Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth
© Hines et al; licensee BioMed Central Ltd. 2012
Received: 16 December 2011
Accepted: 30 January 2012
Published: 30 January 2012
During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.
To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.
This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.
During the construction of neural circuits, growing axons of developing neurons extend long distances en route to the appropriate synaptic targets. New membrane and materials are added to extending axons and increase the plasma membrane surface area by 10 to 1,000-fold . A typical 1-μm-diameter axon extends at the rate of 0.5 mm per day, necessitating the insertion of new membrane at the rate of 1.1 μm2 per minute. The body of evidence indicates that new membrane is incorporated primarily at the motile tip of the axon, the nerve growth cone [2–5]. Cytoplasmic vesicles, derived in the neuronal cell body, undergo anterograde transport along the length of the axon and are added by local exocytosis within the growth cone [6–9].
In addition to a role in axon extension, numerous studies have demonstrated that regulated membrane trafficking is intricately involved in growth cone chemotaxis. Axon pathfinding toward the synaptic target requires dynamic interactions with the extracellular matrix (ECM) and the detection of spatial guidance cues within the local environment. The ability of growth cones to adapt to a wide range of guidance cue concentrations may involve regulated vesicle trafficking . Moreover, attractive growth cone turning toward a locally applied gradient of nerve growth factor requires asymmetric membrane insertion at the attractant side, or leading edge . In contrast, repulsive growth cone turning requires endocytic pathways and correlates with asymmetric endocytosis [12–14]. These findings have led to the notion that the balance of exocytic and endocytic activities across the growth cone serves to control local membrane protrusion versus membrane removal and drives bidirectional axon guidance . Further support for this idea comes from the finding that growth cone collapse is associated with regulated membrane retrieval [16, 17].
During axon extension, the growth cone surface membrane is also retrieved at rates sufficient to turn over completely within 30 minutes . At first glance, these energetically demanding processes appear to counteract the substantial membrane addition that must occur in order to drive axon extension. Numerous reports indicate that local membrane retrieval and recycling play important roles in axonal growth [19–24]. The precise regulators of these endocytic routes are incompletely understood, but likely include actin, cholesterol, Pincher, phosphoinositide 3-kinase (PI3K) and Rac1 . Despite these functional insights, the mechanisms by which local membrane retrieval and recycling facilitate nerve growth and guidance are relatively unknown . In chemotaxing cells, mounting evidence indicates that regulated vesicle trafficking, both endocytic and exocytic, is critical for directed migration [26–29]. The emerging view from multiple studies suggests that polarized endocytosis and exocytic recycling can spatially polarize receptor signaling, cytoskeletal regulators, and focal adhesion turnover in order to drive cell motility [30, 31].
How endocytic processes regulate membrane dynamics and remodel the growth cone surface membrane to support nerve growth remain outstanding issues that await further characterization of the membrane retrieval processes in the growth cone. Previous ultrastructural studies have provided high-resolution snapshots, revealing coated and noncoated vesicles, membrane-contiguous vacuoles, elongated tubules and stacks of lumenless membrane disks with largely unknown functions [32–36]. However, the identification and discrimination of parallel structures in live growth cones by fluorescence and DIC microscopy has been unyielding [18, 35, 37–40]. This may reflect the temporal limitations of pulse-chase endocytic assays, which effectively track the fate of endosomes minutes after internalization but fail to capture early events. Here, we have overcome these limitations by local and transient application of lipophilic membrane dyes to single growth cones, combined with high-speed confocal microscopy, in order to monitor the initial formation of single nascent endocytic vesicles. This approach provides high spatiotemporal resolution and has revealed individual modes of rapid endocytic membrane retrieval with distinct spatial distribution and temporal dynamics in actively extending Xenopus spinal neuron growth cones. We discovered hot-spots of concentrated single vesicle membrane retrieval events in the growth cone that are actin-dependent, and internalization by much larger endocytic tubules. Furthermore, we have uncovered evidence for unexpectedly rapid recycling of endocytic compartments, where nascent vesicles and tubules disappear within seconds of forming. Finally, we provide evidence that substrate-stimulated outgrowth enhances the rate of bulk endocytosis in the growth cone and requires the function of Rho GTPases. This set of findings and the optical imaging approach may serve as an important foundation for future studies aimed at elucidating the molecular regulators, specific cargo and functional significance of distinct membrane retrieval and recycling pathways in axonal growth and guidance.
Detection of single-vesicle retrieval events
A similar focal labeling approach applied fluorescent dextran, which is taken up with fluid into the lumen of endocytic vesicles during membrane retrieval (Figure 1B). After the dextran labeling, a second micropipette delivered buffered saline to wash away uninternalized dextran. This attenuated the background fluorescence significantly while leaving the internalized signal unaffected. The consequent increased signal to noise ratio permitted the detection of nascent endocytic vesicles and tubules by time-lapse confocal imaging. When co-applied from the same micropipette, both FM 5-95 and fluorescent dextran revealed numerous nascent vesicles in the growth cone following the brief application period (Figure 1C, D). These findings highlight the rapid kinetics of membrane retrieval in the growth cone by single endocytic vesicles.
Hot-spots of rapid membrane retrieval in the growth cone
To determine the spatial and temporal dynamics of membrane retrieval during growth cone migration, we used high-resolution confocal microscopy and collected images at 1 Hz in real time during the focal endocytic assay after FM dye labeling. Remarkably, we discovered that single vesicle membrane retrieval events often clustered at hot-spots, defined as focal regions of the growth cone where multiple vesicles formed in near synchrony. The frequency of vesicle formation concentrated within endocytic hot-spots greatly exceeded that seen in surrounding regions of the growth cone surface membrane. By carefully observing endocytic events in multiple growth cones, we were able to identify discrete endocytic modes based on the spatial distribution and temporal dynamics (Figures 2, 3, 4, 5, 6, 7, 8). The nascent endocytic vesicles associated with hot-spots were small (< 0.5 μm diameter). Temporally, the initiation of hot-spots was stochastic. Furthermore, single endocytic vesicles formed at hot-spots during only a short time period lasting up to several seconds following initiation, after which endocytic activity abruptly terminated. In many instances, the induction of endocytic hot-spots correlated with regions of self-contact or occurred at regions undergoing rapid membrane remodeling.
Membrane retrieval at dorsal ridges and lamellipodial margins
Membrane retrieval at sites of cell-cell contact
Membrane retrieval by tubules and vacuoles
Might FM dye-labeled vacuoles represent distinct endocytic compartments within the growth cone cytoplasm or are they contiguous with the plasma membrane? By performing the sequential FM dye membrane labeling assay, we found that in all instances (12/12), vacuoles immediately labeled by the initial FM 2-10 pulse also incorporated dye upon a subsequent FM 5-95 pulse (Figure 10C). In contrast, small endocytic vesicles labeled by the initial dye pulse were rarely labeled by the second dye pulse. These findings illuminate an important distinction between small endocytic vesicles and vacuolar structures. Small vesicles, which can incorporate both lipophilic and fluid-phase markers and exclude subsequent dye pulses, are rapidly internalized endocytic compartments. In contrast, vacuoles appear to be contiguous with the plasma membrane for extended durations that can be 10s of seconds to minutes. Although membrane-contiguous vacuoles may eventually undergo endocytosis, it is likely that the dye-labeled vacuoles seen by our membrane labeling assay rarely represent internalized compartments.
Rapid recycling of internalized membrane
Rates of membrane retrieval in central and peripheral regions of the growth cone
Positive correlation between the rate of endocytosis and axon outgrowth
To further probe the relationship between axonal growth rate and membrane retrieval, we next asked whether inhibiting Rho GTPases, which attenuates axon outgrowth, could affect the endocytic rate in the growth cone. Treatment of spinal neuron cultures with Toxin B, a general inhibitor of Rho GTPases , caused a modest but significant decrease in the rate of axon outgrowth (Figure 13C). Higher concentrations had a dramatic effect on substrate attachment and neurite formation that precluded their use (data not shown). Importantly, fluid-phase endocytosis was also inhibited significantly by Toxin B treatment (Figure 13D). Collectively, these findings provide further support for a positive correlation between the rate of axonal growth and membrane retrieval in the growth cone.
Comparison of spatial endocytic modes: potential regulators and functions
The use of focal endocytic assays in this study has revealed the spatial dynamics of endocytic vesicle formation in actively extending growth cones for the first time. Using this approach, we have discovered several distinct modes of rapid membrane retrieval based on the spatial and temporal characteristics of vesicle formation, as summarized in Figure 12. Collectively, endocytic hot-spots elicited by self-contact between peripheral processes were the most frequent means of membrane retrieval. These include contacts among adjacent filopodia, lamellipodia, or between a filopodium and nearby lamellipodium. The disappearance of filopodia upon contact with the growth cone body or with other filopodia correlated with hot-spots of endocytic membrane retrieval. We found that vesicle formation at self-contact sites was sensitive to cytochalasin D treatment, implicating a role for F-actin polymerization in this process. Although the precise nature of these membrane remodeling events awaits further investigation, these observations suggest that membrane fusion and endocytic retrieval may represent additional mechanisms for removing filopodia and reshaping growth cone morphology. Furthermore, it is possible that these processes may allow spatially distant environmental information, at the tips of filopodia, to be rapidly transmitted to the growth cone body. For instance, receptor activation and second messenger signals in the filopodia may be relayed to the growth cone, cooperating with previously reported means for spatial transmission of second messenger signals [52, 53].
Small-vesicle hot-spots at membrane ridges were the second most frequently observed endocytic route. This includes vesicle formation at dorsal ridges, as well as ridges that form at the lateral margins of lamellipodia as the growth cone changes shape. These regions undergo considerable membrane remodeling and are very transient in nature, forming and disappearing within seconds. It is likely that actin dynamics and membrane curvature are integrally involved in ridge formation, ruffling and vesicle formation. The actin regulator Rac1 and the WAVE complex have been closely linked with forms of macropinocytosis [16, 45, 54]. Furthermore, BAR-domain proteins, which facilitate membrane curvature, also participate in membrane ruffling and macropinocytosis . Endocytic hot-spots within membrane ridges at the dorsal surface and lateral margins of the growth cone may be regulated by these same effectors.
We observed an abundance of large stationary vacuoles in the growth cone central domain. Most vacuoles incorporated FM dye immediately upon surface membrane labeling, suggesting that these structures were pre-existing and continuous with the plasma membrane at the time of labeling. This conclusion was further supported by our sequential FM dye labeling experiments, which showed that the same vacuolar structures could be labeled by dual dye pulses separated by 40 seconds. It is likely that the vacuoles seen in this study are synonymous to reverse shadow-cast vacuoles previously observed by correlative DIC and electron microscopy, which often contained an orifice that contacted the plasma membrane . By tracking vacuoles with DIC video microscopy, Dailey and Bridgman also found that vacuoles in the growth cone central domain were much more stable (10- to 20-minute lifetime) than endocytic compartments that formed in the peripheral domain.
Finally, we also found evidence for internalization and rapid recycling of elongated tubules in both the central and peripheral regions of the growth cone. Although tubule formation was relatively infrequent, the size of these compartments (up to 12 μm in length) suggests that tubules account for the majority of bulk membrane retrieval by surface area. This process may be similar to the high-capacity membrane retrieval system described in non-neuronal cells, which is regulated by Cdc42 and GRAF1 and may facilitate adhesion receptor recycling [56–58]. The focal endocytic assays used in the present study provided direct observation of endocytic tubules within seconds after internalization. This rapid detection was critical, because peripheral tubules typically fused with other compartments or were recycled within one to two minutes. In all instances, nascent peripheral tubules were processively transported toward the central domain of the growth cone at rates near 10 μm/minute. This is comparable to the reported rates for actin retrograde flow, measured at 5 to 6 μm/minute in cultured neurons isolated from other species [59–61]. In contrast, central tubules and vacuoles were most often stationary, but the infrequent transport of these structures proceeded at more rapid rates consistent with microtubule-based axonal transport [62, 63].
Use of FM dyes to monitor endocytosis in the growth cone
The use of FM dyes to monitor vesicle dynamics in neurons was pioneered by Betz and colleagues in the 1990s and has since been utilized by numerous studies that have greatly advanced our understanding of synaptic function . Membrane labeling with FM dyes has also been used in the growth cone in order to track the fate of endocytic compartments [11, 18, 19, 37]. In this study, we have optimized an approach to rapidly image the initial formation and early trafficking of nascent endocytic structures locally within the growth cone. The ability of this technique to reveal single vesicle formation with high spatial and temporal resolution is likely due to three main attributes of this assay. First, the fluorescence emission of FM dyes is at least two orders of magnitude brighter when bound to the plasma membrane than in aqueous solution. Second, the brief focal dye pulse allows the free FM dye that remains non-bound to the surface membrane to be rapidly diluted into the surrounding buffered saline. Furthermore, FM 5-95 and FM 2-10 are slightly less lipophilic than the more commonly used FM 1-43 and FM 4-64, and consequently de-stain from the plasma membrane in a relatively quick manner , allowing the transient labeling and imaging of rapid membrane retrieval events. In our hands, FM 2-10 de-stained even more rapidly than FM 5-95.
The amphiphilic nature of FM dyes implies that they bind to the plasma membrane, are internalized by vesicular processes, and become trapped in nascent cytoplasmic vesicles as the dye is unable to cross the lipid bilayer. Recent studies have validated the ability of FM dyes to selectively label endocytic vesicles. First, fluorescence resonance energy transfer (FRET) studies detected no interaction between membrane-bound FM dyes and a cytoplasmic-GFP under physiological conditions . Furthermore, FM dyes microinjected into the cytoplasm of cells fail to label intracellular organelles and vesicles . Thus, the dye labeling emanating from intracellular membranes is unlikely to come from dye that was somehow able to traverse the plasma membrane.
Our use of the focal membrane labeling assay, combined with confocal microscopy, demonstrates that structures associated with both the dorsal and ventral (apical and basal) surface membranes can be labeled (Figure 10B). However, due to the close apposition of the ventral membrane with the underlying substrate, it is possible that dye labeling is non-uniform. Our own findings, combining the focal labeling assay with total internal reflection microscopy (TIRF), support this notion, as ventral membrane labeling lags slightly behind the dorsal surface (data not shown). Therefore, this assay may be inherently biased toward measuring membrane retrieval at the apical surface of the growth cone. This property, which could be a potential advantage or impediment depending on the assay, should be considered by investigators utilizing the focal membrane labeling assay in future studies.
Functions for high-capacity membrane retrieval and recycling systems
Taken together, the present findings provide further insight into rapid and high capacity membrane retrieval and recycling systems in the growth cone. Although the functions of these energetically demanding processes are yet to be understood, similar membrane recycling systems in non-neuronal cells appear to be driving factors for cell polarization. For example, in migrating fibroblasts, clathrin-independent carriers (CLICs) internalize the vast majority of membrane and extracellular fluid at the leading edge. This recently defined endocytic mechanism, previously considered macropinocytotic, is now recognized to enrich specific molecular cargo such as the adhesion proteins β1-integrin, Thy-1 and CD44, and is critical for optimal cell migration [56, 57]. Furthermore, clathrin-mediated endocytosis of specific cargo is also polarized to the front of migrating cells. For instance, the endocytic adaptor proteins Numb, Dab2, and ARH cooperatively facilitate endocytosis of integrin receptors, which need to be subsequently recycled in order to polarize focal adhesion turnover to the leading edge [68–71]. Similar vesicular processes can spatially localize cytoskeletal activity (Rac1, Cdc42) and receptor- and nonreceptor- tyrosine kinase signaling to the leading edge of migrating cells [27, 29, 72, 73].
In the growth cone, cytoskeletal protrusion, adhesion complex turnover, and tyrosine kinase signaling are all polarized to the leading edge [74, 75]. It is possible that one or more of the endocytic modes described in this study contribute to these processes in order to optimize axon extension. In an over-simplified model, membrane addition (exocytosis) would facilitate axon extension, whereas membrane retrieval (endocytosis) would attenuate extension by removing bulk membrane. However, this model is contradicted by recent findings that show the rate of endocytic membrane retrieval positively correlates with the dynamic remodeling of growth cone shape . The results of the present study further extend this notion by showing that substrate-stimulated outgrowth also correlates positively with increased endocytic membrane retrieval. Local modes of endocytosis may promote axon outgrowth by polarizing signaling, cytoskeletal dynamics or adhesion turnover to the leading edge. Further characterization of endocytic routes in the growth cone will enable the rigorous testing of these models in future studies.
In this study, we have utilized live-cell confocal microscopy and a transient membrane-labeling assay to reveal the spatiotemporal dynamics of rapid membrane retrieval and turnover in extending spinal neuron growth cones. This approach demonstrated that endocytic events are stochastic and occur at hot-spots initiated at sites of active membrane remodeling or self-contact between peripheral extensions of the growth cone, with unique spatial and temporal properties. The rate of these bulk endocytic processes correlates with the rate of axon outgrowth and requires the function of Rho-family GTPases, suggesting that one or more distinct endocytic modes has important roles in growth cone motility. Future characterization of the molecular regulators and functional cargo associated with these endocytic modes will uncover the functional contributions of these processes to growth cone motility and chemotactic guidance.
Primary neuron culture and immunofluorescence labeling
Spinal neuron cultures from stage 22 Xenopus laevis (Xenopus 1, Dexter, MI, USA) embryos of either sex were prepared by methods previously described [76, 77]. All experiments and animal housing were conducted according to National Institutes of Health (NIH, Bethesda, MD, USA) guidelines for animal care and safety, with the approval and under the auspices of the Mayo Clinic Institutional Animal Care and Use Committee. Unless indicated, spinal neuron cultures were grown on non-coated coverglass at room temperature (20 to 22°C) and experiments were performed 12 to 20 h after plating. Coating with poly-D-lysine (PDL, 0.5 mg/ml; Sigma, St. Louis, MO, USA) and fibronectin (FN, 20 μg/ml; Sigma) was performed in Dulbecco's phosphate-buffered saline (D-PBS) for one hour (37°C) followed by repeated washes in calcium- and magnesium-free PBS. Culture medium consisted of Leibovitz medium (87.1% vol/vol, GIBCO, Grand Island, NY, USA), fetal bovine serum (0.4% vol/vol, HyClone, Logan, UT, USA), and saline solution (12.5% vol/vol; 10 mM D-glucose, 5 mM sodium pyruvate, 1.26 mM calcium chloride (CaCl2), and 32 mM HEPES, pH 7.5). Cultured spinal neurons were chemically fixed (20 minutes; 2.5% formaldehyde, 0.01% glutaraldehyde), permeabilized with Triton X-100 (0.1%) and processed for immunofluorescence labeling as described [12, 78]. Microtubules were labeled with polyclonal anti-β-tubulin (0.4 μg/ml; Abcam, Cambridge, England, UK) and an Alexa488 conjugated secondary antibody (2 μg/ml; Invitrogen, Carlsbad, CA, USA). Filamentous actin was labeled with Alexa555-conjugated phalloidin (260 nM; Invitrogen).
Image acquisition and processing
We acquired digital time-lapse DIC images using a Zeiss (Jena, Germany) Axiocam CCD camera mounted on a Zeiss Axiovert 200 M inverted microscope (100 × oil immersion objective, 1.4 numerical aperture, 1.6 × optical zoom). For rapid time-lapse imaging of endocytic membrane retrieval, we used a Zeiss LSM 5LIVE confocal microscope equipped with a 63 × water immersion objective (1.2 numerical aperture, 2 × optical zoom). Individual frames were acquired at a rate of 100 ms per capture. We generated all representative movies using Image J software (NIH, LSM toolbox plugin) by exporting time-lapse stacks to a QuickTime format (MOV, MPEG4 compression, three frames per second) . Images of immunolabeled growth cones were captured on a Zeiss LSM 5LIVE confocal microscope using a 63 × oil immersion objective (1.4 numerical aperture, 1.6 × optical zoom).
Focal endocytic assays
All focal endocytic assays were performed in a serum-free modified Ringers (MR) solution (120 mM sodium chloride (NaCl), 2.2 mM potassium chloride (KCl), 2 mM CaCl2, 1 mM magnesium chloride (MgCl2), 5 mM HEPES, 2 mM sodium pyruvate; pH 7.6, 20 to 22°C). Spinal neuron cultures on glass-bottomed uncoated dishes were positioned over an inverted confocal microscope. Using a micromanipulator stabilized by a floatation table, we positioned a micropipette 100 μm in front of the leading edge of the growth cone in the direction of neurite extension. We fabricated micropipettes to an approximate 1-μm opening by heat-pulling capillary glass (1 mm OD, 0.58 mm ID, Warner Instruments, Hamden, CT, USA) with a micropipette puller (Flaming/Brown, Sutter Instruments, Novato, CA, USA; and PC-10, Narishige, East Meadow, NY, USA). A stock solution of FM 5-95 or FM 2-10 (10 mM in H20, Invitrogen) was diluted to 1 mM or 2 mM, respectively, in MR and 2 to 4 μL were loaded into each micropipette. A picospritzer (Picospritzer III, Parker Instrumentation, Huntsville, AL, USA) controlled focal dye application by applying four repetitive pulses (2 Hz, 400 ms pulse duration, 2.5 p.s.i.) immediately after the onset of confocal imaging. Cytochalasin D (30 nM; Sigma) was added 30 minutes before dye application and confocal imaging. For focal application of fluorescent dextran, the micropipette was loaded with fluorophore-conjugated dextran (Alexa488 or tetramethylrhodamine-labeled, 10,000 MW, neutral charge, Invitrogen; 1 mM in MR) and positioned 80 μm in front of the growth cone in the direction of neurite extension. A second micropipette containing MR was used to focally wash away uninternalized dextran. A picospritzer controlled both micropipettes by delivering 10 to 20 repetitive pulses of fluorescent dextran (2 Hz, 120 ms duration, 2.5 p.s.i.) and subsequently washing away uninternalized dextran with the second micropipette (2 Hz, 120 ms duration, 2.5 p.s.i.) until the background fluorescence intensity subsided and internal vesicles could be visualized (approximately 5 to 10 s). For co-internalization of FM 5-95 (100 μM in the micropipette) and fluorescent dextran (Alexa-488 conjugated; 500 μM in the micropipette), we simultaneously applied both dyes for 10 s from the same micropipette (2 Hz, 120 ms duration, 2.5 p.s.i.). A second micropipette was used to wash away uninternalized fluorescent dextran as previously described.
Determination of endocytic density
We determined the distribution of endocytic vesicles in the peripheral and central regions of the growth cone by counting individual vesicles within the defined regions of interest. All analyses were performed within ImageJ software (Bio-Formats ZVI plug-in, Madison, WI, USA). Individual vesicles were identified 15 s after the initial focal FM 5-95 application. Vesicles that had originated within 1 μm of the outline of the growth cone were considered peripheral. In order to determine the area of individual growth cones, we set fluorescence thresholds slightly above the background fluorescence levels and generated binary images (background fluorescence = 0, membrane fluorescence = 1). We then selected the outline of the growth cone as a region of interest in order to measure the total area. In order to determine the area of the central domain, we eroded the peripheral region of the binary growth cone image (1-μm diameter), redefined the region of interest outlining the new growth cone (central region), and measured the area within. The area of the peripheral region was determined by subtracting the central area from the total growth cone area. Endocytic density values were determined by dividing the number of endocytic events by the area of the respective region. Data from multiple growth cones was then averaged to determine the mean endocytic density (the number of vesicles per μm2).
Quantitative fluid-phase endocytic assay
For comparisons of the rate of membrane retrieval, we incubated spinal neuron cultures with fluorescent dextran (150 μM; Texas Red conjugated, 3000 MW, lysine fixable, Invitrogen) for 10 minutes at room temperature followed by consecutive rinses (10 minutes) at reduced temperature (10°C). Neurons were then chemically fixed with 5% formaldehyde in a cytoskeleton-stabilizing buffer for 20 minutes and mounted with Prolong Gold (Invitrogen) . Clostridium difficile Toxin B (20 ng/ml, Calbiochem, Gibbstown, NJ, USA) was applied at the time of plating. Culture medium was used for all dye incubations and washes. We acquired digital fluorescence images using a Zeiss Axiocam mounted on a Zeiss Axiovert 200 M inverted microscope (20 ×, 0.8 numerical aperture, 1.6 × optical zoom). Identical acquisition parameters were used for all experimental groups and the original 14-bit images were analyzed using ImageJ software. A region of interest encompassing the entire growth cone (defined as the distal 40 μm of the axon) was used to determine the mean fluorescence intensity of dextran-labeled endocytic vesicles in the growth cone. A threshold was set above the background intensity, identical for all conditions, and the fluorescence intensity of the region of interest was measured. Data were background subtracted and the final corrected intensity value for each growth cone was normalized to the appropriate mean control.
Axonal growth assays
To determine the effect of increasing doses of cytochalasin D on axon outgrowth, we measured the rate of axon extension during a 1-h assay performed 12 to 14 h after plating. Cytochalasin D (10 to 100 nM) or dimethyl sulfoxide (DMSO) was added 30 minutes prior to the growth assay. For measurements of neurite length on different substrates, spinal neurons were plated on PDL or PDL + FN substrates. After 14 h in vitro, cultures were chemically fixed and phase-contrast digital images were captured using a cooled CCD camera (ProgRes C10 plus, Jenoptik, Jupiter, FL, USA) mounted on a Zeiss (Axiovert 40CFL) inverted microscope (10 × objective). Axon lengths were determined using the ImageJ plug-in NeuronJ . We measured only the longest neurite, or branch of each neurite, and only axons > 50 μm in length were included in the analysis. To determine the effect of Toxin B on axon outgrowth, we measured the rate of axon extension during a 1-h assay performed 12 to 20 h after plating . Toxin B (20 ng/ml) was added at the time of plating.
Statistical analyses were performed using Graphpad Prism software (v5, La Jolla, CA, USA). The D'Agostino and Pearson omnibus test was used to assess the data for normality. Statistical comparisons with normal distributions used either a two-tailed t-test or one-way analysis of variance (ANOVA; Tukey post-test), as indicated in the figure legends. All other comparisons utilized the non-parametric Mann-Whitney U-test.
- DIC imaging:
differential interference contrast imaging
Dulbecco's phosphate-buffered saline
fluorescence resonance energy transfer
- TIRF microscopy:
total internal reflection fluorescence microscopy
- Toxin B:
Clostridium difficile toxin B
We thank Allan Bieber, Bruce Horazdovsky, Charles Howe, Mark McNiven, Fredric Meyer, the late Richard Pagano (Mayo Clinic) and members of the Henley lab for critical comments. We also thank Anthony Windebank for sharing lab space at the beginning of these studies, and Jim Tarara, Ellen Liang and Jarred Nesbitt for technical assistance. This work was supported by a John M. Nasseff, Sr. Career Development Award in Neurologic Surgery Research from the Mayo Clinic (JRH), career development funds from the Craig Neilsen Foundation (JRH), and the US National Institutes of Health (JRH). A Robert D. and Patricia E. Kern Predoctoral Fellowship award supported JHH. A Langan MD/PhD Predoctoral Fellowship award supported LPC.
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