Membrane

trafficking

in neurons

Eric Holtzman Columbia

Neurons variety

possess an unusually of active

endocytic ways,

University,

endocytic-like

phenomena

both

to the genesis

and

New York, New York, USA

extensive

Colgi

processes.

contribute,

fate

of the

apparatus

The Colgi

and

apparatus

probably

in multiple

membrane

systems

exhibit and

a the

overlapping in axons

and

terminals.

Current

Opinion

in Neurobiology

Introduction

in PC12 cells. The synaptophysin is sulfated, indicating its likely passage through trunsGolgi systems on its way to the vesicles; this adds to the evidence for involvement of trunsGolgi elements in the genesis of synaptic vesicle membrane [ 1,5-8].

The facets of neuronal membrane trafhcking that receive the most attention are the supply of materials to presynaptic terminals, the cycling of membranes involved in neurotransmission, and the retrograde movement of membrane-delimited compartments from terminals to cell bodies. These topics, in particular the first two, in which important progress has been made in the past year, will be the central concern of the review. I will, however, also consider advances in other areas, including the properties of axonal compartments and the uses of endocytosis by nerve cells. For a more comprehensive review see [l].

The origins of synaptic

1992, 2:607-612

As judged by accessibility to extracellular biotinylation, much of the newly made synaptophysin in PC12 cells reaches SLMVs only after it has been exposed at the cell surface and retrieved by endocytosis [4**I. This resurrects the proposal that endoqtosis is involved in the maturation of ‘new’ synaptic vesicles as well as in the recycling of old ones. (Earlier versions of this proposal, in which the putative precursor structures were large densecored vesicles, remain under attack [9].> These findings could open the way for comparable studies on the SSVs of neurons, synaptic vesicle proteins other than synaptophysin, and synaptic vesicles other than SSVs. Such studies should clarify how much attention must be paid to the differences-some obtious, some more subtle [lo]-between the behavior of true synaptic vesicles and that of other closely related secretory bodies. For now, one must bear in mind the numerous observations suggesting that structures much like new synaptic vesicles can originate along axons or in cell bodies, seemingly by budding from larger structures (for an example, see [S] >, without known involvement of endocytosis. It will be important, for example, to determine the nature of the synaptic vesicle-like structures that accumulate in neuronal cell bodies of C: elegans mutants lacking kinesin-related transport motors [ 11 I.

vesicles

New macromolecular constituents of synaptic vesicles move in membrane-bound compartments from the Golgi apparatus in the cell body to the nerve terminals. Key details of this movement are still obscure; even the turnover times for vesicle membrane components are too sketchily known to permit the calculation of rates of supply for the terminals. In part because ‘mature’ (e.g. transmitter-filled) vesicles of the small clear variety (small synaptic vesicles; SSVs) that abound at many synapses, are sparse along normal axons and at interruptions, it has been proposed (see [ 1,2] > that the transport of vesicle components may involve Golgi-derived precursor structures. Some of these might be larger than the SSVs. Conceivably, the precursors could contain machinery to mediate maturational processes analogous to the proteolytic cleavages and other modifications of secretory material seen in neurosecretory granules [ 1 ,?I}. Probable precursor structures for synaptic vesicle-like microvesicles (SLMVs) in the neuron-related PC12 cell line have now been identified by virtue of their content of the synaptic vesicle protein, synaptophysin [4*-l. In their sedimentation on sucrose gradients, and also in other respects (microscopy was not reported), these structures behave like the constimtive secretory vesicles that had previously been described

Synaptophysin sometimes turns up in ‘unexpected locations in neurons, such as in dendrites or at the apical tip of the photoreceptor cell body [8,12**]. This suggests that neurons may normally produce synaptophysincontaining vesicles that are in addition to the vesicles or precursors thought to move more or less directly to the terminals. One can imagine, for example, the existence of a population that recycles in the cell body, perhaps via the Golgi apparatus [13] or endosomes. These vesicles could retrieve synaptic vesicle components that had

Abbreviations ER-endoplasmic

reticulum;

SLMV-synaptic

@

Current

vesicle-like

Biology

microvesicle;

Ltd ISSN 0959-4388

SSV-small

synaptic

vesicle.

607

608

Neuronal and glial cell biology

moved initially to the ‘wrong’ domain, or participate in as yet unrecognized local phenomena. Recycling of synaptic vesicles The two major contending classes of models for recycling of SSVs-retrieval by rapid reversal of exocytosis versus delayed retrieval involving clathrin-coated vesicles and the intermediate endocytic sacs, vacuoles and tubules called ‘cistemae’ [ 14]-remain to be reconciled [1,6,14-161. It seems best at present simply to recognize that both types of processes may well coexist and to try to understand their possible physiological or developmental inter-relations. The possibility that vesicles establish only a minute transient pore in the plasma membrane, though certainly not ruled out as important for neurotransmission [ 171, has still to be definitively meshed with the uptake of large tracers into recycling synaptic vesicles [1,12**,14,15]. In either model, the finding that several major proteins of synaptic vesicles form complexes with one another that resist dispersal with detergents [18**] affords an attractive explanation for the ‘fidelity’of retrieval-the fact that despite their diminutive size, recycled vesicles acquire the requisite panel of transporters, proton pumps and other molecules needed to accumulate transmitters and to prepare for exocytosis. Popular versions of both models envisage some budding of synaptic vesicles without an obligatory direct involvement of clathrin, either from the cell surface or from intracellular cisternae. (The latter might form by separation of modest-sized invaginations from the cell surface, or by fusions involving clathrin-coated vesicles.) There is little clear evidence yet on the pertinence of nonclathrin coats to synaptic vesicle recycling, but several lines of investigation on non-neuronal cells are worth watching. For example, vesicle-like invaginations of the cell surface (‘caveolae’) with distinctive coats [19], undergo rapid cycles of closing and reopening reminiscent of ‘rapid reversal’ recycling models for synaptic vesicles. On the other hand, brefeldin A, which interferes with certain non-clathrin coats (e.g. those containing P-COP), as well as with certain clathrin-containing ones, reversibly alters the behavior of specific intracellular and endocytic membranes, such that vesicle formation is suppressed in favor of the production of larger structures [20]. This change calls to mind the modulations in retrieval of synaptic vesicles under varying experimental circumstances [1,14,15], especially the alterations in the relative prominence of newly recycled vesicles as compared with cell surface invaginations or cisternae. In the presynaptic terminals of retinal photoreceptors, brefeldin A increases the abundance of clustered tubules and vacuoles some of which are endocytic (M SantaHernandez and E Holtzman, unpublished data). Endosomes Endocytic bodies with the morphology of endosomes of other cell types-vacuoles, tubules and multivesicular structures---can be found in most regions of neurons

(see Fig.1). Some of these bodies, studied thus far in neurons that use acidic amino acids as neurotransmitters, behave as though they have acidified interiors, which is one of the usual hallmarks of endosomes [ 1,211. Neuronal endosome-like structures carry membranes and endocytosed materials toward the lysosomal system. In dendrites, cell bodies, and axonal growth cones they participate in such traditional endosomal functions as the cycling of transferrin [ 12**,22]. Perhaps they also move toxins, viruses, and membrane-associated receptor systems activated by growth factors or other ligands, from the cell surface towards sites where such agents exert their effects [ 11. The extent to which endosome-related bodies participate in the life history of synaptic vesicles or other membrane systems in- mature nerve terminals is uncertain, however, despite the attractive analogies one can draw between synaptic vesicle recycling and the likely generation of small non-clathrin coated vesicles by non-neuronal endosomes. One complication is that ‘endosome’ is still an elastic category as there are no universally applicable markers or other identification criteria. Another is the fact that synaptic vesicles have proton pumps similar to those of conventional endosomes. The most obviously endosome-like of the morphologi tally identifiable participants in synaptic vesicle recycling are the cisternae mentioned above. They are endocytic and are among the structures thought to be acidified. But cisternae are not always evident during recycling, and acidification may not be required (Fig.1). Moreover, if one accepts that genuine endosomes can be small, coated, or otherwise atypical [23], then bodies smaller than cisternae, grading down even to some newly recy cled synaptic vesicles that have yet to fill with transmitters, nucleotides and the like, could be candidates [ 11. Perhaps endosomes participate in the life history of synaptic vesicles chiefly when sorting of vesicle components from other material becomes necessary. The differential distribution of lumenal contents and membrane molecules to later destinations-intracellular stores, degradative sites, or the plasma membrane-is a principal function of endosomes in many cells. The terminallike varicosities of maturing cultured hippocampal neu rons have been found to retain endocytosed antibodies directed against the synaptic vesicle protein synaptotagmin, while sending endocytosed wheat germ agglutinin along the axon [24]. It will be instructive to determine whether both types of molecules occupy common initial endocytic compartments [25], or whether they traverse different paths from the start. Several cultured ‘neuroendocrine’ cell lines, including PC12 cells, form both synaptophysin-containing SLMVs and other structures in which endocytic receptors colocalize with synaptophysin [ 12**,26*~]. Although kinetic and morphological analyses still need to be strengthened, a reasonable supposition is that these cells use endosomes to sort synaptophysin from endocytic receptors and other membrane components inappropriate for synaptic vesicles. The sorting machinery may depend on vesicle proteins or other cell type-specific molecules in addition to synaptophysin as certain transfected non-neuroendocrine cells that express synaptophysin do not segregate synaptophysin from endocytic receptors [ 12**,26”]. (Cultured

Membrane

trafficking in neurons Holtzman

Fig.1. Electron micrograph of a terminal-like axonal varicosity from a cultured rat hippocampal neuron that had been exposed to the endocytic tracer, horseradish peroxidase, in a high K+ medium containing ammonium chloride. The tracer is demonstrable in synaptic vesicles and in larger endocytic structures that are probably comparable to the endosomes of non-neuronal cells. Synaptic vesicles can be labeled despite the presence of weak bases even at concentrations that abolish staining with acidotropic (weak base) vital dyes. This could mean that passage through a markedly acidified compartment is not obligatory for synaptic vesicle recycling (see [211). Reproduced with permission from E Augenbraun (author’s laboratory).

hepatoma cells may have the capacity for such segregation; B Wiedenmann, personal communication.) In appropriate cell types, an endosome-dependent sort ing route traversed by synaptophysin could serve both in recycling and in the passage of new membrane molecules to synaptic vesicles [4**]. If so, the already blurry distinctions between ‘new’ and ‘recycled’ vesicles will need careful examination [ 1,8] The report that the carboxyterminal cytoplasmic domain of synaptophysin is needed for efficient endocytosis of the protein foreshadows the detailing of the targeting information used to control the several phases of vesicle genesis and cycling [27*]. It is possible that certain vesicle proteins possess most of the operative targeting information, with other proteins being carried along in multiprotein complexes or in membranes [ 18**]. Regulation Styryl dyes, whose behavior suggests that they enter the membranes of synaptic vesicles during the endocytic phase of the cycle, and can subsequently be released from the terminals when the vesicles are reused in neurotransmission (Fig.2), provide a means for studying the population dynamics of recycling vesicles in living ter minals [ 28..,291. One conclusion is that newly recycled vesicles become confined quite rapidly to the collections of vesicles that group near active zones. In these populations, newly recycled vesicles mingle at random among the other vesicles within a few minutes of acquiring the dyes. Presumably the ‘caging’ of vesicle populations and the mobility of individual vesicles are iniluenced by interactions with the cytoskeleton. The capacities of synapsins both to affect the state of actin networks and to control vesicle interaction with these networks could be highly relevant [ 30*]. The puzzle as to how the flows of vesicle membrane to and from the terminal might be controlled by mi-

Fig.2. The upper panel shows a fluorescence micrograph of a frog neuromuscular junction after stimulation of synaptic transmission in the presence of the fluorescent dye FMl-43. Betz and colleagues [2Bgo,291 propose that the bright spots correspond to clusters of synaptic vesicles aligned with postsynaptic membrane regions rich in acetylcholine receptors. Vesicles evidently acquire the dye during the endocytic phase of their cycle and then rapidly rejoin the clusters. The lower panel shows the neuromuscular junction after subsequent restimulation has depleted the dye from the clusters, presumably as a result of release of the dye during the exocytic phase of neurotransmission. Reproduced with permission from W Betz.

crotubule ‘motor’ proteins has been highlighted by the report that cell fractions enriched in synaptic vesicles contain cytoplasmic dynein, the presumed retrograde motor [ 311. If dynein proves to be present on many of the synaptic vesicles, then why do they not move retrogradely along the axon, as some of the larger bodies thought to participate in vesicle turnover [1,6,32] probably do? Perhaps the answer lies in geometric factors (e.g. access to microtubules) along with the influences of molecules that regulate dynein [ 331, Current models for regulation of the exocytic/endocytic vesicle cycle argue for participation of diverse proteins. Included are vesicle membrane proteins that interact with the cytoskeleton or the plasma membrane, such as synaptotagmin, which has properties suggesting it could mediate a Ca2 + -responsive association of vesicles with plasma membrane locales at which transmitters are released [34*,35]. (But see also [36] for evidence that synaptotagmin may not always be required for the functioning

609

610

Neuronal and glial cell biology

of synaptic vesicle related secretory organelles.) Other regulatoty proteins, such as the synaptic vesicle associated GTF-binding protein rab3A [37*] and the protein kinase p+m [38], may cyclically bind to and dissociate from the vesicle membrane. Work on the sb&ire mutation in Drrs@ika points towards participation of the protein dynamin in the endocytic phase of the cycle [39,401. recent insight Into molecular orchestration is the observation that rab3A may undergo polyisoprenylation, a modification that could help promote association with membranes [41]. How release of rab3A from the vesicles occurs is not yet known. (It would be simpler if release did not require severing rab3A’s links to the poly isoprenyls during repeated cycles.) Another interesting observation is that synaptic vesicle membranes contain some proteins similar to those found in the membranes of other rapidly cycling membrane systems, such as vesicles that transport glucose transporters between the cell interior and the plasma membrane of the adipocyte [42].

A

Other facets of membrane

traffic

local synthesis

synthesis of membrane constituents in neuronal processes merits renewed exploration, especially in view of the evidence that neurons deliver different mRNAs to different intracellular zones. Dendrites commonly contain ribosomes and endoplasmic reticulum (ER), and although the complexities of synaptosomal preparations always give pause for thought, membrane proteins tenta tively identified as postsynaptic plasma membrane components are among those synthesized by synaptosomes [43-l. Axon hillocks and distal axons of some neurons also have ribosomes and some rough ER, but little protein synthesis is seen in axons from most vertebrate preparations. Still, the occurrence of specific mRNAs coding for neurosecretoty materials in axons [44,45*1 suggests that it would be wise to suspend judgement as to whether local synthesis ever makes significant contributions of proteins to axonal membrane-delimited compartments such as the axonal ER. This is even more the case for lipids. New membrane lipids in neurons behave quite differently from new membrane proteins (for references, see [l]), as is emphasized by the report that axons of cultured rat neurons can incorporate choline and ethanolamine into phospholipids [46-l. The local

The neuronal endoplasmic

Endocytosis in cell-cell

signaling and surface

reorganization

Microscopists working on nervous tissue occasionally encounter endocytic vesicles containing formed elements, such as membranes seemingly internalized from adjacent cell surfaces. Such configurations could reflect little-studied biologically important uses of endocytosis [ 1 I. They are prominent, for example, in cultured Aplysia neurons at times when the cells are endocytosing cell adhesion molecules during reorganization of cell surfaces and intercellular relations [53-l. Perhaps similar configurations mediate processes such as the endocytic entry of specific plasma membrane proteins from one cell into endosomes or other compartments of a neighboring cell. Such transfer has recently been found to accompany inductive interactions between adjacent developing Drosophila retinal neurons [54-l. Perspectives Researchers have progressed toward delineating roles of synaptic vesicle proteins (see [55]) and also seem to be on the verge of a markedly better understanding of how endosome-like compartments contribute to the life histories of neuronal membranes. Comparable progress is badly needed on detailing the functional relations among the ER, Golgi apparatus, and the poorly understood sets of smooth surfaced sacs, tubules and vesicles that mediate much of axonal transport. Acknowledgements in my laboratoty is supported by NIH Betz, P De Camille, P Greengard, WB Huttner, Parton, K Simons, F Valtorta, B Wiedenmann, L Zipursky provided useful reprints, preprints, information.

Work

grant NE1 03168. W R Jahn, R Kelly, RG H Zimmermann and manuscripts or other

reticulum

it extends throughout much of the neuron, the ER offers potential routes for transport of diverse materials, including axonal membrane constituents [ 1,7]. Partly because axons contain several types of membrane-bound compartments that are easy to confuse with one an other, little is known of such transport, except that it has been reported not to be based on the rapid movement of large segments of the reticulum [47], and that elongate axonal compartments maintain close associations with the cytoskeleton (see [48] >. Evidence continues to ac-

As

cumulate that local differentiation of the ER, typified by the extensive smooth regions in axons, extends to the differential distribution of components of the neuron’s intracellular Caz+ -storage system among different ER regions and derivatives [49,50]. In the case of the sarcoplasmic reticulum, one such component, calsequestrin, may pass from the Golgi apparatus to the ER 151,521. If this proves true in neurons as well, questions will arise about routes and mechanisms of sorting, targeting and transport. The answers could resolve prevailing uncertainties about the relations among the ER, the Golgi apparatus and axonal compartments [ 1,7].

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