INTRODUCTION: The giant squid axon is a large axon located within the neural system of the squid. These axons are quite large, making them a great model to manipulate and study transport and neuronal migration. They are approximately one millimeter in diameter, which does not sound GIANT!, but, in comparison to axons of other organisms, including amphibians and mammals, they are several hundred times larger. The squid giant axon carries information to the muscles of a squid’s mantle, which allows for contraction, and extremely fast movement. Within the giant squid axon, bidirectional transport is key. The coordination between microtubules associated molecular motors that work in opposing directions is important. Cytoplasmic dynein and conventional kinesin are two of the major molecular motors that are involved in bidirectional transport within the squid giant axon. Proper brain development is imperative to the development of the axon. Impaired neuronal development will cause neurodegenerative disorders and prevent correct molecular motor activity. Segal et al. used the squid giant axon as a model organism to examine the modulation of bidirectional transport by peptides, proteins, and genes. By testing the way in which Ndel1, a cytoplasmic dynein binding protein, can help regulate transport, they were able to find the effects of such proteins on transport within the axon. The following video gives a brief, yet informative, overview of how experiments prior to Segal et al, led to the discovery and understanding of the squid giant axon and how it works.
TRANSPORT IN THE SQUID GIANT AXON: Within the squid giant axon, there are several key components to aiding and modulating transport. Some of the key factors involved in transport are: microtubules, cytoplasmic dynein, kinesin, and retrograde and anterograde transport. A simple review of cell biology will aid in the understanding of these topics.
NDE1 & NDEL1 – REGULATION OF BIDIRECTIONAL TRANSPORT: In their experiment on the squid giant axon, Segal et al. examined Ndel1 may play a part in the regulation of bidirectional transport. In order to test this, they injected soluble Ndel1 protein, Ndel1-derived proteins, and fluorescent beads into the squid giant axon. Because of the similarities between the squid axon and mammalian axons, mammalian Ndel1 proteins were used to study the neuronal transport. Ndel1 proteins and peptides were chosen because of their full-length structure within a fifty amino acid sequence near the 250th amino acid. Segal et al. were able to generate two peptides from Ndel1, including DID and GST, to evaluate the bidirectional transport within the axon.
Figure A shows the predicted domains within the proteins that could be derived for peptides. Segal et al. used carboxylated fluorescent beads and confocal microscopy to determine these folded and unfolded regions of the protein. Injecting peptides mixed with carboxylated beads into the giant axon allowed for the tracking of beads. Thousands of beads were tracked and separated based on whether or not they did one of several things. The beads could: move continuously in one direction, change directions while traveling with or without intervening pauses, or remain stationary at a zero velocity. Figure B shows the injection procedure using a glass slide and a suspension of peptides and beads between two oil droplets, as well as an image of the beads within the axon.
After conducting their experiments, Segal et al. were able to determine much about the transport within the squid giant axon including the bidirectional nature of axons and how Ndel1 plays a role in the modulation of peptides.
CONCLUSION: Segal et al. performed an experiment in which Ndel1-derived peptides were tested to determine whether or not they have the ability to direct transport in the squid giant axon. One of the first things the team did was to inject peptides mixed with carboxylated beads into the axon. They found that both anterograde and retrograde movement could be found within the axon. Movements both into and out of the cells within the axon. This can be seen in Figure 3. The panels show sequences of time frames capturing individual beads moving in the anterograde and retrograde directions.
The final conclusion for this experiment showed that Ndel1 proteins increased instantaneous velocities in the anterograde direction; whereas the instantaneous velocities were increased in both directions when DID and Nde1-peptides were used. By tracking the velocities of beads this could be seen. Figure 4 shows the directed velocity of tracks versus time. There are both fast and slow movements in the anterograde direction (the positive sign) and in the retrograde direction (the negative sign); however, there is much more activity in the anterograde direction. In order to explain the way in which bidirectional transport works, Segal et al. uses the tug of war model as a generalization model. Figure 5 from the experiment depicts this model and allows it to explain the various ways in which bidirectional transport can be generalized and viewed.
This figure does a great job of showing the cell biology of bidirectional transport and the ways in which dynein and kinesin interact with microtubules to modulate bidirectional transport. Segal et al. suggests that active bidirectional transport can be regulated using motor-binding proteins.
Remarks: This study by Segal et al. was not the first of its kind, nor will it be the last. The squid giant axon is a great model to study because its results can be translated into various categories of invertebrates and vertebrates alike. The experiment brings up new questions about transport within axons and how it is carried out; however, it does a good job of determining the ways in which motor activities and cell biology work together to modulate such happenings. Further examination of axon transport in the squid giant axon would definitely be worthwhile.
- Segal, Michal, Ilya Soifer, Heike Petzold, Jonathan Howard, Michael Elbaum, Orly Reiner. “Ndel1-derived peptides modulate bidirectional transport of injected beads in the squid giant axon.” Biology Open. 1.10 (2012): 220-231. Print.
- Haydon, D.A, J.R. Elliot, B.M. Hendry. “The Squid Axon.” Current Topics in Membranes and Transport. 22.1 (1984): 445-478. Print.
- Marsh, Stuart, dir. The Squids Giant Axon. Youtube, 2007. Web. 1 May 2013. <http://www.youtube.com/watch?v=omXS1bjYLMI>.
- Lisieski, Mike, ed. “The Squid Giant Axon.” Cephalove: A Many Armed Love Affair. Southern Fried Science Network, 26 05 2010. Web. 1 May 2013. <http://cephalove.southernfriedscience.com/?p=51>.