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GAP-43 Potentiates Cytoskeletal Reorganization in Raft Domains
Molecular and Cellular Neuroscience 14, 85–97 (1999) Article ID mcne.1999.0775, available online at http://www.idealibrary.com on
MCN
B-50/GAP-43 Potentiates Cytoskeletal Reorganization in Raft Domains Lambertus H. J. Aarts,1 Paul Verkade,*,1 Jacqueline J. W. van Dalen, Andrea J. van Rozen, Willem Hendrik Gispen,† Loes H. Schrama,2 and Peter Schotman Rudolf Magnus Institute for Neurosciences, Department of Physiological Chemistry, and †Department of Medical Pharmacology, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands; and *Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, and Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany
B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts.
INTRODUCTION Axonal pathfinding occurs through detection of soluble and cell surface-associated attractive or repulsive guidance cues. These environmental cues are detected by receptors located at the leading edge of the 1
These authors made equal contributions to the study. To whom correspondence should be addressed at the Department of Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Fax: ⫹31–30–2539035. E-mail: [email protected]. 2
1044-7431/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
neuritic growth cone and are ultimately translated into changes in growth, such as acceleration, turning, branching, or collapse (Goodman, 1996). One of the proteins that has been implicated as having an important role in growth cone morphology and function is the neural growth-associated protein B-50 (GAP-43) (for review see Oestreicher et al., 1997). The expression of B-50 has been shown to be dramatically upregulated during periods of axonal growth and regeneration. Overexpression of B-50 in a variety of cell types evoked spontaneous formation of filopodia and blebs (Nielander et al., 1993; Widmer and Caroni, 1993; Meiri et al., 1996; Aarts et al., 1998), whereas depletion of B-50 expression in neuronal cells attenuated neurite outgrowth and reduced growth cone complexity and adhesiveness (Aigner and Caroni, 1993; Jap Tjoen San et al., 1995). B-50 is highly expressed in outgrowing neurons where it is associated with the cytoplasmic face of the plasma membrane in axons and growth cones. After synthesis on free ribosomes in the cytosol, B-50 is attached to membranes via palmitoylation of two cysteines in the N-terminus of the protein (Skene and Vira´g, 1989). This palmitoylation presumably occurs somewhere along the secretory pathway since pharmacological interference with transport prevented incorporation of [3H]palmitate as well as membrane binding (Gonzalo and Linder, 1998). The membrane-associated protein is transported to the axonal compartment via kinesin-dependent, fast axonal transport on Golgi-derived vesicles (Skene and Willard, 1981; Ferreira et al., 1992; Benowitz and Lewis, 1983). Mutation of the N-terminal cysteines prevented
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86 the sorting of B-50 to the Golgi apparatus as well as to the plasma membrane (Zuber et al., 1989b; Liu et al., 1994; Aarts et al., 1995). Palmitoylation is a posttranslational lipid modification that is limited to a specific subset of proteins which are mostly found associated with the plasma membrane and include G-protein-coupled receptors, subunits of heterotrimeric G-proteins, Ras, adenylcyclase, and nonreceptor tyrosine kinases (Milligan et al., 1995). Increasing evidence suggests that double acylation may serve to direct proteins to detergent-insoluble glycolipidenriched membranes (DIGs) that are enriched in specific lipids and proteins such as glycosylphosphatidylinosi-
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tol (GPI)-anchored proteins (Harder and Simons, 1997; Arni et al., 1998). In the cellular milieu, these highly ordered lipid–protein assemblies are hypothesized to represent microdomains in membranes (called rafts) involved in specific cellular functions such as signal transduction, apical membrane sorting, adhesion, and morphogenesis (Simons and Ikonen, 1997). Recently, B-50 has been demonstrated to be enriched in DIGs of rat brain homogenates (Joliot et al., 1997; Maekawa et al., 1997) and of PC12 cells (Arni et al., 1998), suggesting that B-50 might be localized to and exert its function in raft domains. In this study we show that B-50 is accumulated at the
FIG. 1. Immunolocalization of B-50 in Rat1 fibroblasts stably transfected with B-50. Fixed and permeabilized cells were labeled for B-50 immunoreactivity (IR) and observed by confocal laser scanning microscopy as described under Experimental Methods. Note the difference in distribution of B-50 IR between the various cells: whereas the cells in the upper right corner display a cytosolic distribution, the more isolated cells at the left and bottom exhibit accumulation of B-50 on large vesicles. Scale bar, 10 µm.
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FIG. 2. Double labeling of stably transfected Rat1 fibroblasts for B-50 and various organelle markers. Fixed and permeabilized cells were double stained for (a–e, left) B-50 and (middle) WGA-FITC (a), TGN-38 (b), rab5 (c), rab7 (d), or Thy-1 (e) and observed by confocal laser scanning microscopy as described Experimental Methods. Images were merged yielding a yellow color in case of colocalization (right). Scale bar, 10 µm.
FIG. 3. Time-lapse confocal microscopy of living Rat1 fibroblasts expressing a B-50/EGFP fusion construct. Transfected cells were grown for 24 h in the presence of serum before observation by confocal laser scanning microscopy. (a) Rapid pinocytic uptake and retrograde transport of vesicles covered with B-50/EGFP from ruffling membranes. Note the generation of a large vesicle at 28309 and of two small vesicles after 68. (b) Ruffling and pinocytosis in a transfected cell and in an adjacent untransfected cell. The top row shows the B-50-GFP fluorescence, the bottom row shows the corresponding transmission images. Note the generation of a cluster of vesicles in the transfected cell at 20 s (arrows) that have fused at 60 s (arrowheads, top). In the bottom pinocytotic vesicles (arrows) are also visible in the untransfected cell. (c) Local accumulation of B-50/EGFP at sites of membrane ruffling. Note the events occurring in the region of the arrow: local accumulation of B-50/EGFP in the period from 30 to 90 s is followed by an increase in membrane activity and surface area in the period from 60 to 150 s. Scale bars, 10 µm.
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leading edge of ruffling membranes that are enriched in F-actin, implicating a localized role for B-50 in membrane motility. In fibroblasts these membrane ruffles lead to an enhanced membrane uptake through pinocytes positive for Thy-1, a GPI-anchored raft marker, and negative for rab5, an early endosome marker. The number of Thy-1-positive pinocytes is three times higher in B-50-transfected fibroblasts. In primary hippocampal cells B-50 also completely colocalized with Thy-1. Moreover, B-50 could be clustered by Thy-1 antibodyinduced patching. These patches were negative for syntaxin, used as a nonraft marker protein. Our data indicate that B-50 is present in rafts in intact cells and is taken up by pinocytosis, rather than endocytosis. Moreover, actin polymerization shows that the presence of B-50 leads to cytoskeletal rearrangements.
RESULTS To investigate the role of B-50 in the modulation of cell surface activity, we have generated stable lines of
Rat1 fibroblasts that constitutively express high levels of B-50. Immunolabeling of stably transfected cells with a monoclonal anti-B-50 antibody NM4 revealed two different patterns of intracellular distribution of B-50 (Fig. 1). Cells in contact with neighboring cells (see the flattened cells at the upper right half of Fig. 1) displayed diffuse labeling of B-50 immunoreactivity (IR), whereas in more isolated cells (at the lower left of Fig. 1) B-50 IR was accumulated at large vesicular structures that often filled the entire cytoplasm at one site of the cell (Fig. 1, left lower half). A similar distribution of B-50 IR was observed in another stably transfected Rat1 fibroblast cell line (not shown). To establish the nature of the B-50-immunoreactive vesicles, we performed a series of double-labeling experiments using several specific organelle markers. The Golgi apparatus of cultured cells can be labeled and localized by wheat germ agglutinin (WGA), a lectin that binds to N-acetylglucosamine and N-acetylneuramic acid residues predominantly found in the Golgi complex (Virtanen et al., 1980). Double labeling with antiB-50 antibodies (Fig. 2a, left) and FITC-conjugated WGA
90 (Fig. 2a, middle) showed a clear overlap in a region juxtaposing the nucleus, characteristic of the Golgi apparatus (Fig. 2a, right, yellow color). However, considerable B-50 IR was located on vesicular structures that lacked WGA-binding sites (Fig. 2a, right, red color). Double labeling with the trans-Golgi network marker TGN-38 (Fig. 2b; Luzio et al., 1990) and with CTR433, which is localized to the cisternae of the medial compartment of the Golgi apparatus (not shown; Jasmin et al., 1992), showed a minor colocalization with B-50 IR (Fig. 2b, right). Immunolabeling of the endoplasmic reticulum using antibody 1D3 was clearly distinct from the B-50 staining pattern (not shown). To establish whether the B-50-immunoreactive vesicles represented endocytic structures, we double labeled cells with anti-B-50 antibodies together with either anti-rab5 or anti-rab7 antibodies, which mark early and late endosomes, respectively (Chavrier et al., 1990). Some colocalization was observed between B-50 and rab5 (Fig. 2c, right), whereas all staining for rab7 coincided with B-50 IR (Fig. 2d, right). However, the majority of the B-50-IR vesicles were devoid of either rab-5 or rab-7. A major uptake pathway for GPI-anchored proteins present in detergentinsoluble complexes (so-called rafts) is via pinocytosis (Bamezai et al., 1992; Brown and Waneck, 1992; Poussin et al., 1998). Upon double labeling with Thy-1, a GPIanchored cell adhesion molecule that is sorted to the apical/axonal plasma membrane of polarized cells (Ledesma et al., 1998), we observed a near complete colocalization of B-50 IR (Fig. 2e, left) with Thy-1 (Fig. 2e, right). Codistribution of the two proteins in the region of the Golgi apparatus was found, at the plasma membrane as well as at pinocytes. Little if any red color was left in the merged images (Fig. 2e, right), indicating that all of the detectable B-50-immunopositive structures colocalized with Thy-1 IR. To further establish the presence of B-50 in DIGs we have transfected Rat1 fibroblasts with B-50-GFP and the nonraft marker syntaxin. No colocalization of B-50 and syntaxin could be observed in these cells after Triton X-100 permeabilization (data not shown). To establish the origin of the B-50-IR vesicles, we followed their formation and fate in time-lapse studies on live, wild-type Rat1 fibroblasts transiently transfected with a B-50/enhanced green fluorescent protein (EGFP) fusion construct (Fig. 3). The B-50/EGFP signal appeared partly associated with the Golgi apparatus but accumulated predominantly at the leading edge of lamellae and at pinocytes. Upon time-lapse imaging, transfected cells displayed intense membrane ruffling that resulted in the rapid generation of pinocytotic vesicles that could be followed during their retrograde
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transport (Fig. 3a). The amount of generated vesicles was correlated with the activity of the ruffling membrane, since the highest concentration of vesicles was found between the ruffling membrane and the corresponding perinuclear space. Newly generated B-50-GFPlabeled pinocytic vesicles (Fig. 3b, upper left, arrows) moved in the direction of the nuclear region where they often fused with other vesicles (Fig. 3b, upper right, arrowheads). Pinocytotic events were not restricted to transfected cells but rather coincided with spots of membrane ruffling. Adjacent untransfected fibroblasts also displayed pinocytic vesicle uptake, visible in the corresponding phase-contrast image, but much less pronounced (Fig. 3b, bottom, the arrows point to vesicles taken up in the untransfected cell). The number of vesicles in transfected versus untransfected cells was determined by counting Thy-1-immunolabeled vesicles in transiently B-50-GFP-transfected fibroblasts. In B-50positive cells we counted 6.85 ⫾ 0.84 (mean ⫾ SEM, n ⫽ 21) Thy-1-positive vesicles, whereas in B-50-negative cells 2.27 ⫾ 0.56 (mean ⫾ SEM, n ⫽ 17) vesicles were present. Thus the introduction of B-50 increased the number of pinocytes threefold (P ⬍ 0.005, t test). Occasionally, we observed active recruitment of B-50/ EGFP toward sites on the plasma membrane of a live fibroblast (Fig. 3c). The gradual accumulation of B-50/ EGFP at these specific sites was followed by a gradual increase in membrane ruffling, indicating that these events are causally linked (Fig. 3c, arrows). One of the underlying events in membrane ruffling is polymerization of actin. Comparison of the doublestaining patterns of the stably transfected Rat1 fibroblasts for B-50 (Fig. 4a) and F-actin (Fig. 4b) shows that both B-50-IR and F-actin binding phalloidin-FITC are specifically high at sites on the plasma membrane involved in ruffling (Figs. 4c and 4d, yellow color). The accumulation of pinocytic vesicles (with intense B-50 staining but devoid of F-actin) appears especially high in cells with high levels of B-50 and intense F-actin plasma membrane staining (Figs. 4c and 4d). The colocalization of the neural protein B-50 with Thy-1 in Rat1 fibroblasts prompted us to examine whether B-50 and Thy-1 are colocalized in neuronal cells as well. In outgrowing dissociated primary hippocampal neurons, B-50 IR (Fig. 5a, left) completely colocalized with Thy-1 IR throughout the neuritic extensions (Fig. 5a, middle). Both B-50 and Thy-1 have been reported to be enriched in Triton-insoluble fractions (Brown and Rose, 1992; Joliot et al., 1997; Maekawa et al., 1997; Schroeder et al., 1998). Permeabilization of neurons with 0.05% Triton X-100 after fixation with 4% paraformaldehyde resulted
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FIG. 4. Accumulation of both B-50 and actin filaments at ruffling membranes of stably transfected fibroblasts. Fixed and Triton X-100permeabilized cells were stained for B-50 IR (a) and F-actin binding phalloidin-FITC (b). Images were merged yielding a yellow color in case of colocalization (c, scale bar, 10 µm). (d) An enlargement of the boxed area in c (scale bar, 2.5 µm).
in a redistribution of B-50 (Fig. 5b, left) and Thy-1 (Fig. 5b, middle). Both proteins colocalized in discrete punctate structures resembling varicosities (van Lookeren Campagne et al., 1992), presumably detergent-insoluble plasma membrane patches (Fig. 5b, right, yellow color). To study the coalescence of B-50 and Thy-1 in specific
membrane domains we examined the copatching behavior of both proteins after Thy-1 antibody-induced clustering. GPI-anchored proteins have been reported to be redistributed into discrete patches following immunolabeling when proteins are not properly fixed (Mayor et al., 1994). Recently, Harder and co-workers (1998) showed
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FIG. 5. Colocalization of B-50 and Thy-1 in primary hippocampal neurons. Paraformaldehyde-fixed hippocampal cells were double stained for B-50 (left) and Thy-1 (middle) and merged (right) after permeation with saponin (a) or Triton X-100 (b), as described under Experimental Methods. Scale bar, 10 µm.
that this property can be used in unfixed cells to demonstrate the coaccumulation of raft-associated proteins into discrete patches. To avoid patching resulting from improper fixation, we fixed hippocampal neurons using brief formaldehyde fixation and subsequent incubation in methanol at ⫺20°C. Using this fixation protocol both Thy-1 and B-50 were evenly distributed along the plasma membrane (data not shown). Copatching of B-50 and Thy-1 was studied by incubating the cells with Thy-1 antibodies followed by cross-linking using Cy-5labeled secondary antibodies. After fixation and subsequent B-50 immunolabeling, both Thy-1 IR and B-50 IR (Fig. 6) were redistributed into discrete, membraneassociated patches. Patches of Thy-1 and B-50 appeared to coincide completely (Fig. 6, merged turquoise color), indicating that antibody-induced clustering of Thy-1 at the extracellular leaflet of the plasma membrane caused a dramatic redistribution of the intracellular protein B-50, resulting in a concentration of B-50 in membrane domains with patched Thy-1. To demonstrate that this
phenomenon is specific for raft-associated proteins, we also stained the cells for syntaxin, a nonraft marker (Fig. 6). The staining of syntaxin (indicated by arrows) was mainly excluded from the Thy-1/B-50 clusters and only occasionally overlapped as indicated by the arrowheads (Fig. 6, right). Thus we conclude that syntaxin is excluded from Thy-1 clustered membrane patches and that most of the B-50 is included in DIGs.
DISCUSSION Recently, we have demonstrated that transient transfection of B-50 in Rat1 fibroblasts caused formation of filopodial extensions during initial spreading (Aarts et al., 1998). In those studies, we frequently observed membrane ruffling and pinocytotic activity in transfected cells with a lamellipodial or mixed morphology (L. H. J. Aarts, unpublished data). In the present study, we have examined the cell surface activity of B-50 in
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FIG. 6. Accumulation of B-50 in membrane domains formed by patched Thy-1 in primary hippocampal neurons. Thy-1 was antibody cross-linked and after brief washing in PBS cross-linking was performed with donkey anti-mouse-conjugated Cy5 (blue) antibodies at 4°C for 1 h. After fixation with 4% paraformaldehyde/methanol the hippocampal neurons were immunolabeled in the absence of detergent for B-50 (labeled with FITC, green) and syntaxin (labeled with TRITC, red). Shown from left to right are Thy-1 labeling after clustering, B-50 labeling, syntaxin labeling, and the merge of the three leftmost images. The four right images are enlargements of the areas indicated by the rectangles in the preceding four images, presented in the same order. Arrows point to the position of syntaxin (red) which is excluded from the Thy-1/B-50 patches. The arrowheads point to a membrane area where colocalization of Thy-1, B-50, and syntaxin is observed (white).
stably transfected Rat1 fibroblasts at later time points of growth. In some of these cells B-50 accumulates at discrete membrane domains that are actively involved in membrane ruffling and pinocytosis, the endocytotic pathway for plasma membrane raft domains (Brown and Waneck, 1992; Bamezai et al., 1992; Poussin et al., 1998). The difference in intracellular location of B-50 IR in active versus resting cells (Fig. 1) suggests that B-50 is actively targeted to specific, motile domains at the plasma membrane. This was indeed observed in living B-50/EGFP-transfected cells, in which recruitment of B-50/EGFP was correlated with an increase in local membrane ruffling (Fig. 3c). The high levels of B-50/ EGFP were shown to correlate with the generation of pinocytic vesicles (Fig. 3b), a process that is closely linked with high membrane activity (i.e. ruffling, BarSagi and Feramisco, 1986; Keller et al., 1990). This implies that B-50 potentiates both membrane ruffling and pinocytosis in Rat1 fibroblasts, as was confirmed by counting the number of pinosomes in B-50-transfected cells in comparison with untransfected cells. The molecular events that underlie formation of membrane ruffles are largely unknown but involve polymerization of actin immediately underneath the plasma membrane (Ridley and Hall, 1992). Indeed, intense F-actin staining was present at the membrane ruffles in B-50-transfected cells (Fig. 4), suggestive of a local increase in actin filament formation at sites with high levels of B-50. This corroborates our recent finding that the B-50-induced formation of filopodia in spreading Rat1 fibroblasts was accompanied by a profound increase in the concentra-
tion of cortical actin filaments, a process that was dependent on Rho-GTPase function (Aarts et al., 1998). Manipulation of the expression levels of B-50 in a variety of neuronal and nonneuronal cells has been shown to induce distinct morphological changes depending on cell type, expression level, and experimental conditions. Transfection of B-50 in nonneuronal cells resulted in the formation of filopodia during initial spreading (Zuber et al., 1989a; Widmer and Caroni, 1993; Aarts et al., 1998). This was also observed in the stably transfected Rat1 fibroblasts of this study in which more than 80% of the cells adopted a filopodial morphology during spreading (not shown). Enhanced expression of B-50 causes formation of filopodia, blebs, or even neuritelike extensions, probably depending on the cell type and the level of expression (Nielander et al., 1993; Verhaagen et al., 1994; Wiederkehr et al., 1997). In transgenic mice, overexpression of B-50 provoked the development of highly enlarged terminal fields and increased injuryinduced sprouting (Aigner et al., 1995). Using immunoelectron microscopy, Holtmaat and co-workers observed that overexpression of B-50 in distinct transgenic mice as well as in adenoviral vector-infected mice caused formation of large membranous labyrinths in olfactory axonal endings (Holtmaat et al., 1995, 1997). Interestingly, the B-50-related myristoylated alaninerich C-kinase substrate (MARCKS) has been demonstrated to influence membrane ruffling and cell spreading as well (Myat et al., 1997). Like B-50, MARCKS has been implicated in assembly of actin structures at the plasma membrane as well as in the assembly of adhesive structures (Aderem, 1995). Moreover, transfection
94 of B-50, MARCKS, and CAP-23 in fibroblasts induced similar dynamic structures at the cellular periphery (Wiederkehr et al., 1997). The ERM family of actinmembrane cross-linking proteins may represent functionally related proteins since they were shown to be enriched in and to influence the formation of adhesive structures and actin-based cell surface protrusions such as microvilli, filopodia, and membrane ruffles (Amieva and Furthmayr, 1995; Martin et al., 1995; Sainio et al., 1997). Although it is largely unknown how these functionally related proteins influence actin-membrane dynamics, they may make use of a common mechanism involving the Rho-GTPase (Hirao et al., 1996; Mackay et al., 1997; Aarts et al., 1998). In this study, we show that in transfected fibroblasts, B-50 is colocalized and coconcentrated with the GPIanchored cell adhesion molecule Thy-1 in the Golgi apparatus, at ruffling membranes, and on pinocytic vesicles (Fig. 2e). This may imply that Thy-1 and B-50 are cotransported from the Golgi apparatus to ruffling membranes and are subsequently retrieved together by pinocytic vesicles. Although retrograde transport of vesicular B-50 (Li et al., 1993) has been demonstrated in cultured neurons, it is not known whether these vesicles are generated by a pinocytic-like event in ruffling membranes within growth cones. Nevertheless, B-50 IR and Thy-1 appeared to be completely colocalized in both the cell bodies and the neurites of primary hippocampal neurons (Fig. 5a). Following treatment with Triton X-100, a significant fraction of B-50 IR remained clustered with Thy-1 in detergent-resistant membrane remnants (Fig. 5b), suggesting that both proteins are associated with the same Triton-insoluble membrane skeleton fraction in situ. Recently, B-50 was shown to be enriched in DIGs (Joliot et al., 1997; Maekawa et al., 1997). In the cellular milieu, DIGs have been proposed to represent membrane microdomains (rafts) that are involved in protein sorting, signal transduction, adhesion, and morphogenesis by concentrating a specific subset of proteins, including cell surface molecules such as GPI-anchored proteins and various, mostly doubly acylated, signal transduction proteins (Simons and Ikonen, 1997). However, detergent-induced in vitro or in situ clustering does not provide evidence for concluding a native association with rafts (Mayor and Maxfield, 1995). Therefore, we have studied the copatching behavior of B-50 and Thy-1 following Thy-1 antibody-induced clustering. It is assumed that antibody-mediated crosslinking causes multimerization of membrane microdomains as a consequence of specific lipid preferences of its constituents (Harder et al., 1998). Copatching may thus result from the coalescence of lipid microdomains
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containing cross-linked raft membrane components (Harder et al., 1998). The coclustering of the intracellular protein B-50 with the GPI-anchored extracellular membrane protein Thy-1 following antibody-mediated crosslinking (Fig. 6) indicates that both proteins share a preference for raft lipids. Syntaxin, a membraneassociated SNARE protein involved in exocytosis, does not cocluster with Thy-1 and B-50 in rafts (Fig. 6). It is tempting to speculate that B-50 is concentrated in the cytoplasmic leaflet of raft domains in vivo following clustering of specific GPI-anchored molecules (such as Thy-1, NCAM120, axonin/TAG-1) at the extracellular site of raft domains. The accumulation of B-50 would subsequently result in local alterations in the underlying actin cytoskeleton, ultimately leading to changes in neuritic growth.
EXPERIMENTAL METHODS Cell Culture and Transfection Experiments Wild-type Rat1 fibroblasts were cultured in DMEM (high glucose, 4.5 g/L) supplemented with 10% fetal calf serum (FCS), 100 U penicillin/streptomycin, and L-glutamine (2 mM) in a humidified atmosphere at 37°C and 7% CO2. Stable transfection was performed by cotransfection of a B-50 expression construct (in pCDNA1; Invitrogen, San Diego, CA; Nielander et al., 1993) with the puromycin-resistance plasmid pBabePuro at a concentration ratio of 1:10 in a 6-cm dish using lipofectin (Life Technologies). The day after transfection, puromycin was added to the culture to a final concentration of 1 µg/ml. Resistant clones were visible within 6 days of transfection. Among several stably transfected cell lines one cell line, designated B14, was selected for further research. After being seeded on 12-mm acidcleaned glass coverslips, cells were grown for 30 h in the presence of 10% FCS before fixation with 4% paraformaldehyde (20 min at 4°C). Transfection of Rat1 fibroblasts with syntaxin cDNA was performed with the plasmid pCMV-HPC-1a (a gift from M. Verhage) using calcium phosphate precipitation. Hippocampal cell cultures were obtained by dissection of rat hippocampi (embryonal day 19) as described (Banker and Cowan, 1977). Briefly, hippocampi were dissociated by trypsinization and trituration and plated on poly-L-lysine-coated glass coverslips in B27-supplemented Neurobasal medium (Life Technologies). After attachment of cells to the substrate, coverslips were transferred to dishes containing monolayer cultures of astroglia in chemically defined medium (DMEM (high glucose, 4.5 g/L), 5 µg/ml insulin, 0.5 µg/ml transfer-
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rin, 20 µg/ml progesterone, 100 µM putrescine, 30 µM SeO2, 90 mg/L bovine serum albumin, and penicillin/ streptomycin (each 100 IU/ml)), without being in physical contact. Cells were grown for 14 days before antibody cross-linking or fixation with 4% paraformaldehyde (20 min at 4°C).
BSA) and incubated for 1 h at 4°C with anti-Thy-1 antibodies diluted in PBS⫹⫹ (monoclonal Ox-7, 1:20; Biotrend). After brief washing in PBS, further crosslinking was performed with donkey anti-mouse-conjugated Cy5 antibodies at 4°C for 1 h. Cells were fixed for 5 min in 4% paraformaldehyde at 4°C and subsequent incubation in methanol at ⫺20°C for 5 min before B-50 and syntaxin immunolabeling.
Immunocytochemistry and Microscopy Following fixation, cells were washed with phosphatebuffered saline (PBS) and incubated in 1 M NH4Cl in PBS for 10 min. Subsequently, cells were incubated in a blocking buffer (0.5% BSA, 2% normal goat serum, and 0.1% saponin in PBS (or instead 0.05% Triton X-100 or no detergent, when indicated)) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the cells overnight at 4°C. After washing and incubation with secondary antibodies (1 h, 37°C), cells were embedded in Dabco/Mowiol and observed with an upright Leica TCS NT confocal laser scanning microscope (Heidelberg, Germany). B-50 immunoreactivity was visualized with the specific monoclonal antibody NM4 (1:4000; Mercken et al., 1992) or with the polyclonal antibody 8613 (1:50 or 1:300; Verkade et al., 1996). Phalloidin–FITC (Sigma Chemical, St. Louis, MO; 2 µg/ml) and WGA–FITC (Sigma; 40 µg/ ml) were included along with the secondary antibody incubation. Antibodies directed against organellespecific markers include anti-CTR433 (monoclonal, 1:100; a gift from Dr. Van der Sluijs), anti-TGN-38 (polyclonal, 1:100; a gift from Dr. Banting), anti-rab5 (monoclonal, 1:20), anti-rab7 (polyclonal, 1:50; a gift from Dr. Chavrier), antibody 1d3, specifically detecting the ER (monoclonal, 1:50; a gift from Dr. Fuller), and anti-syntaxin antibody I378 (polyclonal, 1:100; a gift from Dr. Verhage; McMahon et al., 1995). Monoclonal anti-Thy-1 (1:100) was obtained from Sera-Lab for immunolabeling (Sussex, England). Time-lapse recordings of living Rat1 fibroblasts were performed at 37°C and 7% CO2 in a microchamber mounted on an inverted Leica TCS NT confocal laser scanning microscope (Heidelberg, Germany). Rat1 fibroblasts were transfected with pB-50/EGFP, a fusion construct bearing the open reading frame of B-50 cloned directly upstream of EGFP in pEGFP-N1 (Clontech, Palo Alto, CA; Aarts et al., 1998). After 24 h of growth in 10% FCS, time-lapse recordings were started with time intervals of 10 s.
Antibody-Induced Patching Hippocampal cells were washed twice in PBS⫹⫹ (PBS supplemented with 0.9 mM Ca2⫹, 1 mM Mg2⫹, and 0.2%
ACKNOWLEDGMENTS We thank Drs. Van der Sluijs, Banting, Chavrier, Verhage, and Fuller for providing the antibodies that made it possible to detect various intracellular vesicles and Bianca Hellias for culturing hippocampal cells. Moreover we thank Ruud F. G. Toonen for the syntaxin transfection of the fibroblasts and Liselijn A. B. Wisman for the subsequent immunocytochemistry. This work was supported by NWO Grant 903–42–006 and by the Prinses Beatrix Fonds, Grant 95–1008.
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B-50/GAP-43 Potentiates Cytoskeletal Reorganization in Raft Domains Lambertus H. J. Aarts,1 Paul Verkade,*,1 Jacqueline J. W. van Dalen, Andrea J. van Rozen, Willem Hendrik Gispen,† Loes H. Schrama,2 and Peter Schotman Rudolf Magnus Institute for Neurosciences, Department of Physiological Chemistry, and †Department of Medical Pharmacology, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands; and *Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, and Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany
B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts.
INTRODUCTION Axonal pathfinding occurs through detection of soluble and cell surface-associated attractive or repulsive guidance cues. These environmental cues are detected by receptors located at the leading edge of the 1
These authors made equal contributions to the study. To whom correspondence should be addressed at the Department of Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Fax: ⫹31–30–2539035. E-mail: [email protected]. 2
1044-7431/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
neuritic growth cone and are ultimately translated into changes in growth, such as acceleration, turning, branching, or collapse (Goodman, 1996). One of the proteins that has been implicated as having an important role in growth cone morphology and function is the neural growth-associated protein B-50 (GAP-43) (for review see Oestreicher et al., 1997). The expression of B-50 has been shown to be dramatically upregulated during periods of axonal growth and regeneration. Overexpression of B-50 in a variety of cell types evoked spontaneous formation of filopodia and blebs (Nielander et al., 1993; Widmer and Caroni, 1993; Meiri et al., 1996; Aarts et al., 1998), whereas depletion of B-50 expression in neuronal cells attenuated neurite outgrowth and reduced growth cone complexity and adhesiveness (Aigner and Caroni, 1993; Jap Tjoen San et al., 1995). B-50 is highly expressed in outgrowing neurons where it is associated with the cytoplasmic face of the plasma membrane in axons and growth cones. After synthesis on free ribosomes in the cytosol, B-50 is attached to membranes via palmitoylation of two cysteines in the N-terminus of the protein (Skene and Vira´g, 1989). This palmitoylation presumably occurs somewhere along the secretory pathway since pharmacological interference with transport prevented incorporation of [3H]palmitate as well as membrane binding (Gonzalo and Linder, 1998). The membrane-associated protein is transported to the axonal compartment via kinesin-dependent, fast axonal transport on Golgi-derived vesicles (Skene and Willard, 1981; Ferreira et al., 1992; Benowitz and Lewis, 1983). Mutation of the N-terminal cysteines prevented
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86 the sorting of B-50 to the Golgi apparatus as well as to the plasma membrane (Zuber et al., 1989b; Liu et al., 1994; Aarts et al., 1995). Palmitoylation is a posttranslational lipid modification that is limited to a specific subset of proteins which are mostly found associated with the plasma membrane and include G-protein-coupled receptors, subunits of heterotrimeric G-proteins, Ras, adenylcyclase, and nonreceptor tyrosine kinases (Milligan et al., 1995). Increasing evidence suggests that double acylation may serve to direct proteins to detergent-insoluble glycolipidenriched membranes (DIGs) that are enriched in specific lipids and proteins such as glycosylphosphatidylinosi-
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tol (GPI)-anchored proteins (Harder and Simons, 1997; Arni et al., 1998). In the cellular milieu, these highly ordered lipid–protein assemblies are hypothesized to represent microdomains in membranes (called rafts) involved in specific cellular functions such as signal transduction, apical membrane sorting, adhesion, and morphogenesis (Simons and Ikonen, 1997). Recently, B-50 has been demonstrated to be enriched in DIGs of rat brain homogenates (Joliot et al., 1997; Maekawa et al., 1997) and of PC12 cells (Arni et al., 1998), suggesting that B-50 might be localized to and exert its function in raft domains. In this study we show that B-50 is accumulated at the
FIG. 1. Immunolocalization of B-50 in Rat1 fibroblasts stably transfected with B-50. Fixed and permeabilized cells were labeled for B-50 immunoreactivity (IR) and observed by confocal laser scanning microscopy as described under Experimental Methods. Note the difference in distribution of B-50 IR between the various cells: whereas the cells in the upper right corner display a cytosolic distribution, the more isolated cells at the left and bottom exhibit accumulation of B-50 on large vesicles. Scale bar, 10 µm.
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FIG. 2. Double labeling of stably transfected Rat1 fibroblasts for B-50 and various organelle markers. Fixed and permeabilized cells were double stained for (a–e, left) B-50 and (middle) WGA-FITC (a), TGN-38 (b), rab5 (c), rab7 (d), or Thy-1 (e) and observed by confocal laser scanning microscopy as described Experimental Methods. Images were merged yielding a yellow color in case of colocalization (right). Scale bar, 10 µm.
FIG. 3. Time-lapse confocal microscopy of living Rat1 fibroblasts expressing a B-50/EGFP fusion construct. Transfected cells were grown for 24 h in the presence of serum before observation by confocal laser scanning microscopy. (a) Rapid pinocytic uptake and retrograde transport of vesicles covered with B-50/EGFP from ruffling membranes. Note the generation of a large vesicle at 28309 and of two small vesicles after 68. (b) Ruffling and pinocytosis in a transfected cell and in an adjacent untransfected cell. The top row shows the B-50-GFP fluorescence, the bottom row shows the corresponding transmission images. Note the generation of a cluster of vesicles in the transfected cell at 20 s (arrows) that have fused at 60 s (arrowheads, top). In the bottom pinocytotic vesicles (arrows) are also visible in the untransfected cell. (c) Local accumulation of B-50/EGFP at sites of membrane ruffling. Note the events occurring in the region of the arrow: local accumulation of B-50/EGFP in the period from 30 to 90 s is followed by an increase in membrane activity and surface area in the period from 60 to 150 s. Scale bars, 10 µm.
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leading edge of ruffling membranes that are enriched in F-actin, implicating a localized role for B-50 in membrane motility. In fibroblasts these membrane ruffles lead to an enhanced membrane uptake through pinocytes positive for Thy-1, a GPI-anchored raft marker, and negative for rab5, an early endosome marker. The number of Thy-1-positive pinocytes is three times higher in B-50-transfected fibroblasts. In primary hippocampal cells B-50 also completely colocalized with Thy-1. Moreover, B-50 could be clustered by Thy-1 antibodyinduced patching. These patches were negative for syntaxin, used as a nonraft marker protein. Our data indicate that B-50 is present in rafts in intact cells and is taken up by pinocytosis, rather than endocytosis. Moreover, actin polymerization shows that the presence of B-50 leads to cytoskeletal rearrangements.
RESULTS To investigate the role of B-50 in the modulation of cell surface activity, we have generated stable lines of
Rat1 fibroblasts that constitutively express high levels of B-50. Immunolabeling of stably transfected cells with a monoclonal anti-B-50 antibody NM4 revealed two different patterns of intracellular distribution of B-50 (Fig. 1). Cells in contact with neighboring cells (see the flattened cells at the upper right half of Fig. 1) displayed diffuse labeling of B-50 immunoreactivity (IR), whereas in more isolated cells (at the lower left of Fig. 1) B-50 IR was accumulated at large vesicular structures that often filled the entire cytoplasm at one site of the cell (Fig. 1, left lower half). A similar distribution of B-50 IR was observed in another stably transfected Rat1 fibroblast cell line (not shown). To establish the nature of the B-50-immunoreactive vesicles, we performed a series of double-labeling experiments using several specific organelle markers. The Golgi apparatus of cultured cells can be labeled and localized by wheat germ agglutinin (WGA), a lectin that binds to N-acetylglucosamine and N-acetylneuramic acid residues predominantly found in the Golgi complex (Virtanen et al., 1980). Double labeling with antiB-50 antibodies (Fig. 2a, left) and FITC-conjugated WGA
90 (Fig. 2a, middle) showed a clear overlap in a region juxtaposing the nucleus, characteristic of the Golgi apparatus (Fig. 2a, right, yellow color). However, considerable B-50 IR was located on vesicular structures that lacked WGA-binding sites (Fig. 2a, right, red color). Double labeling with the trans-Golgi network marker TGN-38 (Fig. 2b; Luzio et al., 1990) and with CTR433, which is localized to the cisternae of the medial compartment of the Golgi apparatus (not shown; Jasmin et al., 1992), showed a minor colocalization with B-50 IR (Fig. 2b, right). Immunolabeling of the endoplasmic reticulum using antibody 1D3 was clearly distinct from the B-50 staining pattern (not shown). To establish whether the B-50-immunoreactive vesicles represented endocytic structures, we double labeled cells with anti-B-50 antibodies together with either anti-rab5 or anti-rab7 antibodies, which mark early and late endosomes, respectively (Chavrier et al., 1990). Some colocalization was observed between B-50 and rab5 (Fig. 2c, right), whereas all staining for rab7 coincided with B-50 IR (Fig. 2d, right). However, the majority of the B-50-IR vesicles were devoid of either rab-5 or rab-7. A major uptake pathway for GPI-anchored proteins present in detergentinsoluble complexes (so-called rafts) is via pinocytosis (Bamezai et al., 1992; Brown and Waneck, 1992; Poussin et al., 1998). Upon double labeling with Thy-1, a GPIanchored cell adhesion molecule that is sorted to the apical/axonal plasma membrane of polarized cells (Ledesma et al., 1998), we observed a near complete colocalization of B-50 IR (Fig. 2e, left) with Thy-1 (Fig. 2e, right). Codistribution of the two proteins in the region of the Golgi apparatus was found, at the plasma membrane as well as at pinocytes. Little if any red color was left in the merged images (Fig. 2e, right), indicating that all of the detectable B-50-immunopositive structures colocalized with Thy-1 IR. To further establish the presence of B-50 in DIGs we have transfected Rat1 fibroblasts with B-50-GFP and the nonraft marker syntaxin. No colocalization of B-50 and syntaxin could be observed in these cells after Triton X-100 permeabilization (data not shown). To establish the origin of the B-50-IR vesicles, we followed their formation and fate in time-lapse studies on live, wild-type Rat1 fibroblasts transiently transfected with a B-50/enhanced green fluorescent protein (EGFP) fusion construct (Fig. 3). The B-50/EGFP signal appeared partly associated with the Golgi apparatus but accumulated predominantly at the leading edge of lamellae and at pinocytes. Upon time-lapse imaging, transfected cells displayed intense membrane ruffling that resulted in the rapid generation of pinocytotic vesicles that could be followed during their retrograde
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transport (Fig. 3a). The amount of generated vesicles was correlated with the activity of the ruffling membrane, since the highest concentration of vesicles was found between the ruffling membrane and the corresponding perinuclear space. Newly generated B-50-GFPlabeled pinocytic vesicles (Fig. 3b, upper left, arrows) moved in the direction of the nuclear region where they often fused with other vesicles (Fig. 3b, upper right, arrowheads). Pinocytotic events were not restricted to transfected cells but rather coincided with spots of membrane ruffling. Adjacent untransfected fibroblasts also displayed pinocytic vesicle uptake, visible in the corresponding phase-contrast image, but much less pronounced (Fig. 3b, bottom, the arrows point to vesicles taken up in the untransfected cell). The number of vesicles in transfected versus untransfected cells was determined by counting Thy-1-immunolabeled vesicles in transiently B-50-GFP-transfected fibroblasts. In B-50positive cells we counted 6.85 ⫾ 0.84 (mean ⫾ SEM, n ⫽ 21) Thy-1-positive vesicles, whereas in B-50-negative cells 2.27 ⫾ 0.56 (mean ⫾ SEM, n ⫽ 17) vesicles were present. Thus the introduction of B-50 increased the number of pinocytes threefold (P ⬍ 0.005, t test). Occasionally, we observed active recruitment of B-50/ EGFP toward sites on the plasma membrane of a live fibroblast (Fig. 3c). The gradual accumulation of B-50/ EGFP at these specific sites was followed by a gradual increase in membrane ruffling, indicating that these events are causally linked (Fig. 3c, arrows). One of the underlying events in membrane ruffling is polymerization of actin. Comparison of the doublestaining patterns of the stably transfected Rat1 fibroblasts for B-50 (Fig. 4a) and F-actin (Fig. 4b) shows that both B-50-IR and F-actin binding phalloidin-FITC are specifically high at sites on the plasma membrane involved in ruffling (Figs. 4c and 4d, yellow color). The accumulation of pinocytic vesicles (with intense B-50 staining but devoid of F-actin) appears especially high in cells with high levels of B-50 and intense F-actin plasma membrane staining (Figs. 4c and 4d). The colocalization of the neural protein B-50 with Thy-1 in Rat1 fibroblasts prompted us to examine whether B-50 and Thy-1 are colocalized in neuronal cells as well. In outgrowing dissociated primary hippocampal neurons, B-50 IR (Fig. 5a, left) completely colocalized with Thy-1 IR throughout the neuritic extensions (Fig. 5a, middle). Both B-50 and Thy-1 have been reported to be enriched in Triton-insoluble fractions (Brown and Rose, 1992; Joliot et al., 1997; Maekawa et al., 1997; Schroeder et al., 1998). Permeabilization of neurons with 0.05% Triton X-100 after fixation with 4% paraformaldehyde resulted
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FIG. 4. Accumulation of both B-50 and actin filaments at ruffling membranes of stably transfected fibroblasts. Fixed and Triton X-100permeabilized cells were stained for B-50 IR (a) and F-actin binding phalloidin-FITC (b). Images were merged yielding a yellow color in case of colocalization (c, scale bar, 10 µm). (d) An enlargement of the boxed area in c (scale bar, 2.5 µm).
in a redistribution of B-50 (Fig. 5b, left) and Thy-1 (Fig. 5b, middle). Both proteins colocalized in discrete punctate structures resembling varicosities (van Lookeren Campagne et al., 1992), presumably detergent-insoluble plasma membrane patches (Fig. 5b, right, yellow color). To study the coalescence of B-50 and Thy-1 in specific
membrane domains we examined the copatching behavior of both proteins after Thy-1 antibody-induced clustering. GPI-anchored proteins have been reported to be redistributed into discrete patches following immunolabeling when proteins are not properly fixed (Mayor et al., 1994). Recently, Harder and co-workers (1998) showed
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FIG. 5. Colocalization of B-50 and Thy-1 in primary hippocampal neurons. Paraformaldehyde-fixed hippocampal cells were double stained for B-50 (left) and Thy-1 (middle) and merged (right) after permeation with saponin (a) or Triton X-100 (b), as described under Experimental Methods. Scale bar, 10 µm.
that this property can be used in unfixed cells to demonstrate the coaccumulation of raft-associated proteins into discrete patches. To avoid patching resulting from improper fixation, we fixed hippocampal neurons using brief formaldehyde fixation and subsequent incubation in methanol at ⫺20°C. Using this fixation protocol both Thy-1 and B-50 were evenly distributed along the plasma membrane (data not shown). Copatching of B-50 and Thy-1 was studied by incubating the cells with Thy-1 antibodies followed by cross-linking using Cy-5labeled secondary antibodies. After fixation and subsequent B-50 immunolabeling, both Thy-1 IR and B-50 IR (Fig. 6) were redistributed into discrete, membraneassociated patches. Patches of Thy-1 and B-50 appeared to coincide completely (Fig. 6, merged turquoise color), indicating that antibody-induced clustering of Thy-1 at the extracellular leaflet of the plasma membrane caused a dramatic redistribution of the intracellular protein B-50, resulting in a concentration of B-50 in membrane domains with patched Thy-1. To demonstrate that this
phenomenon is specific for raft-associated proteins, we also stained the cells for syntaxin, a nonraft marker (Fig. 6). The staining of syntaxin (indicated by arrows) was mainly excluded from the Thy-1/B-50 clusters and only occasionally overlapped as indicated by the arrowheads (Fig. 6, right). Thus we conclude that syntaxin is excluded from Thy-1 clustered membrane patches and that most of the B-50 is included in DIGs.
DISCUSSION Recently, we have demonstrated that transient transfection of B-50 in Rat1 fibroblasts caused formation of filopodial extensions during initial spreading (Aarts et al., 1998). In those studies, we frequently observed membrane ruffling and pinocytotic activity in transfected cells with a lamellipodial or mixed morphology (L. H. J. Aarts, unpublished data). In the present study, we have examined the cell surface activity of B-50 in
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FIG. 6. Accumulation of B-50 in membrane domains formed by patched Thy-1 in primary hippocampal neurons. Thy-1 was antibody cross-linked and after brief washing in PBS cross-linking was performed with donkey anti-mouse-conjugated Cy5 (blue) antibodies at 4°C for 1 h. After fixation with 4% paraformaldehyde/methanol the hippocampal neurons were immunolabeled in the absence of detergent for B-50 (labeled with FITC, green) and syntaxin (labeled with TRITC, red). Shown from left to right are Thy-1 labeling after clustering, B-50 labeling, syntaxin labeling, and the merge of the three leftmost images. The four right images are enlargements of the areas indicated by the rectangles in the preceding four images, presented in the same order. Arrows point to the position of syntaxin (red) which is excluded from the Thy-1/B-50 patches. The arrowheads point to a membrane area where colocalization of Thy-1, B-50, and syntaxin is observed (white).
stably transfected Rat1 fibroblasts at later time points of growth. In some of these cells B-50 accumulates at discrete membrane domains that are actively involved in membrane ruffling and pinocytosis, the endocytotic pathway for plasma membrane raft domains (Brown and Waneck, 1992; Bamezai et al., 1992; Poussin et al., 1998). The difference in intracellular location of B-50 IR in active versus resting cells (Fig. 1) suggests that B-50 is actively targeted to specific, motile domains at the plasma membrane. This was indeed observed in living B-50/EGFP-transfected cells, in which recruitment of B-50/EGFP was correlated with an increase in local membrane ruffling (Fig. 3c). The high levels of B-50/ EGFP were shown to correlate with the generation of pinocytic vesicles (Fig. 3b), a process that is closely linked with high membrane activity (i.e. ruffling, BarSagi and Feramisco, 1986; Keller et al., 1990). This implies that B-50 potentiates both membrane ruffling and pinocytosis in Rat1 fibroblasts, as was confirmed by counting the number of pinosomes in B-50-transfected cells in comparison with untransfected cells. The molecular events that underlie formation of membrane ruffles are largely unknown but involve polymerization of actin immediately underneath the plasma membrane (Ridley and Hall, 1992). Indeed, intense F-actin staining was present at the membrane ruffles in B-50-transfected cells (Fig. 4), suggestive of a local increase in actin filament formation at sites with high levels of B-50. This corroborates our recent finding that the B-50-induced formation of filopodia in spreading Rat1 fibroblasts was accompanied by a profound increase in the concentra-
tion of cortical actin filaments, a process that was dependent on Rho-GTPase function (Aarts et al., 1998). Manipulation of the expression levels of B-50 in a variety of neuronal and nonneuronal cells has been shown to induce distinct morphological changes depending on cell type, expression level, and experimental conditions. Transfection of B-50 in nonneuronal cells resulted in the formation of filopodia during initial spreading (Zuber et al., 1989a; Widmer and Caroni, 1993; Aarts et al., 1998). This was also observed in the stably transfected Rat1 fibroblasts of this study in which more than 80% of the cells adopted a filopodial morphology during spreading (not shown). Enhanced expression of B-50 causes formation of filopodia, blebs, or even neuritelike extensions, probably depending on the cell type and the level of expression (Nielander et al., 1993; Verhaagen et al., 1994; Wiederkehr et al., 1997). In transgenic mice, overexpression of B-50 provoked the development of highly enlarged terminal fields and increased injuryinduced sprouting (Aigner et al., 1995). Using immunoelectron microscopy, Holtmaat and co-workers observed that overexpression of B-50 in distinct transgenic mice as well as in adenoviral vector-infected mice caused formation of large membranous labyrinths in olfactory axonal endings (Holtmaat et al., 1995, 1997). Interestingly, the B-50-related myristoylated alaninerich C-kinase substrate (MARCKS) has been demonstrated to influence membrane ruffling and cell spreading as well (Myat et al., 1997). Like B-50, MARCKS has been implicated in assembly of actin structures at the plasma membrane as well as in the assembly of adhesive structures (Aderem, 1995). Moreover, transfection
94 of B-50, MARCKS, and CAP-23 in fibroblasts induced similar dynamic structures at the cellular periphery (Wiederkehr et al., 1997). The ERM family of actinmembrane cross-linking proteins may represent functionally related proteins since they were shown to be enriched in and to influence the formation of adhesive structures and actin-based cell surface protrusions such as microvilli, filopodia, and membrane ruffles (Amieva and Furthmayr, 1995; Martin et al., 1995; Sainio et al., 1997). Although it is largely unknown how these functionally related proteins influence actin-membrane dynamics, they may make use of a common mechanism involving the Rho-GTPase (Hirao et al., 1996; Mackay et al., 1997; Aarts et al., 1998). In this study, we show that in transfected fibroblasts, B-50 is colocalized and coconcentrated with the GPIanchored cell adhesion molecule Thy-1 in the Golgi apparatus, at ruffling membranes, and on pinocytic vesicles (Fig. 2e). This may imply that Thy-1 and B-50 are cotransported from the Golgi apparatus to ruffling membranes and are subsequently retrieved together by pinocytic vesicles. Although retrograde transport of vesicular B-50 (Li et al., 1993) has been demonstrated in cultured neurons, it is not known whether these vesicles are generated by a pinocytic-like event in ruffling membranes within growth cones. Nevertheless, B-50 IR and Thy-1 appeared to be completely colocalized in both the cell bodies and the neurites of primary hippocampal neurons (Fig. 5a). Following treatment with Triton X-100, a significant fraction of B-50 IR remained clustered with Thy-1 in detergent-resistant membrane remnants (Fig. 5b), suggesting that both proteins are associated with the same Triton-insoluble membrane skeleton fraction in situ. Recently, B-50 was shown to be enriched in DIGs (Joliot et al., 1997; Maekawa et al., 1997). In the cellular milieu, DIGs have been proposed to represent membrane microdomains (rafts) that are involved in protein sorting, signal transduction, adhesion, and morphogenesis by concentrating a specific subset of proteins, including cell surface molecules such as GPI-anchored proteins and various, mostly doubly acylated, signal transduction proteins (Simons and Ikonen, 1997). However, detergent-induced in vitro or in situ clustering does not provide evidence for concluding a native association with rafts (Mayor and Maxfield, 1995). Therefore, we have studied the copatching behavior of B-50 and Thy-1 following Thy-1 antibody-induced clustering. It is assumed that antibody-mediated crosslinking causes multimerization of membrane microdomains as a consequence of specific lipid preferences of its constituents (Harder et al., 1998). Copatching may thus result from the coalescence of lipid microdomains
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containing cross-linked raft membrane components (Harder et al., 1998). The coclustering of the intracellular protein B-50 with the GPI-anchored extracellular membrane protein Thy-1 following antibody-mediated crosslinking (Fig. 6) indicates that both proteins share a preference for raft lipids. Syntaxin, a membraneassociated SNARE protein involved in exocytosis, does not cocluster with Thy-1 and B-50 in rafts (Fig. 6). It is tempting to speculate that B-50 is concentrated in the cytoplasmic leaflet of raft domains in vivo following clustering of specific GPI-anchored molecules (such as Thy-1, NCAM120, axonin/TAG-1) at the extracellular site of raft domains. The accumulation of B-50 would subsequently result in local alterations in the underlying actin cytoskeleton, ultimately leading to changes in neuritic growth.
EXPERIMENTAL METHODS Cell Culture and Transfection Experiments Wild-type Rat1 fibroblasts were cultured in DMEM (high glucose, 4.5 g/L) supplemented with 10% fetal calf serum (FCS), 100 U penicillin/streptomycin, and L-glutamine (2 mM) in a humidified atmosphere at 37°C and 7% CO2. Stable transfection was performed by cotransfection of a B-50 expression construct (in pCDNA1; Invitrogen, San Diego, CA; Nielander et al., 1993) with the puromycin-resistance plasmid pBabePuro at a concentration ratio of 1:10 in a 6-cm dish using lipofectin (Life Technologies). The day after transfection, puromycin was added to the culture to a final concentration of 1 µg/ml. Resistant clones were visible within 6 days of transfection. Among several stably transfected cell lines one cell line, designated B14, was selected for further research. After being seeded on 12-mm acidcleaned glass coverslips, cells were grown for 30 h in the presence of 10% FCS before fixation with 4% paraformaldehyde (20 min at 4°C). Transfection of Rat1 fibroblasts with syntaxin cDNA was performed with the plasmid pCMV-HPC-1a (a gift from M. Verhage) using calcium phosphate precipitation. Hippocampal cell cultures were obtained by dissection of rat hippocampi (embryonal day 19) as described (Banker and Cowan, 1977). Briefly, hippocampi were dissociated by trypsinization and trituration and plated on poly-L-lysine-coated glass coverslips in B27-supplemented Neurobasal medium (Life Technologies). After attachment of cells to the substrate, coverslips were transferred to dishes containing monolayer cultures of astroglia in chemically defined medium (DMEM (high glucose, 4.5 g/L), 5 µg/ml insulin, 0.5 µg/ml transfer-
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rin, 20 µg/ml progesterone, 100 µM putrescine, 30 µM SeO2, 90 mg/L bovine serum albumin, and penicillin/ streptomycin (each 100 IU/ml)), without being in physical contact. Cells were grown for 14 days before antibody cross-linking or fixation with 4% paraformaldehyde (20 min at 4°C).
BSA) and incubated for 1 h at 4°C with anti-Thy-1 antibodies diluted in PBS⫹⫹ (monoclonal Ox-7, 1:20; Biotrend). After brief washing in PBS, further crosslinking was performed with donkey anti-mouse-conjugated Cy5 antibodies at 4°C for 1 h. Cells were fixed for 5 min in 4% paraformaldehyde at 4°C and subsequent incubation in methanol at ⫺20°C for 5 min before B-50 and syntaxin immunolabeling.
Immunocytochemistry and Microscopy Following fixation, cells were washed with phosphatebuffered saline (PBS) and incubated in 1 M NH4Cl in PBS for 10 min. Subsequently, cells were incubated in a blocking buffer (0.5% BSA, 2% normal goat serum, and 0.1% saponin in PBS (or instead 0.05% Triton X-100 or no detergent, when indicated)) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the cells overnight at 4°C. After washing and incubation with secondary antibodies (1 h, 37°C), cells were embedded in Dabco/Mowiol and observed with an upright Leica TCS NT confocal laser scanning microscope (Heidelberg, Germany). B-50 immunoreactivity was visualized with the specific monoclonal antibody NM4 (1:4000; Mercken et al., 1992) or with the polyclonal antibody 8613 (1:50 or 1:300; Verkade et al., 1996). Phalloidin–FITC (Sigma Chemical, St. Louis, MO; 2 µg/ml) and WGA–FITC (Sigma; 40 µg/ ml) were included along with the secondary antibody incubation. Antibodies directed against organellespecific markers include anti-CTR433 (monoclonal, 1:100; a gift from Dr. Van der Sluijs), anti-TGN-38 (polyclonal, 1:100; a gift from Dr. Banting), anti-rab5 (monoclonal, 1:20), anti-rab7 (polyclonal, 1:50; a gift from Dr. Chavrier), antibody 1d3, specifically detecting the ER (monoclonal, 1:50; a gift from Dr. Fuller), and anti-syntaxin antibody I378 (polyclonal, 1:100; a gift from Dr. Verhage; McMahon et al., 1995). Monoclonal anti-Thy-1 (1:100) was obtained from Sera-Lab for immunolabeling (Sussex, England). Time-lapse recordings of living Rat1 fibroblasts were performed at 37°C and 7% CO2 in a microchamber mounted on an inverted Leica TCS NT confocal laser scanning microscope (Heidelberg, Germany). Rat1 fibroblasts were transfected with pB-50/EGFP, a fusion construct bearing the open reading frame of B-50 cloned directly upstream of EGFP in pEGFP-N1 (Clontech, Palo Alto, CA; Aarts et al., 1998). After 24 h of growth in 10% FCS, time-lapse recordings were started with time intervals of 10 s.
Antibody-Induced Patching Hippocampal cells were washed twice in PBS⫹⫹ (PBS supplemented with 0.9 mM Ca2⫹, 1 mM Mg2⫹, and 0.2%
ACKNOWLEDGMENTS We thank Drs. Van der Sluijs, Banting, Chavrier, Verhage, and Fuller for providing the antibodies that made it possible to detect various intracellular vesicles and Bianca Hellias for culturing hippocampal cells. Moreover we thank Ruud F. G. Toonen for the syntaxin transfection of the fibroblasts and Liselijn A. B. Wisman for the subsequent immunocytochemistry. This work was supported by NWO Grant 903–42–006 and by the Prinses Beatrix Fonds, Grant 95–1008.
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