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Slow Axonal Transport: Stop and Go As ‘Bell-Shaped’,Which Spread As They Move Along the Axon Towards the Axon Tip (FIG

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Slow Axonal Transport: Stop and Go As ‘Bell-Shaped’,Which Spread As They Move Along the Axon Towards the Axon Tip (FIG PERSPECTIVES action. Science 177, 401–408 (1972). 40. Bonnet, C. Considérations sur les Corps Organisés (Rey, transport represent the movement of 35. Udrisar, D. & Rodbell, M. Microsomal and cytosolic Amsterdam, 1762). fractions of guinea pig hepatocytes contain 100-kilodalton 41. Boyer, P. D. The ATP synthase — a splendid molecular cytoskeletal and cytosolic proteins at much GTP-binding proteins reactive with antisera against alpha machine. Annu. Rev. Biochem. 66, 717–749 (1997). slower rates, and the nature of the carrier subunits of stimulatory and inhibitory heterotrimeric GTP- 42. Haller, A. Elementa Physiologiae Corporis Humani binding proteins. Proc. Natl Acad. Sci. USA 87, 6321–6325 (Bousquet, Lausanne, 1757). structures for these proteins is not known. (1990). 43. Rastogi, V. K. & Girvin, M. E. Structural changes linked to Proteins that associate with neurofilaments 36. Alberts, B. The cell as a collection of protein machines: proton translocation by subunit c of the ATP synthase. preparing the next generation of molecular biologists. Cell Nature 402, 263–268 (1999). and microtubules move in slow component ‘a’ 92, 291–294 (1998). at average rates of roughly 0.3–3 mm day–1 37. Mitchell, P. & Moyle, J. Chemiosmotic hypothesis of Acknowledgements µ –1 oxidative phosphorylation. Nature 213, 137–139 (1967). This article has benefited from discussions with A. Cattaneo of (~0.004-0.04 m s ), and proteins that associ- 38. Malpighi, M. The Viscerum Structura (Montii, Bologna, the International School for Advanced Studies (S.I.S.S.A.) of ate with microfilaments, as well as many other 1666). Trieste, and has been made possible by bibliographical help from 39. Malpighi, M. Dissertatio Epistolica de Formatione Pulli in L. LIannucci of the University of Pisa. I also thank L. Galli-Resta, cytosolic proteins, are transported in slow Ovo (Martyn, London, 1673). A. Pignatelli and B. Pelucchi for critically reading the manuscript. component ‘b’ at average rates of roughly 2–8 mm day–1 (~0.02–0.09 µm s–1) (TABLE 1). No movement en masse OPINION In radioisotopic pulse-labelling experiments, slow components ‘a’ and ‘b’ form unimodal asymmetrical waves, often loosely described Slow axonal transport: stop and go as ‘bell-shaped’,which spread as they move along the axon towards the axon tip (FIG. 1). traffic in the axon Each wave represents the concerted move- ment of many distinct proteins whose indi- vidual waveforms coincide. Early studies on Anthony Brown slow axonal transport stressed the coherence of these transport waves but not the spread- Efforts to observe the slow axonal transport the movement of a unique type of macromol- ing, and this gave rise to the idea that of cytoskeletal polymers during the past ecular structure (TABLE 1). cytoskeletal and cytosolic proteins move decade have yielded conflicting results, and The fast components of axonal transport along the axon en masse, that is, in a slow and this has generated considerable controversy. are now known to represent the anterograde synchronous manner1. The movement of neurofilaments has now and retrograde movement of distinct types of The expectation of a slow and synchronous been seen, and it is rapid, infrequent and membranous organelles along microtubules at movement has had a profound influence on highly asynchronous. This motile behaviour average rates of roughly 50–400 mm day–1 the design of experiments aimed at detecting could explain why slow axonal transport has (~0.5–5 µm s–1), propelled by the action of slow axonal transport. For example, many eluded observation for so long. molecular motor proteins2. Membranous studies have used fluorescence photobleaching organelles can therefore be considered to be or photoactivation strategies in which fluores- Neurons communicate with other cells by the carrier structures for fast axonal transport. cent or caged fluorescent cytoskeletal proteins extending cytoplasmic processes called axons In contrast, the slow components of axonal are injected into nerve cells and then a popula- and dendrites. Remarkably, axons can attain lengths of one metre or more, although they Table 1 | The moving structures of axonal transport* lack ribosomes and Golgi complexes. Axonal Rate class Average rate Moving structures Composition proteins and Golgi-derived vesicles are formed (selected examples) in the neuronal cell body and are shipped Fast components along the axon by a process called axonal transport. This movement is essential for the Fast anterograde 200–400 Golgi-derived vesicles Synaptic vesicle proteins, mm day–1 and tubules kinesin, enzymes of growth and survival of axons, and continues (≈2–5 µm s–1) (secretory pathway) neurotransmitter metabolism throughout the life of the nerve cell. Studies on axonal transport in laboratory Bi-directional 50–100 Mitochondria Cytochromes, enzymes of –1 animals with radioisotopic pulse labelling mm day oxidative phosphorylation (≈0.5–1 µms–1) have shown that there are hundreds of axonal- ly transported proteins, but that these proteins Fast retrograde 200–400 Endosomes, lysosomes Internalized membrane move at a small number of discrete rates, mm day–1 (endocytic pathway) receptors, neurotrophins, ≈ µ –1 which can be categorized as either fast or slow. ( 2–5 m s ) active lysosomal hydrolases Each discrete rate component represents the Slow components movement of a largely distinct subset of pro- Slow component ‘a’ 0.3–3 Neurofilaments, Neurofilament proteins, teins that are transported together throughout mm day–1 microtubules‡ tubulin, spectrin, tau proteins their journey along the axon. To explain these observations, Lasek and colleagues proposed Slow component ‘b’ 2–8 Microfilaments, Actin, clathrin, dynein, the structural hypothesis of axonal transport, mm day–1 supramolecular dynactin, glycolytic which postulates that all axonal proteins move (≈0.02–0.09 µm s–1) complexes of the enzymes by association with, or as integral parts of, sub- cytosolic matrix 1 cellular carrier structures . According to this *Data compiled from REFS 1,41,44. ‡ In some neurons, microtubule proteins are transported in slow hypothesis, each rate component represents component ‘b’ as well as slow component ‘a’. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 1 | NOVEMBER 2000 | 153 PERSPECTIVES tion of these proteins is marked by bleaching in axons has been a vexing problem, but the that the extent of bleaching in the photo- or activating the fluorescence in a narrow band most likely explanation is that cytoskeletal pro- bleaching studies on neurofilament proteins across the axon [see supplementary figure teins do not move en masse in axons after all. was only partial, reducing the fluorescence online]. In these experiments, a slow and syn- intensity in the axon to 20–50% of its initial chronous movement should be manifested as Neurofilaments move in fits and starts value6. If the residual unbleached fluores- a slow translocation of the marked zone A recent breakthrough in the study of slow cence in the bleached region exceeded the flu- towards the axon tip. However, most studies axonal transport has come from observations orescence intensity of a single neurofilament, on tubulin, actin and neurofilament proteins on neurofilament proteins, tagged with green then it is likely that the movement of neuro- using one or both of these techniques showed fluorescent protein (GFP), in cultured rat filaments across the bleached zone could have that the marked zone does not move3–9. sympathetic neurons14,15. These cultured neu- gone unnoticed. This could also apply to the Although gradual recovery of the fluorescence rons contain relatively few neurofilaments and photobleaching studies on actin and tubu- was observed after photobleaching, it had no frequently show discontinuities in their axonal lin3–5,9,12,13. For example, in the study of Okabe obvious directionality and was therefore neurofilament array, resulting in short seg- and Hirokawa12 on tubulin, photobleaching attributed to exchange between the bleached ments of axon that lack neurofilaments14. reduced the fluorescence intensity in the axon polymers and diffusible fluorescent subunits. Time-lapse imaging of these naturally occur- to 10–40% of its initial value and, in other Directional movement of the photobleached ring gaps in the axonal neurofilament array similar studies, researchers have estimated or photoactivated zone was observed in cul- has enabled the observation of axonal trans- that as much as 10–20% of the total tubulin tured frog neurons10–12, but it probably resulted port without the need for photobleaching or in the axon could have moved through the from stretching of the axon owing to the rapid photoactivation approaches. Contrary to photobleached gaps without detection4. growth and poor adhesion of these neurons expectations, neurofilaments move rapidly, Similar detection limits have also been esti- on laminin substrates11,13. The repeated failure with peak rates as high as 3 µm s–1, and these mated for the fluorescence photoactivation of so many efforts to demonstrate slow syn- movements are frequently interrupted by pro- technique8. The ability of the photobleaching chronous movement of cytoskeletal proteins longed pauses14,15 (FIG. 2). The average velocity experiments to detect the movement of excluding the pauses is about 0.2–0.3 µm s–1. cytoskeletal polymers may also have been Assuming an average transport rate in the hampered by the short length of the bleached –1 µ a range of 0.3–3 mm day (TABLE 1), we can esti- regions (3–5 m), and the relatively long mate that individual neurofilaments spend time-lapse intervals (typically five minutes or 83–99% of their time pausing during their more). These considerations suggest that the journey down the axon. photobleaching and photoactivation strate- Radioisotopic pulse labelling studies in the gies should be capable of detecting the slow mouse optic nerve led Nixon and axonal transport of cytoskeletal proteins if Logvinenko17 to propose almost 15 years ago they could be optimized to enable the detec- that there are two kinetically distinct popula- tion of single rapidly moving polymers. b tions of neurofilament proteins in axons, one that moves and one that is stationary.
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