neurite, which becomes specified as the axon. In discussing mechanisms that might regulate the delivery of new membrane needed for growth, Futerman and Banker (23) raised the possibility that the transport of carrier vesicles may be regulated in accordance with the rate of neurite elongation. According to this view, one might expect the transition from stage 2 to 3 to be accompanied by an increase in the number of axonal carrier vesicles entering the axon. Bradke and Dotti (4) have proposed that the transition from stage 2 to 3 is accompanied by a reorganization of intracellular transport, from multidirectional (into all neurites) to unidirectional (into the emerging axon). According to their model, this concerted change in transport affects a broad variety of organelles, including carrier vesicles conveying both axonal and dendritic proteins, as well as mitochondria and peroxisomes. Our analysis did not reveal the changes in transport predicted by either of these models. In the case of carrier vesicles containing the axonal protein NgCAM, we found no significant increase in the amount of transport into stage 3 axons compared with unspecified stage 2 neurites, nor was the amount of transport into the axon of stage 3 cells greater than into their dendrites. In the case of carrier vesicles conveying the dendritic protein TfR, we did not observe the increase in its axonal trafficking predicted by the Bradke and Dotti model. Instead, far fewer TfR carrier vesicles entered the nascent axon than entered the neurites of stage 2 cells; in stage 3 cells, TfR vesicles were preferentially transported to the dendrites, not to the axon. One important limitation in the current study is that the methods we used for expressing GFP constructs do not yield high levels of expression in very young neurons. Thus we were unable to assess transport before 2 days in culture. It is possible that there are changes in transport that occur concomitantly with axonal specification, but that these changes are not maintained throughout developmental stage 3. Alternative methods will be required to address this possibility.
We have previously shown that in mature neurons, the polarization of NgCAM to the axonal plasma membrane does not depend on directed transport but instead involves events at the plasma membrane, most likely the preferential fusion of NgCAM carrier vesicles with the axonal membrane (ref. 1 and unpublished observations). Because cell surface NgCAM is polarized by stage 3, whereas NgCAM carrier vesicles are transported into both dendrites and axons at this stage, it is tempting to speculate that the same mechanism used in mature cells is responsible for the polarization of NgCAM early in development.
What changes occur in neurons between developmental stages 2 and 3 that might initiate the polarization of cell surface proteins? In the case of dendritic proteins like TfR, it is highly likely that these changes involve the establishment of selective microtubule-based transport. We have shown that carrier vesicles containing TfR are preferentially transported into the dendrites at developmental stage 3, although the selectivity of this process is not as great as in mature cells. Moreover, our results show that at the early stages when TfR-containing vesicles are not fully excluded from the axon, TfR is expressed on the axonal surface. This finding indicates that there is no additional quality control mechanism downstream of transport to prevent TfR-containing vesicles from fusing with the axonal membrane. It is thought that the selective microtubule-based transport that prevents the movement of dendritic carrier vesicles into the axon depends on regional biochemical differences within the neuron (1). These differences might take the form of biochemical differences among microtubules in different regions of the cell or of local differences in the regulation of components of the motor protein-carrier vesicle complex. Of the biochemical characteristics that distinguish axonal from dendritic microtubules in mature neurons, some have been shown to arise early in development. For example, although the microtubule-associated protein t is uniformly distributed in stage 3 neurons, it is differentially phosphorylated in dendrites (24). Similarly, phosphorylated MAP1B is expressed in a proximodistal gradient in axons of cortical and sensory neurons (25, 26). These data suggest that a unique complement of kinase and phosphatase activities is present in developing axons. In addition to producing posttranslational differences in microtubule proteins, local differences in kinase or phosphatase activity could also regulate motor activity or the interaction of motor proteins with cargo vesicles, thereby inhibiting the delivery of dendritic carrier vesicles to the axon (27–29). Similarly, local posttranslational modifications could selectively regulate the vesicle fusion machinery, potentially inhibiting the fusion of NgCAM carrier vesicle in the dendritic domain (30).
We thank Hannelore Asmussen, Jon Muyskens, and Barbara Smoody for the preparation of neuronal cultures, and Silvia LaRue, Julie Harp, and Sarah Godsey for excellent technical assistance. This work was supported by National Institutes of Health Grant NS17112.
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