Sldw axonal transport Ralph A. Nixon McLean
Hospital,
Belmont
and Harvard
Medical
School,
Boston,
Massachusetts,
USA
New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.
Current
Opinion
in Cell
Biology
Introduction
components
Strong support for this view has come from analyses of microtubule dynamics using the method of photobleaching (Fig. 1). Using this technique, cy-toskeletal protein subunits, such as tubulin or actin, coupled to a fluorescent marker, are first introduced into living neurons where they incorporate uniformly into the monomer and polymer pool throughout the axon. A highly focused light source is then used to extinguish or bleach the fluorescence of the molecules contained within a discrete segment of the entire axon length. Two types of information can then be obtained. First, the return of Iluorescence to this bleached zone reveals information about the ability of subunits to assemble and disassemble locally and to translocate. Second, if most of the labeled subunits exist in assembled poiymers (e.g. microtubules, actin Iiiamems), the behavior of the bleached zone will indicate whether or hot cytoskeletal polymers move along the axon (Fig. 1). Unlike conventional pulse-labeling methods, the behavior of the entire pool of the cytoskeletal protein in a region of the axon, rather than just the newly synthesized pool, is monitored. Application of this technique to the transport of fluorescently labeled tubuiin in neurites of PC12 cells [ 91 and in axons of cultured sensory neurons from either the chick [ 101 or adult mouse [ 111 has yielded similar results in each case. The zones of bleached microtubules did not move along the axon or widen. This indicates that the microtubules within these regions are essentially stationary, that is, they move at least 100.fold slower than the SCa rate of axonal transport. Actin studied by the same method behaved in a similar way [ 111.
of the
evolution of conceptual models of slow axonal transport has been reviewed recently [ 1,2**,3*,4*]. Several
The
NF-neurofilament; @
Current
Abbreviations SC-slow Biology
4:8-14
years ago, a number of Iindings [S-S] prompted a reconsideration of the prevalent view that the axonal cytoskeleton is a continuously moving, preassembled matrix. Additional studies using different experimental approaches have now begun to reinforce the notion of the axonal cytoskeleton as a principally stationary structure assembled and maintained from newly synthesized precursors, either in subunit or poiymer form, that are delivered by slow axonal transport.
Neurons overcome unique problems to establish and maintain their extreme polar shape. Not the least of these are the delivery of cytoskeletai proteins to distant axonai and synaptic sites and the regulation of cytoskeleton as sembly and turnover at these distant sites. The delivery mechanism for cytoskeletal proteins and various ‘soluble’ enzymes is slow axonal transport, a process involving the outward movement of materials along axons at one of two families of rates, slow component a (SCa; 0. l-l .Omm per day) or slow component b (SCb; 1.0-3.0 mm per day). Slow axonai transport is easily distinguished from the mechanisms involved in translocating vesicular and vesicle-bound constituents at speeds of 100-400 mm per day toward the synapse (fast anterograde transport) or back to the cell body (retrograde transport). Although major advances have been made in understanding the molecular features of fast axonal transport, many fundamental questions about slow axonal transport remain unanswered. For example, what is the nature of the force-generating mechanism and of the substratum along which cytoskeletal proteins and enzymes move? Is more than one motor involved and what is its relationship to the motors of fast anterograde and retrograde transport? Moreover, there is controversy about the nature of different cytoskeletal proteins during transport. This review focuses mainly on this issue and addresses three major questions. Are proteins conveyed as assembled polymers or as precursor structures, such as monomers, oligomers or heterogeneous complexes of unassembled proteins? What proportion of the axonal cytoskeleton is undergoing transport at any given time and how much is stationary? Finally, how are interactions between moving and stationary components regulated?
Moving and stationary axonal cytoskeleton
1992,
Ltd
component. ISSN
0955+74
Slow axonal
(a)
(b)
/ \
IIII kI)
/ \ / \
:e)
/ \
/ \
Fig. 1. Photolabeling techniques for analyzing cytoskeletal protein dynamics in axons are depicted schematically. In photobleaching studies, cytoskeletal protein subunits are linked to a fluorescent tag. (a) The labeled subunits are introduced into neurons by injection into the perikaryon. fb) These subunits gradually incorporate into polymers throughout the axon. Cc) The fluorescent cytoskeletal array within a discrete zone of the axon is then bleached using a laser and the fate of the zone is followed over a period of hours. Three possible results are shown. Cd) If cytoskeletal polymers are translocating distally along the axon, then the zone should move. Spreading of the zone may occur as it moves if there is variation in the transport rates of individual filaments. fe) If polymers are both translocating and exchanging their bleached subunits for fluorescent ones that are free in the cytoplasm, the zone will both translocate and recover its fluorescence. (0 If they are exchanging subunits but not translocating, the zone will recover its fluorescence without moving. The method of photoactivation is similar to this except that subunits are linked to a fluorophor that is made fluorescent only when activated by the laser. Therefore, the cytoskeleton arrays in the zone marked by the laser are distinguished by their fluoresence. Adapted from I421 with permission.
Additional findings from these photobleaching are instructive about the form of transported and actin. A low level of fluorescence returned bleached zone within seconds, reflecting diffusion
studies tubulin to the of free
transport
Nixon
tubulin or actin subunits from the fluorescent regions flanking the bleached zone. The small degree of initial fluorescence recovery confirmed that the pool of free tubulin or actin monomer in axons is relatively small. The further recovery of fluorescence, which occurred slowly (a,, < 15 min), was interpreted as the gradual incorporation into polymers of the fluorescent subunits whose diffusion into the region gave rise to the initial phase of recovery. These results, coupled with other findings discussed later, indicate that tubulin is transported in mammalian axons principally in monomeric form, although the transport of some short microtubules capable of rapidly exchanging subunits cannot be excluded. Recently, however, a conflicting picture of how tubulin is transported was obtained in Xenopus axons using a variant of the photolabeling approach called photoactivation [ 12**]. Although similar in general experimental design to photobleaching, this method involved linking tubulin to a caged fluorophor and visualizing the molecule by photoactivating the tluorophor, rather than by extinguishing its fluorescence. Because the light energy needed to photoactivate a caged Auorophor molecule is several orders of magnitude less than that required to photobleach a convention fluorophor, this method may reduce potential free-radical damage to cells. After a zone of free tubulin and axonal microtubules was photoactivated in the axons of cultured Xf3zopu.s neurons, the fluorescing zone translocated at rates of 0.9-2.8 mm per day, remaining coherent during its movement and diminishing in fluorescence only over relatively long distances. Curiously, the rate of transport close to the growth cone was fourfold faster than near the cell body, a feature quite distinct from transport behavior in mammalian axons. Because the slow coherent movement of the zone differed from the rapid dispersal of photoactivatable dextran (a model soluble molecule), tubulin in the fluorescent zone was considered to be predominantly in an insoluble form, presumably existing as microtubule polymers. The absence of a trailing fluorescent tail behind the moving activated zone and the persistence of full fluorescence during translocation suggested a low rate of exchange of free monomer with tubulin subunits in the microtubules. In embryonic Xenopus axons, therefore, microtubules seem to undergo continuous distal translocation en bloc during axonal elongation. These results were interpreted as evidence supporting the phenomenon of microtubule sliding as the means for transporting the microtubule cytoskeleton and generating new cytoskeleton structure in growing Xenopus axons. Although previous evidence for sliding between cytoplasmic microtubules has been indirect, the recent demonstration of microtubule sliding by digital-enhanced video microscopy in a lower invertebrate system, the freshwater amoeba Reficulomyxu [ 13-1, represents a p recedent for this possible interpretation of the Xfznopur; data. The apparent conflict between findings on Xenopw ax ons using photoactivation methods with those on mammalian axons using the photobleaching technique may have a simple explanation. In very recent studies by the Hirokawa group (Okabe S, Hirokawa N, 31st Annual Meeting, The American Society for Cell Biology, Boston,
9
10
Cytoplasm
and cell motility
Massachusetts, December 1991, abstract 1008) photoactivation methods applied to XenqOus axons and to cultured sensory neurons from adult mice confirmed all of the previous studies by showing that the way in which tubulin is transported differs considerably between these two species. As in the studies of Reinsch et al. [ 12**], microtubules were the principal form of transported tubulin and were in continuous transport along Xenopus axons. By contrast, microtubules were stationary in axons of mammalian sensory neurons and exhibited subunit exchange with moving tubulin subunits, as reported prwiously using photobleaching methods [9-l I]. In future studies, it w-ill be interesting to determine whether the bulk movement of microtubules represents a fundamentally different slow transport mechanism in Xeno pus, or simply the absence of factors that anchor microtubules to stationary elements [ 14.1 or the presence of factors that decrease the affinity of microtubules for the transport motor. In regard to the latter possibility, certain microtubule-associated proteins have been shown to block kinesin-dependent microtubule mobility in z&-o, possibIy by reducing access of the microtubule to the force-generating mechanism [ 15.1. What proportion moving?
of the cytoskeleton
is
The ongoing photobleaching studies indicate that the amount of actin and tubulin being transported at any given time in peripheral fibers is small compared with the preexisting pools of stationary actin filaments and microtubules in the axon, as has been shown already for neurofilaments [6]. Pulse-labeling analyses in rat motor neurons reinforce this conclusion (PN Hoffman, MA Iopata, DF Watson, RF Luduena, personal communication). Conventional pulse-labeling studies in axotomized axons provide evidence that the wave of axonally transported labeled tubulin is a relatively small proportion of the total tubulin content of mature motor fibers. Axotomy was accompanied by a two-threefold increase in the amounts of two neuron-specific P-tubulin isotypes transported into the proximal nerve stump. Despite these substantial increases in the transported fraction, the total content of these isotypes measured in whole nerve by Immunoassay did not change. This result would be expected only if the pool of preexisting, and presumably stationary, tubulin were large compared with the fraction undergoing transport. Dynamics
of microtubule
and actin
assembly
Biotinylated tubulin subunits undergoing axonal transport have been observed to incorporate all along the axon into preexisting microtubules, principally at their plus ends [ 161. These observations have been corroborated and extended recently using a different experimental approach [ 17**], About half of the axonal microtubule mass In cultured sympathetic neurons was considered to be stable on the basis of its slow rate of depolymerization (tiiz - 240 min) in the presence of nocodazole. These stable microtubules were enriched in acetylated
and detyrosinated a-tubulin. The other half of the microtubule mass was rich in tyrosinated a-tubulin and depolymerized rapidly (ti,, - 5 min) after nocodazole treatment. Immunochemicd detection of tyrosinated a-tubulin proved to be a reliable marker for labile microtubules in axons, in that nearly all microtubules immunolabeled with antibodies to tyrosinated a-tubulin were depleted within 15 min of nocodazole treatment. When applied at the ultrastructural level, antibodies to tyrosinated a-tubulin labeled some microtubules uniformly, while others remained unlabeled. At least 40% of the microtubules, however, were labeled densely at their plus end by the antibody to tyrosinated a-tubulin, but not at all at the other end. In view of the rapid nocodazole-induced depolymerization of labile microtubules and the fast recovery when the drug is removed [ 181, these labile domains are likely to be very dynamic in axons. Because the rapid reassembly after drug treatment depends on the preservation of the stable microtubule population, elongation from the plus end of preexisting microtubules appears to be the principal mode of microtubule assembly in axons. Further evidence for the ability of free monomeric subunits to exchange with subunits in assembled polymers comes from studies of actin filaments in growth cones [19*-l. Biotinylated actin subunits were found to incorporate into the distal ends of actin bundles in Philopo &a and at the membrane-associated fringe of the actin filament network. The addition of actin subunits at the membrane-end of the filament was associated with a rearward movement of the actin polymer toward the base of the growth cone. For both actin [ 19.0] and tubulin 191, the recovery of fluorescence in bleached zones occurred much faster in growth cones than in axons, indicating regional variation in cytoskeletal polymer turnover and implying a large degree of local regulation of this process.
Dynamics
of neurofilament
assembly
In view of this picture of microtubule and actin filament assembly in axons, it is reasonable to ask whether or not the axonal neurofilament network may also be maintained by transported subunits. In general, evidence has supported the view that neurofilament polymers are the transported form [2=*]; however, on the basis of recent findings that the Class III neurofilaments polymerize reversibly, and actively exchange subunits along the filament [20*], a reconsideration of this issue for Class IV intermediate filaments may be warranted. So far, however, reversible polymerization and subunit exchange has been demonstrated only for experimental homopolymers of neurofilament-L (NP-L) in uitro (reviewed in [ 200,210] > and not for the doublet (M-L and -M) or triplet (NP-L, -M and -H) neurofilament protein subunit assemblies known to exist in viva (RK Sihag, personal communication). The existence of a pool of highly phosphotylated soluble NF-H subunits in neuroblastoma cells which can incorporate into neurofikunents [22*] may, however, indicate a potential for dynamic exchange of NF-H subunits within certain neuronal compartments and consequent modulation of the kinetic properties and interactive abilities of neurofilaments [21*].
Slow axonal
Regulation of neurofilament phosphorylation
transport
by
The high-molecular-weight subunits of neurofilaments, NP-M and NP-H, begin to be phosphoryiated extensively at their long carboxyi-terminal domains mainly af ter neurofilaments enter the axon. This process continues during transport and is particularly active in distal regions where stationary neurofilaments are most abundant. A series of differentially phosphorylated isoforms of NF-M and NP-H is generated, the more highly phosphotyiated of which are associated preferentially with stationary neurofilaments. These findings have suggested that carboxyl-terminal-domain phosphotylation is part of the mechanism regulating the equilibrium between moving and stationary neurofilaments and, therefore, the rate of neuroftlament transport [2**,21*]. Recent studies provide further support for this idea. In retinal ganglion cells, neurofilament carboxyl-terminal phosphotylation becomes extensive starting 100-200 urn downstream from the cell body. The morphological correlates of this process were examined by comparing the organization of the neurofilament network in axons at seven levels along a l-mm segment of the proximal optic stalk and optic nerve, which included levels before and after extensive carboxyl-terminal phospholylation. At distal levels where neurofilaments were phosphorylated extensively, axon calibers and numbers of neurofilaments were 3.5.fold higher than at proximal levels where hypophosphorylated neurofilaments predominated. Microtubule numbers were roughIy similar at these different levels. Because the rates of neurofilament transport did not change along this length of the axon, the abrupt increase in neurofilament number implies that neuroIiaments begin to accumulate increasingly into a stationary network beginning at the site where carboxyi-termnal phosphotylation of neurofilament subunits becomes extensive (Nixon RA, Paskevich P, Sihag RK, Wheelock T, 31st Annual Meeting, The American Society for Cell Biology, Boston, Massachusetts, December 1991, abstract 2066). Recently, additional phosphotylation sites on NFL and NP-M subunits, which are regulated by kinase systems distinct from those involved at the carboxy-termnal domains, have been shown to include a subpopulation that exhibits rapid phosphate turnover as neurofiaments enter the axon [23*]. Together, the phosphoty lation events at the carboxyl- and amino-terminal ends represent an intricate mechanism for influencing neurofilament transport rate and regulating neurofilament integration into the stationary cytoskeleton [21*]. The influences of phosphorylation on neurofilament transport are also evident from other recent studies. A slowing of neurofilament transport coincided with increased phosphorylation of epitopes on the carboxyterminal domain of NF-H in rats treated with the toxin p,P’-iminodipropionitrile (IDPN) [ 241, whereas accelera tion of neurotiament transport was associated with decreased phosphorylation of these same carboxyl-terminal epitopes [25*]. Changes in both the transport rate of neurofilaments and the equilibrium between moving and stationary neuroIilaments could therefore account for the marked alterations of neurofilament density shown to oc cur in affected axons [26]. Similarly, the marked retar-
transport
Nixon
dation of neuroiilament transport in proximal stumps of axotomized or crushed axons [27*,28*] may be related to earlier observed increases in NP-H phosphorylation [29]. Exposure of cultures of chick sensory neurons to high concentrations of lithium inhibits substantially the phosphorylation of newiy synthesized NP-M but apparently does not affect previously phosphorylated subunits [30-l. When mature neurons were exposed to lithium for three days, no depletion of fully phosphotylated NF-M occurred from either the cell body or the neurite, sug gesting that these phosphotylated filaments are stationary and are replaced very slowly by newly synthesized neuroNaments [30-l. The regional specialization of the axonal cytoskeleton revealed by morphometric analyses also has important implications for how cytoskeletal protein transport occurs and is regulated locally. In addition to proximal-todistal non-uniformities of the distributions of neuroftlaments and microtubules along axons [7,31], striking focal disconhnuities in the size and composition of the cytoskeleton are being found at nodes of Ranvier [ 32*,33*]. At nodal regions along mouse sciatic nerves, axon calibers were found to be fivefold smaller than those at the internodal regions [ 32.1. A fivefold difference In the numbers of neuroftlaments was also seen between nodes and internodes. Microtubules were slightly more abundant in most nodes and, consequently, microtubule packing density was greatly increased. These discontinuities at nodes of Ranvier would imply opposite changes in neuroIilament and microtubule transport rates through the node, if most of these elements were continuously moving. More likely, however, is the possibility that deposition of neurofilaments into a stationary network is partly inhibited at the node. In view of the data discussed above, it is likely that reversible phosphotyiation of neurofilaments mediates such events at the node. In this regard, immunoelectron microscopic studies indicate that NP-H tail regions are considerably less phosphorylated on neurofilaments at nodes of Ranvier than on neurofilaments in intemodal regions [ 349*]. Properties
of stationary
microtubules
On the assumption that pulse-labeled tubulins trailing behind the moving transport wave are present in stationary microtubules, Watson and colleagues [35=*] have investigated the distinctive properties of this tubulin fraction . Over 70% of the pulse-labeled tub&n retained in axons was recovered in the form of cold-stable microtubules, as compared with only 20% of the labeled tubulin moving in the crest of the SCa wave. The retained tubuRns were also more acetylated than those in the moving wave although, on the basis of earlier studies [36], acetyiation was not considered to play a key role in maintaining this stability. In rats, the proportion of total and pulselabeled tubulins assembled into cold-stable microtubules increased progressively during the interval of 4-10 weeks of age [35**,37*] and was reduced in regenerating axons [28*,38*]. The maturational increase in cold-stable microtubules was accompanied by a further trailing of insoluble tubulin behind the moving wave of soluble tubulin [37=] and local degradation of some of this pool
11
12
Cytoplasm
and cell motility
.
[ 37~,39*]. Progressive age-dependent slowing of tubulin transport would be expected if the stability of stationary microtubules increases, and tubulin subunits, once incorporated, are slowly exchanged. These results, then, support the notion that neuronal maturation is associated with an increase in size of the stationary cytoskeleton and a slower rate of cytoskeleton turnover [2**,40=].
Acknowledgements
What confers stability to microtubules? Among the possible factors suggested by studies of the brain microtubules from the Atlantic cod, is the presence of microtubule-associated proteins [41*]. Although cod microtubules are cold-labile, they exhibit other characteristics of stability including insensitivity to calcium-induced disassembly and resistance to colchicine. In studies on the assembly of isolated microtubule proteins from the cod brain, colchicine resistance did not result from the high proportions of acetylated, detyrosinated tubulins that characterized this microtubule population, but from the microtubule-associated proteins in the preparation. Bovine microtubule-associated proteins had the same effects as the unique microtubule-associated proteins in the cod fibers, indicating that the stabilizing effect is a general property of microtubule-associated proteins interacting with an appropriately modified tubulin.
References
Conclusion Recent research, based on a broadened array of experimental methods, reinforces the idea that the cytoskeleton of mammalian axons is composed of both stationary and moving elements and that, in most cases, the fraction of cytoskeletal proteins undergoing transport is relatively small. A major advantage to the neuron of establishing a predominantly stationary cytoskeletal network that turns over slowly is obvious. Because the axon often comprises an enormous, relatively fixed cytoplasmic volume, having to replace only a small percentage of its cytoskeleton at a time greatly reduces the synthetic burden on the neuron. To maintain the axonal cytoskeleton, newly synthesized cytoskeletal proteins and, in some cases, newly assembled polymers advance along the axon and exchange continually with those in the stationary cytoskeletal structures. No single model describes adequately the transport behavior and assembly states of polypeptides that eventually compose the individual fibrous elements of the ax onal cytoskeleton. For example, neurofilament subunits polymerize close to the cell body, but neurofilarnents form a mature lattice only after entering the axon and integrating into a stationary neurofifament network. Tubulin and actin appear to move into the axon principally as unassembled subunits that add mainly to the distal ends of preexisting stationary polymers. Post-translational protein modifications, such as phosphorylation and tyrosination, occur during axoplasmic transport and may direct the assembly and rearrangements of cytoskeletal proteins in axons. The dynamic behavior of cytoskeletal proteins during transport and assembly into stationary structures endows the axon with the potential for both stability and local and rapid reorganization of the cytoskeleton in response to physiological stimuli.
I would like to ful discussions Khan for her also grateful to publication,
thank Drs I Fischer and PJ Hollenbeck for their helpand critical reading of the manuscript, and Johanne instrumental help with manuscript preparation. I am those authors who provided manuscripts before their
and recommended
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PW,
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A substantial proportion of the transported fraction become immobile proximal to a peripheral nerve enter the daughter axon move at normal rates.
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.
Elegant demonstration of the close relationship between tubulin tyrosination and microtubule stability in axons. Stable microtubule domains, composed of detyrosinated a-tubulin subunits, serve as nucleating sites in axons for the assembly of microtubules with the same polarity orientation.
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34.
MATA
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Neurotilament Epitopes vier. J Neunxytol 1992,
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KUPINA
M,
FINK
DJ:
are
Phosphorylationdependent
Reduced
at the
Node
of Ran-
in press. Reduced phosphoqlation of neurofilaments at nodes of Ranvier accompanies nodal constriction and reduced numbers of neurofilaments. These obsetvations are in accordance with hypotheses linking nemoI% ament phosphorylation to the formation of a stationary neuroftlament network.
35. ..
WATSON DF, HOFFMAN PN, GRIFFIN JW: The Cold Stability of Microtubules Increases During Axonal Maturation. J Neurmci 1990, 10:3344-3352.
Pulse.labeled Nbulin remaining behind the peak of slow axonal uans port, which is believed to represent stationary microtubules, is predominantly in the form of cold-stable microtubules. This fraction increases as axons mature.
36.
BOCK
MM,
Bti
tubuIin Deacetylation 9:358-368.
PW.
HUMPHIUF.~
in Intact
S: Dynamics of Neurons. J Neurosci
Alpha1989,
13
14
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and cell motility
TASHIROT, KOMIYA Y: Maturation and Aging of the Axonal Cytoskeleton: Biochemical Analysis of Transported Tubulin. / Neumsci Res 1992, in press. Axonal maturation is associated with slowing of SCa and SCb rates of transport and rising proportions of cold-stable microcubules. 38. TA~HIROT, Kohtw Y: Changes in Organization and Axonal . Transport of Cytoskeletal Proteins During Regeneration. / Neunxbem 1991, 56:1557-1663. The proportions of soluble Nbufin and actin increase and their transport is selectively accelerated in regenerating rat motor axons. KOENIG E: Evaluadon of Local Synthesis of Axonal Proteins 39. . in the Goldfish Mauthner Cell Axon and Axons of Dorsal 37. .
and
Ventral
Roots
of the
Rat in Vitro.
Mo&c
Cell Neunxci
1991 30(1):192-200. This study of the gold&h provides strong evidence for a limited degree of local protein synthesis and Nmover of certain cytoskeletal proteins In axons. 40. ND(ONRA, SHEATB: The Dynamics of Neuronal Intermediate . PRamems a Developmental Perspective. Cell Motii Q&xke/ 1992, in press.
A review of neuronal intermediate fibrnents focusing on their potential for dynamic behaviors similar to those recently shown for Class III intermediate filaments in non-neural cells. 41. .
B~LGERM, STROMBERGE, WU M: Microtubule-associated Proteindependent Colchicine Stability of Acetyfated Coldlabile Brain Microtubules From the Atlantic Cod, Gadus morhua / Ceil Biol 1991. 113:331-338. Microtubule-associated proteins are one of the factors invohed in stabilizing the assembly of acetyiated cold-labile brain microNbNes. 42.
HOUXNBECK PJ: Cytoskeleton
on
the
Move.
Nature
1990,
343:4084x
RA Nixon, &oratories for Molecular Neuroscience, Mailman Research Center, McLean Hospital, 115 Mill Street, Belmont, Massachusetts02178. 9106, USA
Hospital,
Belmont
and Harvard
Medical
School,
Boston,
Massachusetts,
USA
New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.
Current
Opinion
in Cell
Biology
Introduction
components
Strong support for this view has come from analyses of microtubule dynamics using the method of photobleaching (Fig. 1). Using this technique, cy-toskeletal protein subunits, such as tubulin or actin, coupled to a fluorescent marker, are first introduced into living neurons where they incorporate uniformly into the monomer and polymer pool throughout the axon. A highly focused light source is then used to extinguish or bleach the fluorescence of the molecules contained within a discrete segment of the entire axon length. Two types of information can then be obtained. First, the return of Iluorescence to this bleached zone reveals information about the ability of subunits to assemble and disassemble locally and to translocate. Second, if most of the labeled subunits exist in assembled poiymers (e.g. microtubules, actin Iiiamems), the behavior of the bleached zone will indicate whether or hot cytoskeletal polymers move along the axon (Fig. 1). Unlike conventional pulse-labeling methods, the behavior of the entire pool of the cytoskeletal protein in a region of the axon, rather than just the newly synthesized pool, is monitored. Application of this technique to the transport of fluorescently labeled tubuiin in neurites of PC12 cells [ 91 and in axons of cultured sensory neurons from either the chick [ 101 or adult mouse [ 111 has yielded similar results in each case. The zones of bleached microtubules did not move along the axon or widen. This indicates that the microtubules within these regions are essentially stationary, that is, they move at least 100.fold slower than the SCa rate of axonal transport. Actin studied by the same method behaved in a similar way [ 111.
of the
evolution of conceptual models of slow axonal transport has been reviewed recently [ 1,2**,3*,4*]. Several
The
NF-neurofilament; @
Current
Abbreviations SC-slow Biology
4:8-14
years ago, a number of Iindings [S-S] prompted a reconsideration of the prevalent view that the axonal cytoskeleton is a continuously moving, preassembled matrix. Additional studies using different experimental approaches have now begun to reinforce the notion of the axonal cytoskeleton as a principally stationary structure assembled and maintained from newly synthesized precursors, either in subunit or poiymer form, that are delivered by slow axonal transport.
Neurons overcome unique problems to establish and maintain their extreme polar shape. Not the least of these are the delivery of cytoskeletai proteins to distant axonai and synaptic sites and the regulation of cytoskeleton as sembly and turnover at these distant sites. The delivery mechanism for cytoskeletal proteins and various ‘soluble’ enzymes is slow axonal transport, a process involving the outward movement of materials along axons at one of two families of rates, slow component a (SCa; 0. l-l .Omm per day) or slow component b (SCb; 1.0-3.0 mm per day). Slow axonai transport is easily distinguished from the mechanisms involved in translocating vesicular and vesicle-bound constituents at speeds of 100-400 mm per day toward the synapse (fast anterograde transport) or back to the cell body (retrograde transport). Although major advances have been made in understanding the molecular features of fast axonal transport, many fundamental questions about slow axonal transport remain unanswered. For example, what is the nature of the force-generating mechanism and of the substratum along which cytoskeletal proteins and enzymes move? Is more than one motor involved and what is its relationship to the motors of fast anterograde and retrograde transport? Moreover, there is controversy about the nature of different cytoskeletal proteins during transport. This review focuses mainly on this issue and addresses three major questions. Are proteins conveyed as assembled polymers or as precursor structures, such as monomers, oligomers or heterogeneous complexes of unassembled proteins? What proportion of the axonal cytoskeleton is undergoing transport at any given time and how much is stationary? Finally, how are interactions between moving and stationary components regulated?
Moving and stationary axonal cytoskeleton
1992,
Ltd
component. ISSN
0955+74
Slow axonal
(a)
(b)
/ \
IIII kI)
/ \ / \
:e)
/ \
/ \
Fig. 1. Photolabeling techniques for analyzing cytoskeletal protein dynamics in axons are depicted schematically. In photobleaching studies, cytoskeletal protein subunits are linked to a fluorescent tag. (a) The labeled subunits are introduced into neurons by injection into the perikaryon. fb) These subunits gradually incorporate into polymers throughout the axon. Cc) The fluorescent cytoskeletal array within a discrete zone of the axon is then bleached using a laser and the fate of the zone is followed over a period of hours. Three possible results are shown. Cd) If cytoskeletal polymers are translocating distally along the axon, then the zone should move. Spreading of the zone may occur as it moves if there is variation in the transport rates of individual filaments. fe) If polymers are both translocating and exchanging their bleached subunits for fluorescent ones that are free in the cytoplasm, the zone will both translocate and recover its fluorescence. (0 If they are exchanging subunits but not translocating, the zone will recover its fluorescence without moving. The method of photoactivation is similar to this except that subunits are linked to a fluorophor that is made fluorescent only when activated by the laser. Therefore, the cytoskeleton arrays in the zone marked by the laser are distinguished by their fluoresence. Adapted from I421 with permission.
Additional findings from these photobleaching are instructive about the form of transported and actin. A low level of fluorescence returned bleached zone within seconds, reflecting diffusion
studies tubulin to the of free
transport
Nixon
tubulin or actin subunits from the fluorescent regions flanking the bleached zone. The small degree of initial fluorescence recovery confirmed that the pool of free tubulin or actin monomer in axons is relatively small. The further recovery of fluorescence, which occurred slowly (a,, < 15 min), was interpreted as the gradual incorporation into polymers of the fluorescent subunits whose diffusion into the region gave rise to the initial phase of recovery. These results, coupled with other findings discussed later, indicate that tubulin is transported in mammalian axons principally in monomeric form, although the transport of some short microtubules capable of rapidly exchanging subunits cannot be excluded. Recently, however, a conflicting picture of how tubulin is transported was obtained in Xenopus axons using a variant of the photolabeling approach called photoactivation [ 12**]. Although similar in general experimental design to photobleaching, this method involved linking tubulin to a caged fluorophor and visualizing the molecule by photoactivating the tluorophor, rather than by extinguishing its fluorescence. Because the light energy needed to photoactivate a caged Auorophor molecule is several orders of magnitude less than that required to photobleach a convention fluorophor, this method may reduce potential free-radical damage to cells. After a zone of free tubulin and axonal microtubules was photoactivated in the axons of cultured Xf3zopu.s neurons, the fluorescing zone translocated at rates of 0.9-2.8 mm per day, remaining coherent during its movement and diminishing in fluorescence only over relatively long distances. Curiously, the rate of transport close to the growth cone was fourfold faster than near the cell body, a feature quite distinct from transport behavior in mammalian axons. Because the slow coherent movement of the zone differed from the rapid dispersal of photoactivatable dextran (a model soluble molecule), tubulin in the fluorescent zone was considered to be predominantly in an insoluble form, presumably existing as microtubule polymers. The absence of a trailing fluorescent tail behind the moving activated zone and the persistence of full fluorescence during translocation suggested a low rate of exchange of free monomer with tubulin subunits in the microtubules. In embryonic Xenopus axons, therefore, microtubules seem to undergo continuous distal translocation en bloc during axonal elongation. These results were interpreted as evidence supporting the phenomenon of microtubule sliding as the means for transporting the microtubule cytoskeleton and generating new cytoskeleton structure in growing Xenopus axons. Although previous evidence for sliding between cytoplasmic microtubules has been indirect, the recent demonstration of microtubule sliding by digital-enhanced video microscopy in a lower invertebrate system, the freshwater amoeba Reficulomyxu [ 13-1, represents a p recedent for this possible interpretation of the Xfznopur; data. The apparent conflict between findings on Xenopw ax ons using photoactivation methods with those on mammalian axons using the photobleaching technique may have a simple explanation. In very recent studies by the Hirokawa group (Okabe S, Hirokawa N, 31st Annual Meeting, The American Society for Cell Biology, Boston,
9
10
Cytoplasm
and cell motility
Massachusetts, December 1991, abstract 1008) photoactivation methods applied to XenqOus axons and to cultured sensory neurons from adult mice confirmed all of the previous studies by showing that the way in which tubulin is transported differs considerably between these two species. As in the studies of Reinsch et al. [ 12**], microtubules were the principal form of transported tubulin and were in continuous transport along Xenopus axons. By contrast, microtubules were stationary in axons of mammalian sensory neurons and exhibited subunit exchange with moving tubulin subunits, as reported prwiously using photobleaching methods [9-l I]. In future studies, it w-ill be interesting to determine whether the bulk movement of microtubules represents a fundamentally different slow transport mechanism in Xeno pus, or simply the absence of factors that anchor microtubules to stationary elements [ 14.1 or the presence of factors that decrease the affinity of microtubules for the transport motor. In regard to the latter possibility, certain microtubule-associated proteins have been shown to block kinesin-dependent microtubule mobility in z&-o, possibIy by reducing access of the microtubule to the force-generating mechanism [ 15.1. What proportion moving?
of the cytoskeleton
is
The ongoing photobleaching studies indicate that the amount of actin and tubulin being transported at any given time in peripheral fibers is small compared with the preexisting pools of stationary actin filaments and microtubules in the axon, as has been shown already for neurofilaments [6]. Pulse-labeling analyses in rat motor neurons reinforce this conclusion (PN Hoffman, MA Iopata, DF Watson, RF Luduena, personal communication). Conventional pulse-labeling studies in axotomized axons provide evidence that the wave of axonally transported labeled tubulin is a relatively small proportion of the total tubulin content of mature motor fibers. Axotomy was accompanied by a two-threefold increase in the amounts of two neuron-specific P-tubulin isotypes transported into the proximal nerve stump. Despite these substantial increases in the transported fraction, the total content of these isotypes measured in whole nerve by Immunoassay did not change. This result would be expected only if the pool of preexisting, and presumably stationary, tubulin were large compared with the fraction undergoing transport. Dynamics
of microtubule
and actin
assembly
Biotinylated tubulin subunits undergoing axonal transport have been observed to incorporate all along the axon into preexisting microtubules, principally at their plus ends [ 161. These observations have been corroborated and extended recently using a different experimental approach [ 17**], About half of the axonal microtubule mass In cultured sympathetic neurons was considered to be stable on the basis of its slow rate of depolymerization (tiiz - 240 min) in the presence of nocodazole. These stable microtubules were enriched in acetylated
and detyrosinated a-tubulin. The other half of the microtubule mass was rich in tyrosinated a-tubulin and depolymerized rapidly (ti,, - 5 min) after nocodazole treatment. Immunochemicd detection of tyrosinated a-tubulin proved to be a reliable marker for labile microtubules in axons, in that nearly all microtubules immunolabeled with antibodies to tyrosinated a-tubulin were depleted within 15 min of nocodazole treatment. When applied at the ultrastructural level, antibodies to tyrosinated a-tubulin labeled some microtubules uniformly, while others remained unlabeled. At least 40% of the microtubules, however, were labeled densely at their plus end by the antibody to tyrosinated a-tubulin, but not at all at the other end. In view of the rapid nocodazole-induced depolymerization of labile microtubules and the fast recovery when the drug is removed [ 181, these labile domains are likely to be very dynamic in axons. Because the rapid reassembly after drug treatment depends on the preservation of the stable microtubule population, elongation from the plus end of preexisting microtubules appears to be the principal mode of microtubule assembly in axons. Further evidence for the ability of free monomeric subunits to exchange with subunits in assembled polymers comes from studies of actin filaments in growth cones [19*-l. Biotinylated actin subunits were found to incorporate into the distal ends of actin bundles in Philopo &a and at the membrane-associated fringe of the actin filament network. The addition of actin subunits at the membrane-end of the filament was associated with a rearward movement of the actin polymer toward the base of the growth cone. For both actin [ 19.0] and tubulin 191, the recovery of fluorescence in bleached zones occurred much faster in growth cones than in axons, indicating regional variation in cytoskeletal polymer turnover and implying a large degree of local regulation of this process.
Dynamics
of neurofilament
assembly
In view of this picture of microtubule and actin filament assembly in axons, it is reasonable to ask whether or not the axonal neurofilament network may also be maintained by transported subunits. In general, evidence has supported the view that neurofilament polymers are the transported form [2=*]; however, on the basis of recent findings that the Class III neurofilaments polymerize reversibly, and actively exchange subunits along the filament [20*], a reconsideration of this issue for Class IV intermediate filaments may be warranted. So far, however, reversible polymerization and subunit exchange has been demonstrated only for experimental homopolymers of neurofilament-L (NP-L) in uitro (reviewed in [ 200,210] > and not for the doublet (M-L and -M) or triplet (NP-L, -M and -H) neurofilament protein subunit assemblies known to exist in viva (RK Sihag, personal communication). The existence of a pool of highly phosphotylated soluble NF-H subunits in neuroblastoma cells which can incorporate into neurofikunents [22*] may, however, indicate a potential for dynamic exchange of NF-H subunits within certain neuronal compartments and consequent modulation of the kinetic properties and interactive abilities of neurofilaments [21*].
Slow axonal
Regulation of neurofilament phosphorylation
transport
by
The high-molecular-weight subunits of neurofilaments, NP-M and NP-H, begin to be phosphoryiated extensively at their long carboxyi-terminal domains mainly af ter neurofilaments enter the axon. This process continues during transport and is particularly active in distal regions where stationary neurofilaments are most abundant. A series of differentially phosphorylated isoforms of NF-M and NP-H is generated, the more highly phosphotyiated of which are associated preferentially with stationary neurofilaments. These findings have suggested that carboxyl-terminal-domain phosphotylation is part of the mechanism regulating the equilibrium between moving and stationary neurofilaments and, therefore, the rate of neuroftlament transport [2**,21*]. Recent studies provide further support for this idea. In retinal ganglion cells, neurofilament carboxyl-terminal phosphotylation becomes extensive starting 100-200 urn downstream from the cell body. The morphological correlates of this process were examined by comparing the organization of the neurofilament network in axons at seven levels along a l-mm segment of the proximal optic stalk and optic nerve, which included levels before and after extensive carboxyl-terminal phospholylation. At distal levels where neurofilaments were phosphorylated extensively, axon calibers and numbers of neurofilaments were 3.5.fold higher than at proximal levels where hypophosphorylated neurofilaments predominated. Microtubule numbers were roughIy similar at these different levels. Because the rates of neurofilament transport did not change along this length of the axon, the abrupt increase in neurofilament number implies that neuroIiaments begin to accumulate increasingly into a stationary network beginning at the site where carboxyi-termnal phosphotylation of neurofilament subunits becomes extensive (Nixon RA, Paskevich P, Sihag RK, Wheelock T, 31st Annual Meeting, The American Society for Cell Biology, Boston, Massachusetts, December 1991, abstract 2066). Recently, additional phosphotylation sites on NFL and NP-M subunits, which are regulated by kinase systems distinct from those involved at the carboxy-termnal domains, have been shown to include a subpopulation that exhibits rapid phosphate turnover as neurofiaments enter the axon [23*]. Together, the phosphoty lation events at the carboxyl- and amino-terminal ends represent an intricate mechanism for influencing neurofilament transport rate and regulating neurofilament integration into the stationary cytoskeleton [21*]. The influences of phosphorylation on neurofilament transport are also evident from other recent studies. A slowing of neurofilament transport coincided with increased phosphorylation of epitopes on the carboxyterminal domain of NF-H in rats treated with the toxin p,P’-iminodipropionitrile (IDPN) [ 241, whereas accelera tion of neurotiament transport was associated with decreased phosphorylation of these same carboxyl-terminal epitopes [25*]. Changes in both the transport rate of neurofilaments and the equilibrium between moving and stationary neuroIilaments could therefore account for the marked alterations of neurofilament density shown to oc cur in affected axons [26]. Similarly, the marked retar-
transport
Nixon
dation of neuroiilament transport in proximal stumps of axotomized or crushed axons [27*,28*] may be related to earlier observed increases in NP-H phosphorylation [29]. Exposure of cultures of chick sensory neurons to high concentrations of lithium inhibits substantially the phosphorylation of newiy synthesized NP-M but apparently does not affect previously phosphorylated subunits [30-l. When mature neurons were exposed to lithium for three days, no depletion of fully phosphotylated NF-M occurred from either the cell body or the neurite, sug gesting that these phosphotylated filaments are stationary and are replaced very slowly by newly synthesized neuroNaments [30-l. The regional specialization of the axonal cytoskeleton revealed by morphometric analyses also has important implications for how cytoskeletal protein transport occurs and is regulated locally. In addition to proximal-todistal non-uniformities of the distributions of neuroftlaments and microtubules along axons [7,31], striking focal disconhnuities in the size and composition of the cytoskeleton are being found at nodes of Ranvier [ 32*,33*]. At nodal regions along mouse sciatic nerves, axon calibers were found to be fivefold smaller than those at the internodal regions [ 32.1. A fivefold difference In the numbers of neuroftlaments was also seen between nodes and internodes. Microtubules were slightly more abundant in most nodes and, consequently, microtubule packing density was greatly increased. These discontinuities at nodes of Ranvier would imply opposite changes in neuroIilament and microtubule transport rates through the node, if most of these elements were continuously moving. More likely, however, is the possibility that deposition of neurofilaments into a stationary network is partly inhibited at the node. In view of the data discussed above, it is likely that reversible phosphotyiation of neurofilaments mediates such events at the node. In this regard, immunoelectron microscopic studies indicate that NP-H tail regions are considerably less phosphorylated on neurofilaments at nodes of Ranvier than on neurofilaments in intemodal regions [ 349*]. Properties
of stationary
microtubules
On the assumption that pulse-labeled tubulins trailing behind the moving transport wave are present in stationary microtubules, Watson and colleagues [35=*] have investigated the distinctive properties of this tubulin fraction . Over 70% of the pulse-labeled tub&n retained in axons was recovered in the form of cold-stable microtubules, as compared with only 20% of the labeled tubulin moving in the crest of the SCa wave. The retained tubuRns were also more acetylated than those in the moving wave although, on the basis of earlier studies [36], acetyiation was not considered to play a key role in maintaining this stability. In rats, the proportion of total and pulselabeled tubulins assembled into cold-stable microtubules increased progressively during the interval of 4-10 weeks of age [35**,37*] and was reduced in regenerating axons [28*,38*]. The maturational increase in cold-stable microtubules was accompanied by a further trailing of insoluble tubulin behind the moving wave of soluble tubulin [37=] and local degradation of some of this pool
11
12
Cytoplasm
and cell motility
.
[ 37~,39*]. Progressive age-dependent slowing of tubulin transport would be expected if the stability of stationary microtubules increases, and tubulin subunits, once incorporated, are slowly exchanged. These results, then, support the notion that neuronal maturation is associated with an increase in size of the stationary cytoskeleton and a slower rate of cytoskeleton turnover [2**,40=].
Acknowledgements
What confers stability to microtubules? Among the possible factors suggested by studies of the brain microtubules from the Atlantic cod, is the presence of microtubule-associated proteins [41*]. Although cod microtubules are cold-labile, they exhibit other characteristics of stability including insensitivity to calcium-induced disassembly and resistance to colchicine. In studies on the assembly of isolated microtubule proteins from the cod brain, colchicine resistance did not result from the high proportions of acetylated, detyrosinated tubulins that characterized this microtubule population, but from the microtubule-associated proteins in the preparation. Bovine microtubule-associated proteins had the same effects as the unique microtubule-associated proteins in the cod fibers, indicating that the stabilizing effect is a general property of microtubule-associated proteins interacting with an appropriately modified tubulin.
References
Conclusion Recent research, based on a broadened array of experimental methods, reinforces the idea that the cytoskeleton of mammalian axons is composed of both stationary and moving elements and that, in most cases, the fraction of cytoskeletal proteins undergoing transport is relatively small. A major advantage to the neuron of establishing a predominantly stationary cytoskeletal network that turns over slowly is obvious. Because the axon often comprises an enormous, relatively fixed cytoplasmic volume, having to replace only a small percentage of its cytoskeleton at a time greatly reduces the synthetic burden on the neuron. To maintain the axonal cytoskeleton, newly synthesized cytoskeletal proteins and, in some cases, newly assembled polymers advance along the axon and exchange continually with those in the stationary cytoskeletal structures. No single model describes adequately the transport behavior and assembly states of polypeptides that eventually compose the individual fibrous elements of the ax onal cytoskeleton. For example, neurofilament subunits polymerize close to the cell body, but neurofilarnents form a mature lattice only after entering the axon and integrating into a stationary neurofifament network. Tubulin and actin appear to move into the axon principally as unassembled subunits that add mainly to the distal ends of preexisting stationary polymers. Post-translational protein modifications, such as phosphorylation and tyrosination, occur during axoplasmic transport and may direct the assembly and rearrangements of cytoskeletal proteins in axons. The dynamic behavior of cytoskeletal proteins during transport and assembly into stationary structures endows the axon with the potential for both stability and local and rapid reorganization of the cytoskeleton in response to physiological stimuli.
I would like to ful discussions Khan for her also grateful to publication,
thank Drs I Fischer and PJ Hollenbeck for their helpand critical reading of the manuscript, and Johanne instrumental help with manuscript preparation. I am those authors who provided manuscripts before their
and recommended
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A substantial proportion of the transported fraction become immobile proximal to a peripheral nerve enter the daughter axon move at normal rates.
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RA Nixon, &oratories for Molecular Neuroscience, Mailman Research Center, McLean Hospital, 115 Mill Street, Belmont, Massachusetts02178. 9106, USA
