Cell, Vol.

62, 63-72,

July 13, 1990, Copyright

0 1990 by Cell Press

Polarized Sorting of Viral Glycoproteins to the Axon and Dendrites of Hippocampal Neurons in Culture
Carlos G. Dotti and Kai Simons Cell Biology Program European Molecular Biology Laboratory Postfach 10.2209 Heidelberg, D-6900 Federal Republic of Germany ing have demonstrated that two different scenarios are used. In Madin-Darby canine kidney (MDCK) cells, segregation of newly synthesized glycoproteins takes place in the trans-Golgi network (TGN) (Griffiths and Simons, 1986; Hughson et al., 1988). Apical and basolateral proteins are sorted into two different vesicular carriers, one for the apical surface and another for the basolateral one (Bennett et al., 1988). Sorting in the apical direction is specific, and little missorting of basolateral proteins is observed. Apical proteins, on the other hand, may be delivered in the wrong direction to the basolateral side, and this has led to the postulation of the basolateral route as a default pathway, analogous to what has been proposed for the constitutive biosynthetic pathway from the TGN to the plasma membrane in fibroblasts (Pfeffer and Rothman, 1987; Wandinger-Ness and Simons, 1990). In hepatocytes, sorting is accomplished in a different way (Bartles et al., 1987). In these epithelial cells, segregation of the surface glycoproteins is delayed, taking place at the plasma membrane. Both newly synthesized apical and basolateral proteins seem to travel together from the Golgi complex to the basolateral domain. Sorting occurs after endocytosis into endosomes. The apical proteins are delivered by vesicular carriers across the cell by transcytosis to the apical side. To explore whether surface protein sorting in neurons follows any of the epithelial models, we have used in this study viral glycoproteins as tools to investigate protein transport to the neuronal cell surface. Several enveloped RNA viruses turn cells into virus factories after infection. Host cell protein synthesis is inhibited; instead, virus proteins are translated to assemble new virus particles. The surface glycoproteins of Semliki Forest virus, vesicular stomatitis virus (VSV), and influenza virus are assembled in the endoplasmic reticulum (ER), transported to the Golgi complex, and delivered from the TGN to the cell surface to direct budding of the nucleocapsid from the plasma membrane (Simons and Warren, 1984; DuboisDalcq et al., 1984). These viral glycoproteins have been used as probes to study biosynthetic protein traffic to the cell surface in both cultured fibroblasts and epithelial cells. In MDCK cells, the VSV glycoprotein is basolateral, whereas the influenza hemagglutinin (HA) is apical (Rodriguez-Boulan and Pendergast, 1980). In neurobiology, enveloped viruses have been mainly used as transneuronal tracers (Kuypers and Ugolini, 1990) and as tools for neuropathological analysis (Dubois-Dalcq et al., 1982). The question that we have asked in this study is whether neuronal cells in culture infected with VSV or influenza virus distribute the virus glycoproteins in a polarized fashion. For this purpose, we have used rat hippocampal neurons in culture. These cells undergo a well-defined sequence of events from a nonpolarized to a polarized stage (Dotti et al., 1988). After initially extending lamellipodia (developmental stage l), the cells send out short neurites (stage 2). After a period of 6-24 hr, the cells start to become morphologically polarized. One of the cellular pro-

Summary Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types. Introduction A neuronal cell has two different types of neurites: a single axon and several dendrites. The axon typically transmits signals, whereas the dendrites generally receive them. To perform these different functions, axons and dendrites have to contain different membrane proteins (G. A. Banker and J. E. Mazurkiewicz, Sot. Neurosci. abstract, 1982; Angelides et al., 1988; Jones et al., 1989). How the neuron targets proteins to their specific dendritic and axonal locations is an interesting neurobiological problem that so far has attracted little attention (see Kelly, 1988; Black and Baas, 1989). Neurons are not the only cells that polarize their cell surfaces and have to target surface proteins to different plasma membrane domains. Epithelial cells are also polarized into distinct apical and basolateral cell surface domains (Simons and Fuller, 1985; RodriguezBoulan and Nelson, 1989). These two membrane domains are segregated by tight junctions that function as a fence to block lateral diffusion of lipids (of the exoplasmic leaflet of the bilayer) and proteins between the apical and basolateral sides of the plasma membrane (Gumbiner, 1987). Although surface polarities in neurons and epithelia have little in common functionally and morphologically, the molecular mechanisms of surface protein sorting may share common features. Studies on epithelial protein sort-

Cdl 64

cesses begins to grow rapidly, and when it has grown 10 pm longer than any of the others (Goslin and Banker, 1989), this process is destined to become the final axon (stage 3). A few days later, the remaining neurites also begin to grow to form the dendrites (stage 4). By lo-14 days in culture the axon and the dendrites have acquired all the functional and molecular properties that characterize a mature neuron (stage 5) (reviewed in Banker and Waxman, 1988). Synaptogenesis starts when dendrites develop, and during stage 5 extensive synapse formation has occurred. Many of the axons run along the length of the dendrites, forming numerous synapses en passant (Rothman and Cowan, 1981; Barlett and Banker, 1984b). Furthermore, morphological, functional, and immunocytochemical criteria have been defined to differentiate the axons from the dendrites (G. A. Banker and J. E. Mazurkiewicz, Sot. Neurosci. abstract, 1982; Caceres et al., 1984,1986; Barlett and Banker, 1964a, 1984b; Shaw et al., 1985; Dotti et al., 1987; Goslin et al., 1988; Banker and Waxman, 1968; Baas et al., 1988). In this study, we infected the hippocampal neurons at stages 3 and 5 with VSV and with influenza virus and studied the distribution of the virus glycoproteins by immunofluorescence microscopy. At stage 3, when the axon had been extended, the VSV and the influenza glycoproteins were delivered to the entire cell surface, including the short neurites and the axon. After synaptogenesis had occurred, the VSV glycoprotein was exclusively routed to the dendrite and the cell body, but no axonal delivery was observed. In contrast, the influenza HA was sorted preferentially, although not exclusively, to the axon. These findings pave the way for the use of viral glycoproteins as probes for studying polarized sorting in neurons. Results Newly Synthesized VSV Glycoprotein Is Delivered Exclusively to the Dendritic Domain In the first series of experiments, we analyzed the intracellular distribution of the VSV glycoprotein in cultured hippocampal neurons (stage 5) infected with the temperature-sensitive mutant VSV ts045. In cells infected with this virus the transport of the VSV glycoprotein to the cell surface is critically dependent on temperature (Bergmann et al., 1981; Bergmann and Singer, 1983; Griffiths et al., 1985). At the nonpermissive temperature of 39OC, viral infection occurs, but the VSV glycoprotein is arrested in the ER. Changing the temperature to 20C permits the transport of the glycoprotein to the Golgi complex where it accumulates in the TGN. Synchronous transport from the Golgi to the cell surface occurs after switching to the permissive temperature of 32C. When VSV t&45-infected hippocampal neurons were fixed in methanol after 3.5 hr at 39OC and 90 min at 20C and labeled with the anti-VSV glycoprotein antiserum, positive immunoreactivity was limited to the perinuclear region (Figures lA-1C). The labeling had a reticular appearance, resembling the staining of the Golgi complex obtained with wheat germ agglutinin (data not shown). This pattern of staining was observed in 95% of all neu-

rons. In approximately 20% of the infected cells, labeled Golgi-like elements extended beyond the perinuclear region into one of the cells dendrites (data not shown). This finding supports previous data suggesting the presence of Golgi elements in dendrites (De Camilli et al., 1986). Axons and dendrites were distinguished following morphological and immunological criteria. Morphologically, axons are very long, thin, and untapering. Dendrites are short, typically no longer than 100-200 pm, thick, and tapering (Rothman and Cowan, 1981; Barlett and Banker, 1984b). Immunologically, dendrites but not axons react positively with anti-microtubule-associated protein 2 (antiMAP2) antibodies (Caceres et al., 1984, 1986). After 3.5 hr at 39C and 90 min at 20C using the anti-MAP2 antibodies in the VSV ts045-infected cells, neither the thin and untapering, MAPSnegative processes (axons) nor the more distal segments of the MAP2 positively labeled processes (dendrites) appeared stained. We next analyzed the intracellular distribution of the VSV glycoprotein at different times after releasing the cells from the 20C block (Figures lD-1F). After 1 hr at the permissive temperature of 32OC, VSV glycoprotein was still seen in the cell body, both intracellularly and at the cell surface (see below). In some neuronal processes, VSV glycoprotein appeared in the form of tiny dots. This dotty pattern appeared along the entire length of the neurites. Not all of the numerous processes evident in the phasecontrast micrographs (Figure 1D) showed VSV glycoprotein labeling (Figure 1F). By double labeling the infected cells with an anti-MAP2 antiserum, the VSV glycoproteinlabeled neurites could be identified as dendrites (compare labeled processes in Figures 1E and 1F). Quantitation of how many MAPP-positive processes had VSV glycoprotein immunoreactivity revealed that all the dendrites of infected cells were positive. Axons were usually devoid of VSV glycoprotein immunoreactivity (compare Figure 1D [phase-contrast] with Figure 1F). Of 615 VSV glycoprotein-positive processes, only 29 were MAP2 negative. The absence of axonal labeling could not be attributed to a difference in the thickness of the neurites because numerous thick processes (bundles of axons) were negative. We also ruled out that the lack of axonal labeling was due to a delay in the transport of newly synthesized VSV glycoprotein to this territory, by fixing cells at prolonged times postinfection. Even after 7 hr at permissive temperature, axonal labeling could not be detected (data not shown). In our next series of experiments we analyzed the insertion into the plasma membrane of the newly synthesized VSV glycoprotein (Figure 2). For this purpose two different protocols were used. Infected cells were incubated in the presence of anti-VSV glycoprotein antibodies at 4OC for 20 min and then paraformaldehyde fixed. Alternatively, infected cells were fixed directly in 4% paraformaldehyde and 0.25% glutaraldehyde followed by incubation with primary antibodies. With both methods similar results were obtained. Surface labeling was in the form of small clusters of immunoreactivity (Figures 2B-2C). The labeling was restricted to the cell body and dendrites, as judged by MAP2-positive labeling (Figure 2A). Most (91%) of the

Sorting 65

of Viral Glycoproteins

to Axon

and Dendrites

Figure 1. Intracellular Distribution of the VSV NO45 Glycoprotein in Infected Mature (Stage 5) Hippocampal Neurons Cells were fixed in cold methanol after a 1 hr infection at 32% followed by 2.5 hr at 39% and then incubated for 90 min at 20% (A-C) or after an additional 60 min at the permissive temperature of 32% (D-F). Axons (arrows) and dendrites (arrowheads) are differentiated by their morphology, phase-contrast micrographs (A and D), and MAP2 immunoreactivity (only dendrites are positive) (B and E). Incubation with the anti-VSV glycoprotein antiserum revealed accumulation of G protein in the ceil body at the end of the 20% block (C) and in the cell body and dendrites at the end of the 32% incubation (F). Axons are devoid of labeling. Bar: 15 urn.

Figure 2. Distribution of the VSV ts045 Glycoprotein in Infected Mature (Stage 5) Hippocampal Neurons (A-C) After 1 hr at permissive temperature (conditions as in Figures lD-1F) cells were fixed in 4% paraformaldehyde-0.25% glutaraldehyde and incubated with the anti-VSV glycoprotein antiserum. Anti-MAP2 antiserum was added after permeabilization with Triton X-100. Dendrites and cell body are labeled with the anti-MAP2 antibodies. VSV glycoprotein is exclusively present on the surface of all the MAPPlabeled processes in the form of small dots (arrowheads) (B). The punctate staining is seen also on the cell body surface of the same cell 63 (D-F) Double labeling with anti-VSV glycoprotein and anti-synaptophysin antibodies of ts045-infected cells. Comparison of the antiVSV glycoprotein (E) and anti-synaptophysin (F) antibody labeling shows partial colocalization of both proteins (arrows). Arrowheads in (E) show expression of VSV glycoprotein on the dendritic and cell body surface at sites not labeled with synaptophysin. Bar: 10 urn.

Cdl 66

Figure 3. Intracellular Protein of FPV-Infected

Distribution of the Stage 5 Neurons

HA

Cells were fixed in cold methanol 4 hr (A-C) or 5 hr (D-F) after infection, incubated with an anti-HA antiserum, and processed for immunofluoreescence. An anti-MAP2 antiserum was used to distinguish axons from dendrites (B and E). Four hours postinfection the HA protein appears restricted to the cell body (C). Five hours postinfection all axons show intense HA labeling in the form of small dots and varicosities (arrowheads). Dendrites d3, d4, d5, and d6 are clearly devoid of anti-HA labeling. Some labeling appears restricted to the initial segment in dendrites d2 and dl (arrows). Bar: 12 pm.

dendrites were positive for VSV glycoprotein. VSV glycoprotein insertion at the plasma membrane was detected at 30-60 min after the temperature shift to 3PC. Axons were devoid of anti-VSV glycoprotein immunoreactivity. Our finding of VSV glycoprotein clustering on the dendritic surface is different from previous findings of uniform VSV glycoprotein distribution on the surface of VSV t&45+fected fibroblasts and ATt20 cells (Bergmann et al., 1983; Rivas and Moore, 1989). The nonneuronal cells (mostly astrocytes) present in the hippocampal neuronal culture also showed uniform surface appearance of the VSV glycoprotein (data not shown). The observed clustering of VSV glycoprotein on the dendritic surface and the lack of axonal surface staining did not change with longer infection times. Since dendrites have specialized sites where synaptic contacts occur, we next investigated whether VSV glycoprotein clustering occurred at those sites. Infected cells were double labeled with anti-synaptophysin, a synaptic vesicle marker, and anti-VSV glycoprotein antibodies (Figures 2D-2F). Synaptophysin is an integral membrane protein of synaptic vesicles (Jahn et al., 1985; Wiedenmann and Franke, 1985). In cultured hippocampal neurons it was demonstrated to accumulate in presynaptic terminals (T. Fletcher, l? Cameron, l? De Camilli, and G. Banker, submitted), thus constituting an excellent marker for synaptic contacts. When neurons, surface stained for the VSV

glycoprotein, were incubated with anti-synaptophysin antibodies after permeabilization with Triton X-100, clusters of VSV glycoprotein colocalizing with synaptophysin were observed (Figures 2E and 2F, arrows). However, synaptophysin and the VSV glycoprotein did not completely overlap (Figures 2E and 2F, arrowheads). Newly Synthesized Fowl Plague Vlrus HA Is Dalivemd Predominantly, but Not Exclusively, to the Axonal Domain In filter-grown MDCK cells, VSV glycoprotein is transported exclusively to the basolateral domain, whereas in MDCK cells infected with influenza virus, the major glycoprotein of the virus, HA, is delivered directly to the apical surface (Rodriguez-Boulan and Pendergast, 1980; Matlin and Simons, 1984). Because of the dendritic delivery of VSV glycoprotein, which could be comparable to the basolateral sorting in epithelial cells, we analyzed whether HA was sorted to the axon. In the next series of experiments, we infected mature hippocampal neurons (stage 5) with a wild-type strain of the avian influenza fowl plague virus (FPV) (Figure 3). When FW-infected neurons were fixed in methanol at 4 hr after infection, immunofluorescence microscopy showed that 46% of the cells had positive immunoreactivity with anti-HA antibodies. In these cells, HA labeling was evident in the neuronal cell body (Figures 3A-3C).

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of Viral Glycoproteins

to Axon

and Dendrites

Figure 4. Surface tein of FPV-Infected Infection

Distribution of the HA ProStage 5 Neurons 5 Hr after

(A-C) Infected cells were incubated with antiHA antiserum 30 min at 4% fixed with paraformaldehyde. and processed for immunofluorescence. Dendrites (arrowheads) and axons (arrows) are distinguished by phase-contrast microscopy (A) and MAP2 immunoreactivity (6). Axons are the only processes labeled with the anti-HA antibodies (C). (D-E) The infected cells were paraformaldehyde-glutaraldehyde fixed before anti-HA labeling. Axons, thin and untapering, are labeled for HA (arrows in [El). None of the thick, tapering processes evident by phase-contrast mi-

The appearance of labeling was similar to that found in VSV ts045-infected cells fixed after the 20C block (Figure 1C). After 5 hr of infection, an increase in the cell body labeling was accompanied by the appearance of punctate HA immunoreactivity in some of the neurites (Figures 3D-3F). The cell in this figure is representative of the vast majority of FPV-infected neurons. Axons (MAP2 negative, ax, arrowheads) showed intense HA labeling (Figure 3F). The staining was present in dots and varicosities along the entire length of the axons. HA labeling of dendrites was variable. In some dendrites HA was clearly absent (d3, d4, d5, d6). Other dendrites showed some HA labeling, restricted usually to the initial segment (dl, d2). This could be derived from Golgi elements. HA labeling can also be seen further into the dendrites in dl and d2. One interpretation of this labeling is that the staining was not in dendrites but was derived from axons running in close proximity, parallel to the dendritic surface. This type of axo-dendritic organization is commonly observed in this culture (Rothman and Cowan, 1981; Goslin et al., 1988). Observation of the dendrite dl (Figure 3F) tends to support this view. The intense labeling of the proximal dendrite becomes punctiform and follows the trajectory of the axon that loops at the end of the dendrite (the axonal looping is evident in the corresponding phase-contrast micrograph). Fixation of cells at later times postinfection did not show any significant change of the described pattern: neither a higher number of dendrites became labeled nor was there an increase in axonal labeling. Quantitation

showed that of 815 HA-positive processes, 77% were axons while 33% were classified as dendrites. The insertion of FW HA into the axonal membrane was investigated next. Cells were either incubated with antiHA antiserum at 4% and then fixed or fixed directly in paraformaldehyde-glutaraldehyde. With the first method, HA labeling appeared in the form of small dots on the axonal surface (Figures 4A-4C). However, in the latter case, when lateral mobility was blocked by fixation, the HA appeared distributed uniformly (Figures 4D and 4E). In either case the labeling was restricted to axons, leaving most of the dendrites unlabeled. The cell bodies were usually, but not always, labeled with the anti-HA antibodies. Only 10.7% of the processes positive for HA were dendrites. In those cells in which dendrites were positive for surface HA, the labeling was only in one or two dendrites and usually restricted to the region close to the cell body (less than 10 mm away). In the cell in Figures 4A-4C, the absence of dendritic labeling is unequivocal. Only axons (MAP2 negative, small arrowheads) appeared stained. The characteristic uniform labeling of axons obtained after aldehyde fixation is also clear on the right side of the same figure (Figures 4D and 4E). Only the very thin and untapering processes running along dendrites and cell body are intensely labeled. Delivery of Viral Glycoproteins during the Establishment of Neuronal Polarity Hippocampal cells establish morphological polarity early

Figure 5. lmmunofluorescence Intracellular VSV Glycoprotein Hippocampal Cells at Stages velopment

Localization of and FPV HA in 2 and 3 of De-

VSV ts045-infected cells were methanol fixed after 60 min at permissive temperature (as in Figures ID-IF). FPWnfected cells were fixed at 5 hr postinfection. VSV glycoprotein appears in all the minor processes of stage 2 cells (B). VSV glycoprotein is clearly present in the axon of stage 3 cells (C). HA protein was also found in all neurites of stage 2 cells (E) and in the axon and dendrites (d) of stage 3 cells (F). Bar: 10 urn.

after plating (Dotti et al., 1988). Already after 24 hr in cuiture an axon can be differentiated from a dendrite on a purely morphological basis. In the final series of experiments, we analyzed whether polarized delivery of the newly synthesized viral glycoproteins also occurred in cells at the early stages of polarization (stage 3). Typical stage 3 cells are shown in Figures 5C and 5F They have only one long neurite (axon) and several short processes (dendrites). Infection of this type of cell with VSV tsO45 or with FPV and subsequent immunofluorescence localization of the intracellular viral proteins revealed no specific delivery to axon and dendrites. Positive HA or VSV glycoprotein staining in the form of fine dots was observed in the single axon and the multiple minor processes. Infected stage 2 cells, which are not morphologically polarized, also showed uniform delivery of VSV glycoprotein and HA into all the cellular processes (Figures 58 and 5E). Discussion One important consequence of this study is that an approach that has proved useful in studies of cell surface glycoprotein sorting in cultured fibroblasts and epithelial cells can now also be used to investigate polarized glycoprotein transport in neurons. A prerequisite for this work was the availability of a neuronal cell culture in which the cells polarize into axons and dendrites. Previous studies using viruses to infect neurons or cells with neuron-like properties have not been able to address the question of surface glycoprotein polarity because the cultures have not been as stably polarized and as well differentiated as the mature hippocampal neurons (Dubois-Dalcq et al., 1982; Rivas and Moore, 1989; Tooze et al., 1989). We observed that when immature hippocampal neurons were infected at the stage when the axon had grown out, delivery of the virus glycoproteins was not yet polarized. When mature neurons were infected, however, with VSV or with influenza virus, the distribution of the viral glycoproteins became polarized. The VSV glycoprotein was delivered to dendrites, whereas the influenza HA was mainly routed to the axon. Where is the sorting of the viral glycoproteins taking

place? We do not have conclusive evidence on this point, but our results suggest that the sorting occurs in the Golgi complex. For the VSV glycoprotein, the data are more compelling than for the influenza HA. When the neurons are infected with VSV ts845 at the nonpermissive temperature and then shifted to 20%, the temperature-sensitive VSV glycoproteins move from the ER to the Golgi complex in the cell body, presumably accumulating in the TGN (Griffiths et al., 1985). After a shift to 32X, the glycoproteins move to the dendrites and the perikaryon cell surface. At no time are the VSV glycoproteins seen in the axon. For the influenza HA, accumulation in the Golgi complex was also observed, followed by preferential delivery to the axon, but there was also some transport into dendrites. Previous results for newly synthesized axonal proteins also showed that they pass through the Golgi complex in the cell body before moving on by fast axonal transport (Hammerschlag et al., 1982; Vale et al., 1988). For endogeneous dendritic glycoproteins, less work has been done. There has been some evidence that glycoproteins might be synthesized locally in dendrites as well as in the cell body, followed by delivery to the cell surface (Kreutzberg et al., 1973; Steward et al., 1988). However, more work is clearly required to substantiate whether some dendritic glycoproteins could bypass the Golgi complex. Our data on VSV and influenza glycoprotein sorting in neurons suggest a vesicular sorting mechanism similar to what has been observed previously in MDCK cells. After sorting in the TGN in MDCK cells, the influenza HA is released into apical carrier vesicles, whereas the VSV glycoproteins are transported in vesicular carriers to the basolateral side (Bennett et al., 1988). The hepatocyte model (see Introduction) for glycoprotein transport seems unlikely for hippocampal neurons. If the influenza HA were first delivered to the somato-dendritic domain and then routed by a transcellular route to the axon, we should have been able to follow the HA from the dendritic side to the axon in the highly polarized neurons. Our data suggest that sorting occurred before surface delivery. However, this question will have to be analyzed further by detailed studies of temperature-sensitive mutants of the influenza HA.

;;rting

of Viral Glycoproteins

to Axon

and Dendrites

The small amount of influenza HA delivered to dendrites could be due to several causes. It may represent missorting either by inclusion of HA into dendritic carrier vesicles or by delivery of axonal carrier vesicles to the wrong side (Pfeiffer et al., 1985). If the dendritic pathway were a default pathway, then this dendritic missorting would be similar to what has been postulated for the basolateral pathway in MDCK cells. Alternatively, it is also possible that viral infection leads to cytopathic effects in some cells that influence sorting fidelity (Fuller et al., 1984). After sorting into axonal and dendritic carrier vesicles in the TGN has occurred, these have to be delivered into the axon and the dendrites. For the axonal vesicular intermediates, it is likely that kinesin-mediated fast axonal transport along microtubules is the mode of delivery (Vale et al., 1985, 1986). We observed a punctate pattern of intracellular influenza HA staining throughout the length of axon, presumably representing the vesicular carriers moving in the anterograde direction. After 4 hr of infection, the HA was still in the Golgi region of the cell body, whereas after an additional 30-60 min, HA had been transported all the way to the end of the axon. Fast axonal transport would easily fill the entire axon with the required speed (Sheetz et al., 1989). Dendritic delivery of VSV glycoproteins may also occur by a microtubule-mediated interaction. The dendritic microtubules are of opposite polarity, about 50% having their plus ends in the cell body and the other half having them in the dendritic shafts (Baas et al., 1988). The dendritic carrier vesicles could use a MAP-1C dynein-like motor (Paschal and Vallee, 1987) to move from the cell body (plus ends) toward the microtubule minus ends in the dendrites, as suggested by Black and Baas (1989) for organelle translocation. This mechanism would prevent dendritic vesicles from entering the axonal traffic. The axonal vesicles could either lack the capacity to bind to the dendritic microtubules with their minus ends in the cell body, or bind and move into the dendrites but then move back to the cell body using the population of microtubules of opposite polarity. These are possibilities that require further study. There was a striking difference in the surface immunofluorescence after glutaraldehyde and paraformaldehyde fixation of the influenza HA and of the VSV glycoprotein. The HA was diffusely spread over the entire axolemmal surface, whereas the surface VSV glycoprotein staining was patched on the cell body and on the dendrites. These patches often corresponded to sites where synaptophysin staining was observed. Synaptophysin is known to accumulate in synaptic vesicles in presynaptic bulbs along the axon (Jahn et al., 1985; Wiedenmann and Franke, 1985; Tixier-Vidal et al., 1988). The VSV glycoprotein might patch after surface delivery in or close to dendritic spines (Dubois-Dalcq et al., 1982) by interactions with cytoskeletal elements (Siman et al., 1985; Lazarides and Nelson, 1985; Jones et al., 1989). The influenza HA seems to be freely diffusing along the axolemmal surface because anti-HA antiserum patched the surface HA if added before fixation. One important question is how the segregation of the vi-

ral glycoproteins in the plasma membrane plane is maintained. If the HA were freely mobile then it should move from the axon (and the cell body) into dendrites by lateral diffusion. Conversely, the VSV glycoprotein could move from the cell body into the axon. For the VSV glycoprotein, segregation could be maintained by anchoring to proteins underlying the plasma membrane. However, we have evidence that VSV progeny are released by budding into the extracellular medium (C. G. D. and K. S., unpublished data). This would imply that at least some of the VSV glycoproteins must be mobile to form the patch for interaction with the VSV nucleocapsid to drive the budding process. Is it possible that there is a fence blocking lateral diffusion in the plasma membrane-for instance, in the axon hillock? The axon hillock is located at the boundary between the axon and the somato-dendritic domain, and it is known to have a distinctive membrane organization (Matsumoto and Rosenbluth, 1985; Angelides et al., 1988). In epithelial cells the tight junction marks the boundary between the apical and the basolateral plasma membrane domains and acts as a fence for both lipid and protein diffusion. In addition, interactions with ankyrin and fodrin are known to organize basolateral proteins (e.g., the anion exchanger and the [Na+,K+] ATPase) in subdomains on the basolateral surface (Drenckhahn et al., 1985; Nelson and Veshnock, 1987; Nelson and Hammerton, 1989). Similar mechanisms are known to anchor sodium channels and glutamate receptors in neurons (Siman et al., 1985; Srinivasan et al., 1988). The final question that has to be answered by further experiments is when the sorting in the TGN becomes polarized during neuronal development. Previous work on GAP43, the fatty acylated growth cone protein (Skene, 1989) has suggested that already at stage 3, when morphological polarity has developed, preferential delivery of GAP43 can be observed to the axon growth cone (Goslin et al., 1990). Also, synaptophysin seems to become polarized at this stage (T. Fletcher, l? Cameron, l? De Camilli, and G. Banker, submitted). These results suggest that all the cellular components necessary for polarized sorting are present in immature neurons and that assembly of the sorting and the delivery mechanisms starts already during axon outgrowth. However, it is not yet clear whether these findings only reflect a massive delivery of protein into a rapidly growing axon (as is the case with the delivery of newly synthesized glycoproteins to the leading edge of a motile fibroblast; Bergmann et al., 1983) or whether polarized sorting in the TGN is indeed taking place. Our own results show that although there are differences among stage 3 neurons, no significant polarity in the delivery of VSV glycoproteins and HA could be observed. At this stage, cell polarity can still be reversed. When the axon of a stage 3 neuron is amputated, anyone of its remaining neurites, which would otherwise become dendrites, have the capacity to differentiate into a new axon (Dotti and Banker, 1987; Goslin and Banker, 1989). Stabilization of neuronal polarity takes place later. If axon outgrowth only requires massive membrane traffic directed into one of the neurites but no polarized sorting, then the differentiation of the immature neuron could require gene

activation and synthesis of new proteins necessary for transforming the TGN from a state making only one type of carrier vesicle to the cell surface (cf. fibroblast) to another in which two types of vesicular intermediates are made (cf. MDCK cells). Alternatively, the carrier vesicles and/or the microtubules could be transformed such that axonal and dendritic segregation becomes possible by differential microtubule movement. This gene activation could either be triggered by synaptogenesis or precede it. Before it is possible to test unequivocally whether either of these two models is correct, more has to be learned about the molecular machinery responsible for generating and maintaining neuronal polarity. One very promising prospect is that the analysis of epithelial glycoprotein sorting might also provide clues to neuronal sorting mechanisms.
Experimental Procedures

cell Cultum Hippocampal cells were prepared as described by Barlett and Banker (1984a). Briefly, the hippocampi of 17day-old rat embryos were microscopically dissected, trypsinized (0.25% for 15 min). and further dissociated by repeated passages through a constricted Pasteur pipette. Cells were plated onto polylysine-coated coverslips in dishes containing MEM supplemented with 10% fetal calf serum and allowed to attach to the substratum for 30 min. The neuronal coverslips were then transferred to culture dishes containing a monolayer of astrocytes that had been kept in serum-free medium (MEM supplemented with the N2 mixture of Bottestein and Sato [lQ7Q]) for at least 24 hr. This medium will be referred to as neuronal medium. The coverslips were next put with the neurons upside down, in close proximity toward the glial monolayer. This coculture, neuron-glie, was previously shown to improve the long-term survival of the neurons (Banker, 1980). Cells were maintained in a humidified incubator at 3pC and 5% COP for lo-14 days before virus infections were performed. For some experiments cells were kept in culture for only 24-48 hr before virus infection (stage 2 and stage 3 neurons). InfectIon wlth V!W tW45 and FPV Stocks of FPV and of VSV tsO45 were obtained as described in Matlin and Simons (1984) and Griffiths et al. (1985) respectively. Coverslips with neurons were removed from the culture dishes, rinsed in Hanks balanced salt solution (HBSS), and infected with 80-100 pfu per cell in neuronal medium. The temperature of infection differed depending on which virus was used: 32% (permissive temperature) was the infection temperature for the VSV mutant tsO45, and 3pC for FPV After 1 hr of infection in a 5% CO? atmosphere the coverslips were rinsed in HBSS and returned to fresh neuronal medium for different times and conditions depending on the virus. VSV ts045-infected neurons were further incubated in a 5% COP incubator at the nonpermissive temperature of 39% for 2.5 hr. Cells were then transferred to dishes containing neuronal medium at 2ooC for 90 min. The temperature was maintained constant by using a temperature-controlled water bath. The pH of the medium was kept constant by having the neuronal medium modified with HEPES (10 mM) and low NaHCOs (4 mM). Finally, the coverslips were transferred to dishes containing medium at the permissive temperature of 32oC and kept in a 5% COs atmosphere for various times. Coverslips with FPVinfected neurons were briefly rinsed in HESS and transferred to fresh neuronal medium at 3pC in a 5% COs incubator. Infection was carried out for different times before fixation and processing for immunofluorescence. Immunofluomecence To label intracellular spike VSV glycoproteins or FPV HA, coverslips were briefly rinsed in phosphate-buffered saline (PBS) and fixed in -20C methanol for 6 min. After fixation, coverslips were allowed to air dry and then were rehydrated with PBS. Unspecific primary antibody binding was prevented by a 30 min incubation in PBS containing

10% horse serum. The cells were then incubated in the presence of primary antibodies for either single or double immunofluorescence. The following primary antibodies were used: rabbit anti-VSV glycoprotein (Matlin et al., 1982) rabbit anti-FPV HA (Matlin et al., 1981) mouse monoclonal anti-MAP2 (gift from Dr. Lester Binder), and mouse monoclonal anti-synaptophysin (gift from Dr. Wiedenmann). Incubation with the primary antibodies was for 1 hr at room temperature. The reaction was stopped by three 5 min rinses in PBS followed by a 30 min incubation with the corresponding secondary antibodies (FITC-conjugated goat anti-rabbit IgG and RlTCconjugated rat anti-mouse IgG, Dianova Gmbh, FRG), previously immunopurified on nitmcetlulose paper. To label viral glycoproteins on the neuronal surface, infected cells were fixed in 4% paraformaldehyde-0.25% glutaraldehyde followed by quenching of the aldehydes with sodium borohydride. After extensive washing the coverslips were incubated with anti-VSV glycoprotein or anti-HA antibodies in PBS without detergent. This incubation was followed by incubation with an FITC-conjugated goat anti-rabbit IgG antiserum. Cells were then permeabilized with Triton X-100 (O.l%, 10 min) and further incubated with anti-MAP2 or anti-synaptophysin antibodies followed by RITC-conjugated rat anti-mouse IgG antibodies, This fixation procedure only labeled the VSV glycoprotein on the cell surface. When the anti-MAP2 and anti-VSV glycoprotein antibodies were added together, no cytoskeleton labeling was seen. A second way to label surface viral glycoproteins was to incubate the infected cells with the anti-viral spike glycoprotein antibodies at 4OC, which is known to block endocytosis (von Bonsdorff et al., 1985). Cells were then fixed in 4% paraformaldehyde and processed for immunofluorescence as before. lmmunofluorescence analysis was performed in a fluorescence microscope (Axiophot, Zeiss, FRG). Photographs were taken using high sensitivity film (TMax 3200, Kodak). Acknowledgments We thank Hilkka Virta for providing virus stocks, Dr. Lester Binder for giving us the anti-MAP2 antiserum, and Dr. Bertram Wiedenmann for the anti-synaptophysin antiserum. We are also grateful to Drs. Gary Banker, Jan De Mey, and Wieland Huttner for critical reading of the manuscript. Dr. Carlos G. Dotti is the recipient of an Alexander von Humboldt Stiftung Fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 USC. Section 1734 solely to indicate this fact. Received References Angelides, K. J., Elmer, L. W., Loftus, D.. and Elson, E. (1988). Distribution and lateral mobility of voltage-dependent sodium channels in neurons J. Cell Biol. 106, 19111925. Baas, I? W., Deitch, J. S., Black, M. M., and Banker, G. A. (198F1). Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc. Natl. Acad. Sci. USA 85, 8335-8339. Banker, G. (1980). pocampal neurons Trophic interactions in culture. Science between glial cells 209, 809-810. and hip February 20, 1990; revised May 7, 1990.

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