towards
the cell body. After you have become oriented to the figure, click
on the image below to start the movie.
The video sequence contains 50 frames which were captured at 0.9
sec intervals, for a total filming time of 45 sec. The duration
of the QuickTime movie is 5 sec, and hence the vesicular movements
are speeded up 9 times. The width of the video frame is about 100
mm, and for reference purposes, the lower axon running diagonally
across the frame, with two red and two green
vesicles depicted, is also about 100 mm in length.
Play the movie several times, at normal speed and one frame at
a time, to follow the movements of the two kinds of vesicles and
answer the following questions.
[For more information about fast axonal transport, please consult
the research article by Kaether, C. et al., 2000, Mol. Biol. Cell.
11:1213-1224. PMID10749925.
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Questions
A. Characterize the vesicular movements carefully. For example,
do they always occur in the same direction? Or do some reverse direction?
B. What cytoskeletal organelle likely guides the movement of these
vesicles?
C. How could you test this hypothesis?
D. How fast are the vesicles moving towards the axon extremities?
Towards the cell body?
E. What molecular motors might be responsible for the vesicular
movements? Discuss briefly the basis
for your answer.
F. How could you test your hypothesis? To examine the results of
one experiment designed to identify a molecular motor, turn
the page.
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