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Correspondence to Micha E. Spira: spira{at}cc.huji.ac.il
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Earlier studies revealed that axotomy is associated with massive membrane retrieval along the plasma membrane of the cut axonal end. The retrieved membrane appears to serve as part of the resealing mechanism in a variety of neurons (Ziv and Spira, 1995; Ashery et al., 1996; Fishman and Bittner, 2003; Yoo et al., 2004; Nguyen et al., 2005). Whole cell patch-clamp membrane capacitance measurements revealed that the axotomy of cultured Aplysia californica neurons activates two processes in parallel: membrane retrieval and exocytosis. Surprisingly, it was demonstrated that axotomy-induced membrane retrieval quantitatively dominates exocytosis for >1 h after axotomy. Thus, although vigorous extension of a GC's lamellipodium was visualized, the total membrane surface area of the neuron decreased (Ashery et al., 1996). These observations implied that in order to permit effective extension of a lamellipodium after axotomy, the sites of membrane retrieval and exocytosis must be spatially separate.
Attempts to determine the site of membrane insertion and retrieval in neurons yielded conflicting results. For example, Popov et al. (1993) suggested that membrane is added along the neurites of cultured Xenopus laevis neurons. On the other hand, Dai and Sheetz (1995) and Zakharenko and Popov (1998) concluded that new membrane is added to the GC and that bulk membrane endocytosis occurs in the cell body. To the best of our knowledge, no information is available on the budgeting and spatial distribution of membrane resources or about retrieval and exocytosis in regenerating neurons after mechanical injury.
Using cultured A. californica neurons, we report novel mechanisms that rapidly subdivide the cut axonal end into two structurally and functionally distinct compartments. In one compartment, Golgi-derived anterogradely transported vesicles accumulate and fuse with the plasma membrane in support of GC extension; in the other, retrieved plasma membrane is retained. We demonstrate that formation of the two compartments is generated by reorientation of the MT polarities at the cut axonal end. Our observations suggest that formation of the MT-based vesicle traps optimizes the rapid transformation of an axon into a motile GC after axotomy by sorting and concentrating different membrane resources to restricted sites on the cut axon.
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As soon as MT polarity is reorganized after axotomy (Fig. 2, A1–A8), RH237 fluorescence begins to accumulate within the plus end trap and, to a lesser extent and more slowly, along the minus end trap (Fig. 2, B1–B8 and C1–C8; and Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1). To establish that the accumulated RH237 signal represents the accumulation of vesicles, we compared the distribution of RH237 fluorescence with the ultrastructural composition of the axon at various times after axotomy (n > 10) and confirmed that the RH237 fluorescence signal localizes at regions in which the vesicles and tubular structures concentrate (Sahly et al., 2006).
Sorting of anterogradely transported Golgi-derived vesicles from retrogradely transported pinocytotic vesicles
Labeling of subcellular organelles by RH237 revealed the presence of two distinct zones at which vesicles accumulate (the GCOC and the DZ). However, as RH237 labels lipids in general, it did not allow differentiation between anterogradely and retrogradely transported vesicles. Therefore, we specifically labeled anterogradely transported vesicles using intracellular injections of mRNA encoding superecliptic synaptopHluorin (Sankaranarayanan et al., 2000), enhanced YFP (EYFP)–SNAP-25 (25-kD synaptosome-associated protein; Oyler et al., 1989; Kimura et al., 2003), or cherry (Shaner et al., 2004) –SNAP-25 4–6 h before axotomy and imaging. Retrogradely transported pinocytotic vesicles were labeled by bath application of the fluid-phase pinocytotic maker sulforhodamine 101 (SR101; Teng et al., 1999). In a series of control experiments, we established that (1) the injection of mRNA encoding enhanced GFP, EYFP, or cherry leads to an evenly distributed fluorescent signal that does not concentrate in any of the traps after axonal transection. (2) Vesicles labeled by synaptopHluorins, EYFP–SNAP-25, or cherry–SNAP-25 4–6 h before axotomy are almost exclusively transported anterogradely (Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1), and vesicles labeled by SR101 are transported retrogradely (Video 6).
To illustrate the differential accumulation of anterogradely and retrogradely transported vesicles into the plus and minus end traps, we show simultaneous imaging of the anterogradely transported EYFP–SNAP-25 (Fig. 3 B), the retrogradely transported SR101 (Fig. 3 C), and the distribution of RH237 (Fig. 3 A). For the experiment (n > 20), we first labeled the entire population of vesicles by 30-min incubation of the neuron in RH237; 20 h later, (i.e., 4 h before axotomy), the neuron was injected with mRNA encoding EYFP–SNAP-25. Subsequently, 2 h before transection, the neuron was exposed for 20 min to the pinocytotic marker SR101. After axotomy, under such conditions, anterogradely transported EYFP–SNAP-25–labeled vesicles specifically concentrate within the GCOC (Fig. 3 B), and the retrogradely transported pinocytotic vesicles labeled by SR101 were retained at the DZ (Fig. 3 C). The merged image of both labeling patterns closely matches that of the distribution of RH237 (Fig. 3, compare A with D). As a complementary approach, we also performed experiments in which we simultaneously imaged EB3-GFP and cherry–SNAP-25 or EB3-GFP and SR101. In these experiments, we also observed that anterogradely and retrogradely transported vesicles concentrate in the plus end trap (Fig. 4 B and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1) and minus end trap (Fig. 5 A and Video 9), respectively. Together, these observations demonstrate that the traps formed by MTs lead to the sorting and concentration of vesicles in accordance with their directional movement along MTs.
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For the experiments, the cell body together with a short axonal segment was mechanically removed, leaving a long, isolated axon in the culture (first axotomy; Fig. 4 A2). After various time intervals, which we believe allowed for depletion of the anterogradely transported components from the main axon, the isolated axon was transected again distally to the first transection (second axotomy; Fig. 4 A3). The restructuring of MTs and accumulation of anterogradely or retrogradely transported vesicles under these conditions were then imaged (Figs. 4 and 5, respectively). MTs were imaged by EB3-GFP, and anterogradely transported vesicles were imaged by cherry–SNAP-25 or synaptopHluorins (Figs. 4 and 6 and Videos 7 and 8, available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1). Retrogradely transported vesicles were imaged by SR101 (Fig. 5 and Videos 9 and 10). In a series of 34 experiments in which we first imaged the formation of vesicle traps after axotomy of an intact neuron (first axotomy) and then imaged cytoskeleton reorganization after axotomy of the isolated axon (second axotomy) at different time intervals, we found that in 27/34 experiments, the cut end of the isolated axons (second axotomy) did not form the vesicles traps. However, as indicated by EB3 labeling, the MTs underwent a cycle of depolymerization and repolymerization in response to the second axotomy (Fig. 4 C1, Fig. 5 B1, and Videos 8 and 10). In these experiments, anterogradely transported vesicles did not accumulate (Fig. 4, C2 and C3; and Video 8). These results show that anterogradely transported molecular components, which are driven from the cell body to the axon, participate in the formation of traps.
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To differentiate between the possibilities that Golgi-derived vesicles and tubules or anterogradely transported molecular motors and proteins underlie the trap formation, we disrupted the Golgi system by incubating the neurons for 12 h in 10 µg/ml brefeldin A (BFA; Chardin and McCormick, 1999). (It should be noted that in cultured A. californica neurons, 10 µg/ml BFA disrupts the Golgi apparatus within 30 min of application). Thereafter, neurons expressing EB3-GFP, EYFP–SNAP-25, or cherry–SNAP-25 were transected and imaged (n = 17). We found that both the plus and minus end traps are formed in the presence of BFA (Video 11, available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1), whereas SNAP-25–labeled anterogradely transported vesicles are not detected along the axon and do not accumulate in the traps (see Fig. 7 and Video 11). These observations suggest that formation of the traps does not require the arrival of Golgi-derived anterogradely transported vesicles but requires other anterogradely transported molecular signals.
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-imido]triphosphate– AMP-PNP) as a kinesin inhibitor (Kapoor and Mitchison, 1999; Bananis et al., 2000) and found that in addition to blocking the axoplasmic transport and inhibition of vesicle trap formation after axotomy, it increased the free intracellular calcium levels (as imaged with fura-2) and subsequently led to axonal degeneration. Microinjection of antikinesin antibody (monoclonal mouse antibovine brain kinesin heavy chain; Chemicon) into the neurons did not inhibit anterograde transport and had no effect on formation of the traps.
GC extension after axotomy depends on the accumulation of Golgi-derived vesicles in the GCOC
Subdivision of the cut axonal end into a spatially and functionally distinct plus end trap, which concentrates Golgi-derived membrane resources from the PZ and DZ that continuously retrieve plasma membrane, may enable the extension of a GC lamellipodium on a background of dominating membrane retrieval (Ashery et al., 1996). To evaluate the validity of this hypothesis, we examined (1) whether the vesicles within the GCOC fuse with the plasma membrane and (2) whether the prevention of anterogradely transported vesicle accumulation at the GCOC is sufficient to prevent the extension of a GC's lamellipodium.
The fusion of vesicles that are anterogradely transported and accumulate in the GCOC with the plasma membrane to form the nascent GC was confirmed by injection of mRNA encoding superecliptic synaptopHluorin (Miesenbock et al., 1998) into the cell body 2–5 h before axotomy. The neuron was then axotomized and formed a GCOC. Imaging of synaptopHlourin-labeled vesicles by excitation wavelength of 405 nm revealed the accumulation of vesicles within the GCOC (Fig. 6, A2, B2, and C2). To examine whether these vesicles fuse with the plasma membrane, we imaged the synaptopHluorin-labeled structures by excitation wavelength of 488 nm. This excitation wavelength exclusively activates synaptopHluorins in neutral pH environments (i.e., facing the culture medium). These images revealed that most of the fluorescent signal is localized to the plasma membrane around the GCOC, and part of it is detected within the GCOC cytoplasm (Fig. 6, A1, B1, and C1). These signals are generated by vesicles that fused with the plasma membrane and face the neutral pH of the medium and organelle population with intermediate internal pHs. Confirmation of these conclusions is provided by the experiment in Fig. 6. After control imaging of the GCOC (Fig. 6 A), an acidic culturing solution (pH 5.4) was pressure ejected onto the GCOC. This resulted in an immediate and reversible disappearance of most fluorescent signals generated by excitation at 488 nm from the GCOC's plasma membrane (Fig. 6, compare the A1 control with B1 and note the recovery in C1). These observations clearly demonstrate that anterogradely transported vesicles that concentrate within the GCOC fuse with the surrounding plasma membrane.
To examine whether Golgi-derived vesicles rather than retrieved vesicles are necessary for the promotion of axotomy- induced growth, the supply of anterogradely transported Golgi-derived vesicles was interrupted by bath application of 10 µg/ml BFA. We established that in the presence of BFA, GCs continue to extend for several hours (for as long as vesicles that were processed before BFA application were available; unpublished data). Thus, BFA does not inhibit the fusion of vesicles with the GC plasma membrane in a direct manner (n = 5). We next examined whether the depletion of Golgi-derived vesicles is sufficient to prevent GC formation after axotomy by first incubating the neurons for 12–15 h in 10 µg/ml BFA. Next, EYFP–SNAP-25 mRNA was injected into the cell body, and, 4 h later, the axon was transected. As indicated by the presence of diffuse EYFP fluorescent signal in the axon, we conclude that the injected mRNA was translated (Fig. 7 B). Nevertheless, the fluorescent signal did not concentrate in the GCOC (Fig. 7 B), and a GC lamellipodium did not extend (Fig. 7 A). SR101 imaging revealed that membrane retrieval proceeds normally in the presence of BFA (unpublished data) and that retrogradely transported vesicles are retained by the minus end trap (Fig. 7, C and D), but the formation of a GC's lamellipodium is inhibited. Because of this, we conclude that the accumulation of Golgi-derived vesicles within the plus end trap is essential for promoting the rapid regenerative pattern after axotomy.
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Our results support the hypothesis that reorientation of MT polarity to form the vesicle traps depends on the supply of anterogradely transported molecular components from the cell body to the site of injury. Thus, when an axon is isolated for >.5 h from the cell body, axotomy is not followed by formation of the plus and minus end traps. Rather, the MTs repolymerize with their plus ends pointing toward the tip of the cut axon (Figs. 4 C and 5 B and Videos 8 and 10). Because it is well established that isolated A. californica axons in culture maintain their normal morphology, generate action potentials, and even release neurotransmitters for >24 h (Benbassat and Spira, 1993, 1994; Martin et al., 1997; Oren et al., 1997; Schacher and Wu, 2002), it is reasonable to assume that the aforementioned observations, which were conducted within minutes to 4.5 h after axotomy, are not affected by a general rundown of the axon. We suggest that isolation of the axon from the cell body leads to the depletion of anterogradely transported molecular components that take part in formation of the vesicle traps.
Motor proteins as candidates for vesicle trap formation and maintenance
Previous studies suggested that the formation of polarized MT assemblies can be induced by a variety of cellular structures and molecules, such as membrane compartments, vesicles, cortical and cytoskeletal elements, Rho GTPases, receptors, and molecular motors (Gundersen et al., 2004; Akhmanova and Hoogenraad, 2005; Wu et al., 2006). Radial MT arrays are generally formed by the centrosome, which can induce the outgrowth of MTs with their plus ends directed outwards (Akhmanova and Hoogenraad, 2005). However, much evidence suggests that the centrosome-independent mechanism of MT organization is driven by molecular motors that can substitute for it (Malikov et al., 2005). In fish melanophores, the MT self-organization depends on the minus end–directed motor dynein and occurs through a combination of MT-based granule transport and MT nucleation on the pigment granules (Vorobjev et al., 2001). It was suggested that the dynein molecular complexes interact with more than one MT, and, while propagating toward the minus ends along two MTs, the dynein complex forces the MT plus ends to orient away from the motor (Vorobjev et al., 2001; Cytrynbaum et al., 2004; Malikov et al., 2005). Assuming that dynein remains attached to the MT when it reaches the minus end, the final orientations of the MTs are stabilized such that all minus ends are attached to the dynein complex and all plus ends point away from it. Similar arguments were considered regarding kinesin motors (Serbus et al., 2005).
We found that the inhibition of retrogradely transported SR101-labeled vesicles by EHNA does not interfere with trap formation (Fig. S1). Although this observation cannot rule out with certainty that dyneins are involved in trap generation, the results indicate that formation of the plus and minus end vesicle traps depends on the supply of anterogradely transported molecules from the cell body to the cut axonal end. Because BFA treatment, which disrupts the Golgi apparatus and thus blocks the supply of Golgi-derived vesicles, did not block MT trap formation, we concluded that the restructuring of MT polarity at the cut axonal end is generated by anterogradely transported signals such as the molecular motors themselves or other protein signals (Vorobjev et al., 2001; Cytrynbaum et al., 2004; Serbus et al., 2005).
The results presented in this study suggest but do not prove that plus end–directed molecular motors serve to power the formation of the plus end trap by their accumulation at the severed tips of MTs (Fig. 8, C–E). This process may also contribute indirectly to formation of the minus end trap. This trap is bordered on one side by the minus ends of the MTs that point their plus ends toward the center of the plus end trap (Fig. 8). On the other side of the minus end trap, the MTs point their plus ends toward the plasma membrane. Similar reactions have been demonstrated in both yeast and mammalian cells in which MT stabilization is caused by the interaction of a MT's plus end and proteins localized at the plasma membrane (Allan and Nathke, 2001; Akhmanova and Hoogenraad, 2005). We hypothesize that with time, the trap structures become more robust as the polarity of the MTs direct more proteins and vesicles into the trap, and, as a consequence, more MTs assemble to fit the pattern. Thus, the self-assembled trap structure is reinforced, and the structural and functional subdivision of the cut end into the plus and minus end traps becomes more robust.
The functional role of the vesicle traps and its general implications
An earlier study from our laboratory revealed that the axotomy of cultured A. californica neurons activates membrane retrieval and exocytosis in parallel. Membrane capacitance measurements revealed that the processes of membrane internalization quantitatively dominate exocytosis. Nevertheless, the neuron vigorously extends a GC's lamellipodium (Ashery et al., 1996). This apparent discrepancy is reconciled by assuming spatial dissociation between the site at which Golgi-derived vesicles accumulate and are being inserted into the plasma membrane and the sites from which the membrane is retrieved. It appears that formation of the two vesicle traps isolates the site of growth (GCOC) from the distal compartment that was exposed to very high calcium levels and initially undergoes massive membrane retrieval (Ashery et al., 1996; Fishman and Bittner, 2003). It also provides an efficient cellular mechanism to optimize the accumulation of growth-supporting vesicles and, thereby, facilitates localized growth processes.
It should be noted that earlier studies demonstrated that local disruption of MTs at the middle of an axonal segment either by nocodazole application (Zakharenko and Popov, 1998) or trypsin microinjection (Ziv and Spira, 1998) results in the insertion of transported membranes into the axonal membrane and the initiation of local growth. These observations could be interpreted to support the assumption that vesicles accumulate at the tips of disrupted MTs simply because they cannot move efficiently past that point. However, it is interesting to note that in a series of preliminary experiments (unpublished data), we found (using EB3-GFP) that trypsin microinjection, which leads to the generation of nascent GC extension, is preceded by the formation of a plus end vesicle trap within the middle of the axon. In light of the present findings, it would be interesting to examine with the right molecular tools whether the formation of MT-based vesicle traps serves similar functions in other neurons. It should be noted that the basic phenomenon and mechanisms that operate in generating vesicle traps in transected A. californica neurons have been previously described to serve a variety of functions in other cells (for review see Cytrynbaum et al., 2004).
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Pharmacological reagents
RH237 (N-(4-sulfutyl)-4-(6-(p-dibutylamynophenyl) hexatrenyl)) pyridinum and inner salt (a gift from R. Hildeshiem, Weizmann Institute of Science, Rehovot, Israel; Grinvald et al., 1982) was diluted in ethanol to a concentration of 10 mM and further diluted before use in artificial sea water to a concentration of 10 µM. SR101 (Kodak) was prepared as a stock solution of 10 mM in double-distilled water and further diluted before use in artificial sea water to a final concentration of 40 µM. BFA (Sigma-Aldrich; Chardin and McCormick, 1999) was prepared as a stock solution of 5 mg/ml in methanol and was further diluted to a final concentration of 10 µg/ml in the experimental bathing solution. Retrograde transport of SR101-labeled vesicles was inhibited by bath application of EHNA and HCl (Calbiochem). For the experiments, EHNA was diluted in DMSO to a 1-M stock solution and was further diluted before use to final concentration of 2–3 mM in artificial sea water.
Cell culture
Neurons B1 and B2 from buccal ganglia of A. californica were isolated and maintained in culture as previously described (Schacher and Proshansky, 1983; Spira et al., 1993, 1996). In this study, we refer to these neurons collectively as B neurons. In brief, 1–10 g of juvenile A. californica supplied from the University of Miami's National Resource for A. californica was anesthetized by injecting 380 mM of isotonic MgCl2 solution into the animal's body cavity. Buccal ganglia were dissected and incubated in ms L-15 containing 1% protease (type IX; Sigma-Aldrich) at 34°C for 1.5–2.5 h. After the protease treatment, the ganglia were pinned and desheathed. The neurons were manually pulled out along with their original axon with the aid of a sharp glass microelectrode. The neurons were immediately plated in glass-bottom dishes coated with poly-L-lysine (Sigma-Aldrich) containing culture medium. All experiments were performed 24–48 h after plating at room temperature (21–25°C) after replacing the culture medium with artificial sea water.
Axotomy
Axonal transection was performed by applying pressure on the axon with the thin shaft of a micropipette under visual control as previously described (Spira et al., 1993, 1996, 2003; Ziv and Spira, 1993).
mRNA preparation and injection
mRNAs were in vitro transcribed using the recombinant transcription system as described previously (Sahly et al., 2003). In brief, human protein plus EB3 was prepared as EB3-GFP (Stepanova et al., 2003), and A. californica SNAP-25 (provided by W.S. Sossin, Montreal University, Montreal, Canada) was prepared as EYFP–SNAP-25 or cherry–SNAP-25 (cherry was provided by R.Y. Tsien, University of California, San Diego, La Jolla, CA). These and superecliptic synaptopHluorin (provided by J.E. Rothman, Memorial Sloan-Kettering Cancer Center, New York, NY) were cloned in pCS2+ expression vector. 10 µg of those plasmids were linearized with NotI and purified using a DNA cleanup system (Promega). 1–3 µg of linearized DNA was transcribed using a RiboMax-sp6 kit (Promega). A typical reaction contains 8 µl of transcription buffer, 8 µl rNTP mix containing 25 mM CTP, ATP, UTP, and 12 mM GTP, 4 µl of 15 mM Cap analogue (Roche), 1 µl rRnasin (Promega), and 4 µl of enzyme mix. A final volume of 40 µl was incubated for 2–4 h at 37°C. RNA was purified by using an RNeasy Mini Kit (QIAGEN), and the clean RNA was eluted to a final volume of 25–40 µl and kept at –80°C until use.
The transcribed mRNAs were pressure injected into the cytoplasm of the cultured neurons 4–24 h after plating. In preparing the injections, 3 µl mRNA solution (0.5–5 µg/µl) was diluted in 0.5 µl KCl (0.5 M). We estimated the injected volume to be 
10% of the cell's body volume. Throughout the injection, the input resistance and transmembrane potential of the neuron were recorded by the injection micropipette. At the end of the injection, the micropipette was removed from the cell.
Microscope imaging
Two confocal imaging systems were used: the Radiance 2000/AGR-3 imaging system (Bio-Rad Laboratories) was mounted on an IX70 microscope (Olympus) with a plan-Apo 60X 1.4 NA oil objective (Olympus), and the D-Eclipse C1 imaging system (Nikon) was mounted on an Eclipse TE-2000 microscope (Nikon) with a plan-Apo 60X 1.4 NA oil objective (Nikon). SynaptopHluorin imaging was performed on the Nikon set. The protein was excited at 405 (blue diode laser) and 488 nm (argon laser). The emitted light was collected at 500–530 nm. Images were collected and processed using EZ-C1 software (Nikon). All other imaging was performed using the Bio-Rad Laboratories system. The images were collected and processed using LaserSharp and LaserPix software (Bio-Rad Laboratories), respectively. For simultaneous imaging of GFP fusion proteins and RH237, both chromophores were excited by 488 nm, and the emitted lights were collected at 500–530 nm for GFP and above 660 nm for RH237. For simultaneous imaging of GFP or EYFP fusion proteins and SR101 or cherry fusion proteins, the excitation wavelengths were 488 and 543 nm (green HeNe laser). The emission filter for GFP and EYFP was HQ 500–530 nm, and for SR101 and cherry, the filter was HQ 555–625 nm. Triple imaging of GFP- or EYFP-labeled proteins, SR101, and RH237 was collected by excitation wavelengths of 488 and 543 nm, and the emissions filters were HQ 500–530 nm for GFP or EYFP, HQ 560–580 nm for SR101, and HQ 660LP for RH237. The argon laser excitation intensity was usually lowered to 5–10%. The pinhole was set to 1.6–2.5 mm. Figures were prepared using Photoshop and FreeHand software (both from Adobe).
Online supplemental material
Fig. S1 shows that the effective inhibition of SR101-labeled vesicle retrograde transport by EHNA does not inhibit formation of the plus end trap. Videos 1–3 show the formation of MT-based vesicle traps after axotomy. Videos 4 and 5 show the anterograde transport of EYFP–SNAP-25–labeled vesicles and their accumulation after axotomy. Video 6 shows the retrograde transport of SR101-labeled vesicles. Videos 7 and 8 show that the formation of a plus end trap depends on the anterograde transport of components from the cell body to the axon. Videos 9 and 10 show that the formation of MT-based plus and minus end traps cannot be correlated with retrogradely transported retrieved membrane. Video 11 shows that the formation of MT-based plus and minus end traps does not depend on the supply of Golgi-derived anterogradely transported vesicles. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200607098/DC1.
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Acknowledgments |
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This study was supported by grants from the USA-Israeli Binational Science Research Foundation (grants 2000354 and 2003152). Parts of the work were performed at the Charles E. Smith Family and Prof. Elkes Laboratory for Collaborative Research in Psychobiology. M.E. Spira is the Levi DeViali Professor in Neurobiology.
Submitted: 19 July 2006
Accepted: 6 January 2007
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