We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, -75 Jtm from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and ( + )-ends directed .toward the cell body. At distances within 15 ,um of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.Vertebrate neurons generate and maintain two morphologically and functionally distinct types of neurites, axons and dendrites (1-6). It has long been recognized that axons and dendrites differ in their complements of cytoplasmic organelles (1, 6). Most notable in this regard, ribosomes and Golgi elements are present in dendrites but are absent from axons. What is the basis for the nonuniform distribution of organelles in neurons? Several lines of evidence indicate that the distribution of organelles in a cell reflects active transport processes that selectively convey organelles from their sites of synthesis and assembly to other locations in the cell (7, 8).These observations raise the possibility that many of the differences between the organelle composition of axons and dendrites are produced by differences in the organization of the transport systems that convey materials from the cell body into the axon or dendrite.The transport of organelles is a microtubule-based process; microtubules provide the substrate for organelle translocation and, by virtue of their intrinsic polarity, influence the directionality of transport (7-9). The intrinsic polarity of microtubules is based on the asymmetry of the tubulin subunit and its self-assembly characteristics; the (+)-end is preferred for subunit addition over the (-)-end (10, 11). Microtubule-based translocators convey organelles specifically toward either the ( + )-or the ( -)-end ofthe microtubule (7-9). In the axon, microtubules are uniform with respect to polarity, with the (+ )-ends directed away from the cell body (12-15). Thus, only those organelles that translocate toward (+ )-ends of microtubules will be conveyed from the cell body into the axon.Do microtubules in dendrites have the same polarity orientation as those in axons? To date, information concerning the polarity orientation of dendritic microtubules derives from a few atypical cell types. In the dendrite-like processes of teleost retinal cone cells (16) and frog primary olfactory neurons (17), microtubules are unifo...

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Abstract. We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining in axons after various times in 2 #g/ml nocodazole, we observed that ~48% of the MT mass in the axon is labile, depolymerizing with a t1/2 of "-,5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a tt/2 of r~ 240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated ot-tubulin, whereas the stable MTs contain little or no tyrosinated ot-tubulin and are instead rich in posttranslationally detyrosinated and acetylated o~-tubulin. These results were confirmed quantitatively by immunoelectron microscopic analyses of the distribution of tyrosinated ot-tubulin among axonal MTs. Individual MT profiles were typically either uniformly labeled for tyrosinated ot-tubulin all along their length, or were completely unlabeled. Roughly 48% of the MT mass was tyrosinated, ~52% was detyrosinated, and ~85% of the tyrosinated MTs were depleted within 15 min of nocodazole treatment. Thus, the proportion of MT profiles that were either tyrosinated or detyrosinated corresponded precisely with the proportion of MTs that were either labile or stable respectively. We also observed. MT profiles that were densely labeled for tyrosinated a-tubulin at one end but completely unlabeled at the other end. In all of these latter cases, the tyrosinated, and therefore labile domain, was situated at the plus end of the MT, whereas the detyrosinated, and therefore stable domain was situated at the minus end of the MT, and in each case there was an abrupt transition between the two domains. Based on the frequency with which these latter MT profiles were observed, we estimate that minimally 40% of the MTs in the axon are composite, consisting of a stable detyrosinated domain in direct continuity with a labile tyrosinated domain. The extreme drug sensitivity of the labile domains suggests that they are very dynamic, turning over rapidly within the axon. The direct continuity between the labile and stable domains indicates that labile MTs assemble directly from stable MTs. We propose that stable MTs act as MT nucleating structures that spatially regulate MT dynamics in the axon.

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Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of approximately 0.6-0.7 microm/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.