Correspondence to: Pico Caroni, Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Tel:41-61-697-3727 Fax:41-61-697-3976 E-mail:caroni{at}fmi.ch.
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Abstract |
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The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P2, and promote its retention and clustering. Binding and modulation of PI(4,5)P2 depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P2 modulation. In the neuronlike cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by ED mutants. Agents that globally mask PI(4,5)P2 mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(
ED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P2 modulating proteins, upstream of actin and cell cortex dynamics regulation.
Key Words: neurite outgrowth, cell spreading, anatomical plasticity, actin recruitment, lipid microdomain
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Introduction |
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Signaling at the cell surface integrates specific contextual information from the local environment by recruiting and assembling cell-specific subplasmalemmal protein complexes that regulate cell behavior. To control signal quality and strength, cells assemble signaling platforms and structures of varying composition, complexity, and stability. Actin-based structures are major components of cell-surface signaling complexes such as focal contacts, adherens junctions, caps, and supramolecular activation clusters involved in lymphocyte activation, and synapses in the nervous system (
At the cell surface, actin filament assembly and dynamics are subject to complex temporal and spatial control by signals from the extra- and intracellular environment (
GAP43, myristoylated alanine-rich C kinase substrate (MARCKS),1 and CAP23 (GMC) are major protein kinase C (PKC) substrates associated with the plasma membrane that can bind acidic phospholipids including PI(4,5)P2 (
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In this study, we present experimental evidence that GMC sequester and modulate PI(4,5)P2 at cholesterol-dependent microdomains. Constructs lacking the ED acted as dominant inhibitors of microdomain PI(4,5)P2 accumulation. The effects of GMC constructs on raft PI(4,5)P2, peripheral actin recruitment, and neurite outgrowth were highly correlated, and were comparable to those of agents that directly affect PI(4,5)P2. Based on these results, we propose that GMC are mechanistically related membrane-associated proteins that mediate calcium- and PKC-sensitive modulation of PI(4,5)P2 at plasmalemmal microdomains, upstream of cell cortex and actin dynamics regulation, and neurite outgrowth. In the accompanying paper (Frey et al., 2000), we provide compelling evidence that GAP43 and the related protein CAP23 are related functionally in vivo, where they have common as well as unique functions in neurite outgrowth. Together, these studies establish GMC as members of a family of mechanistically and functionally related proteins. Because of their shared effects on PI(4,5)P2 modulation, we propose the collective designation of pipmodulins.
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Materials and Methods |
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Reagents, Cell Culture, and Transgenic Mice
Inhibitor compounds and growth factors, with their final concentrations, were used as follows. Neomycin (10 mM), NGF (100 ng/ml), cyclodextrin (methyl-ß-cyclodextrin; 5 mM), and cytochalasin D (10 µM) from Sigma Chemical Co.; LiCl (10 mM) and calcimycin (50 µM) from FLUKA AG; and U-73122 (1 µM) from Calbiochem. For transfection, cDNAs were cloned into the eukaryotic expression vector pcDNA3 (Invitrogen). All mutant constructs were generated by conventional PCR techniques and verified by DNA sequencing. The MARCKS cDNA consisted of human MARCKS, with a modified C-term to match the chick sequence SPEGPAEPAE. Chick GAP43(ED) lacked amino acids 3953; pMARCKS(
ED) consisted of the human/chick protein, as described above, which lacked amino acids 152176, and carried an additional Ala3Cys mutation to provide a palmitoylation sequence. Antisera to COOH-terminal peptides of chick MARCKS and GAP43 were as described (
-bungarotoxin, and Alexa-labeled secondary antibodies were from Molecular Probes, Inc.
Cell lines (monkey kidney epithelial cells COS-7, and rat pheochromocytoma PC12 cells, clone B) were from American Type Cell Culture Collection, and cultured in DME supplemented with 10% FCS, or 10% horse serum and 5% FCS, respectively. Hippocampal neurons were isolated from newborn (P0-1) mice. In brief, hippocampi were triturated, cells were washed, resuspended in culture medium (neurobasal; GIBCO BRL), with 0.5 mM L-glutamine, 25 µM glutamate, and 1% B27 supplement (GIBCO BRL), and plated at a density of 20,000 cells per 18-mm poly-L-lysinecoated coverslip.
Transgenic mice expressing chick GAP43(ED), specifically in adult neurons, were generated using the mouse Thy1.2 expression cassette as previously described (
Transfections, Immunocytochemistry, and Analysis of the Actin Cytoskeleton
Liposome-based transfection reagents interfered with PI(4,5)P2 stainings (not shown). Therefore, for transient transfections, COS-7 cells were treated with the nonliposomal reagent Superfect (Qiagen). On the next day, cells were fixed for 30 min at 37°C, followed by 35 h at 4°C with 4% paraformaldehyde in DME with 2 mM EGTA. Subsequently, cells were further processed for immunocytochemistry as described in a previous study (
For quantitative analysis of GMC and PI(4,5)P2 clusters, all cells from randomly selected fields (400x) with substantial levels of transgene expression (3050% of all transgene expressing cells in any given field) were included in the analysis. At least 20 cells from one dish of transiently transfected cells were analyzed for every experiment, and the values are averages from at least two independent experiments. Images from 20 x 20 µm2 bins were captured and all clusters within the bin were analyzed with NIH Image software (see Fig 2).
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PC12B cells were transfected stably using the Fugene 6 reagent from Boehringer. Each experiment shown in the study was carried out with at least three independent clones, with similar results. For process outgrowth assays, 100,000 PC12 cells were plated on collagen-coated (30 µg/ml) 35-mm dishes, and, where indicated, the medium was changed 1 d after plating from DME, 10% horse serum, 5% FCS (growth medium) to DME, 1% horse serum, and 100 ng/ml NGF. Where process formation (>1 cell diameter) in the absence of NGF was monitored, cells were preincubated with or without neomycin for 2 h, replated in the presence or absence of the drug, and analyzed 3 h after replating. No preincubation was carried out when LiCl or the phospholipase C (PLC) inhibitor U-73122 was used.
To analyze the distribution of the actin cytoskeleton in PC12B clones, we determined intensity profiles of RITC-phalloidin labeling across randomly selected cells. Cells were plated on a collagen-coated substratum in the absence of NGF, fixed and stained 3 h after plating, photographed under identical conditions, and were analyzed with Image software. For each analyzed cell, one rectangular bin of 10 pixels in height was placed across the center of the cell (see also schematic in Fig 6 B), and an edge-to-edge labeling intensity profile was collected. For each type of PC12B clone, such profiles had reproducible characteristic features of actin cytoskeleton distribution, as revealed when the profiles were superimposed. To highlight shared features, each of six independent profiles (i.e., six cells) was assigned the same light gray value, and overlapping areas were integrated (see Fig 6 B).
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Subcellular Fractionation and Lipid Analysis
Raft fractions from 2-d hippocampal neuron cultures or adult mouse brain homogenate were isolated according to a standard protocol (
Binding of GMC proteins to phospholipids was determined with a sedimentation assay, as described previously (
Total masses of nonwater-soluble phosphoinositides were determined according to a standard protocol (
Analysis of Peripheral Nerve Regeneration and BotA-induced Sprouting
To analyze peripheral nerve regeneration, the right sciatic nerve of 23-mo-old mice was crushed at the level of the midthigh, by applying pressure for 20 s with a watchmaker tweezer, as previously described (
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Results |
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Codistribution of GMC with PI(4,5)P2 at Plasmalemmal Microdomains
To determine whether subplasmalemmal clusters of GAP43, MARCKS, and CAP23 immunoreactivity may coincide with sites of local PI(4,5)P2 accumulation, we carried out double labeling experiments with an antibody that binds specifically to PI(4,5)P2 (
Evenly patched GMC immunoreactivity patterns can be detected in both paraformaldehyde- or methanol-fixed cells (
To determine whether GMC are raft components, we analyzed corresponding subcellular fractions from neonatal hippocampal neurons, mouse brain homogenates, and neuronlike PC12 cells. The GPI-linked cell-surface protein Thy1 is a well-characterized raft component (
GMC Bind PI(4,5)P2 and Promote PI(4,5)P2 Microdomain Assembly Independent of Actin Cytoskeleton Integrity
To determine whether GMC can influence PI(4,5)P2 -containing domains at the cell surface, we compared PI(4,5)P2 labeling patterns in hippocampal neurons, PC12B cells, and COS cells expressing different levels of these proteins. As shown in Fig 2 A, cells overexpressing GMC exhibited substantially larger macroscopic PI(4,5)P2 clusters. In addition, MARCKS-overexpressing cells (but not those overexpressing GAP43 or CAP23; not shown) also exhibited stronger PI(4,5)P2 cell-surface staining (Fig 2 A). To exclude the possibility that the effects of GMC on the labeling pattern of PI(4,5)P2 were related to the labeling process for these transgenes, we analyzed cells that had been transfected with a bicistronic construct driving the expression of the green fluorescent protein and MARCKS in the same transiently transfected cells. Cells expressing green fluorescent protein, and thus also MARCKS, exhibited larger, more numerous clusters and stronger labeling for PI(4,5)P2 (not shown). Therefore, overexpression of GMC augments the formation of macroscopically detectable plasmalemmal PI(4,5)P2 clusters.
To determine whether GMC proteins bind directly to PI(4,5)P2, we carried out cosedimentation experiments with recombinant proteins and lipid vesicles. Binding of GMC to liposomes depended on the presence of acidic phospholipids such as PI(4,5)P2, and on the presence of the basic ED (Fig 2 B). Mutant GAP43(Ser42Asp), which does not bind calmodulin and codistributes with GMC at plasmalemmal microdomains (
Cell-surface lipid microdomains described so far are highly sensitive to the physical properties of the lipid environment and, in particular, the presence of cholesterol (
GAP43 and MARCKS can interact with actin filaments through their basic ED (
MARCKS and GAP43 Mutants Lacking the Basic Effector Domain Reduce Microdomain Size and Interfere with PI(4,5)P2 Retention at Plasmalemmal Clusters
GMC are acidic proteins, with one unique stretch of exclusively basic residues, that bind to acidic phospholipids like PI(4,5)P2 (Fig 2 A). Because of its palmitoylation, GAP43 does not require the presence of the ED to efficiently associate with the plasma membrane (ED) construct that still associated with the plasmalemma, we introduced an Ala3Cys point mutation to generate palmitoylated pMARCKS(
ED). In spite of the absence of the lipid-binding ED in the mutants, in double-transfected cells overexpressing MARCKS or GAP43, and GAP43(
ED) or pMARCKS(
ED), wild-type and mutant proteins codistributed at the cell surface (Fig 4 A, bottom row). Therefore, accumulation of MARCKS or GAP43 at the characteristic surface-associated clusters, where these proteins colocalize, does not depend on the presence of the ED.
To explore the effects of the ED-free mutants on cluster assembly, we monitored their effects on PI(4,5)P2 clusters. Cells expressing comparatively low levels of pMARCKS (ED) or GAP43(
ED) exhibited cell-surface PI(4,5)P2 immunoreactivity that colocalized with the mutant transgene at small clusters (not shown). In contrast, hippocampal neuron growth cones and COS cells expressing substantial levels of GAP43(
ED) or pMARCKS(
ED) exhibited transgene accumulation at numerous, regularly spaced, small plasmalemmal clusters, but were essentially devoid of plasmalemmal PI(4,5)P2 clusters (Fig 4A and Fig B). These results suggest that the presence of excess ED-free mutant interferes with the function of endogenous components involved in the recruitment of PI(4,5)P2, and cluster coalescence. Since these mutants only differ from MARCKS or GAP43 by the absence of the ED that binds to acidic phospholipids, it seems likely that interference is due to a dominant negative mechanism, involving accumulation of the competing, nonfunctional component at these PI(4,5)P2 sequestering platforms.
PI(4,5)P2 Microdomain Modulation by GMC Does Not Correlate with Alterations in Total Phosphoinositide Contents Nor Bradykinin-induced PI(4,5)P2 Breakdown
We next determined whether overexpression of GMC constructs alters lipid second messenger metabolism. First, we determined the total sizes of the PI, PIP, and PIP2 pools in stably transfected PC12B clones (ED) transgenes (see also Fig 6). These cells express low levels of endogenous MARCKS and CAP23 (not shown) and extremely low levels of endogenous GAP43 (
ED)-overexpressing cells did not exhibit alterations in bulk phosphoinositide levels (Fig 5 A). In contrast, overexpression of MARCKS produced a significant increase in phosphoinositide bulk levels in PC12B cells, a finding that is consistent with the higher PI(4,5)P2 immunoreactivity signals in cells transfected transiently with MARCKS. To determine whether overexpression of the GMC constructs affects signal-induced breakdown of PI(4,5)P2, we analyzed bradykinin-induced PI(4,5)P2 hydrolysis. This well characterized response to the activation of a G proteincoupled receptor involves the activation of PLC to hydrolyze PI(4,5)P2 into inositol triphosphate and diacylglycerol. When compared with wild-type PC12B cells, the levels of stimulus-induced PI(4,5)P2 metabolites were elevated in the presence of MARCKS, but not GAP43 (Fig 5 B) nor pMARCKS (
ED) (not shown). These results are consistent with the interpretation that MARCKS-overexpressing cells contain higher levels of the PLC substrate PI(4,5)P2, whereas GAP43 or pMARCKS(
ED) cells do not. Because GAP43 or pMARCKS(
ED) overexpression did not alter bulk phosphoinositide levels nor stimulus-induced PI(4,5)P2 breakdown, the results show that accumulation at PI(4,5)P2-containing microdomains and augmentation or reduction of domain numbers and size does not, by itself, affect overall levels of PI(4,5)P2 hydrolysis by activated PLC. As discussed below, the additional effects of MARCKS on bulk phosphoinositide levels may be due to the higher density of positive charges of its ED.
Coordinate Regulation of PI(4,5)P2 Microdomains and PI(4,5)P2-sensitive Peripheral Actin Structures by GMC Constructs
GMC proteins and PI(4,5)P2 have both been implicated in regulating the accumulation of actin structures at the plasmalemma (ED) or pMARCKS(
ED) suppressed the accumulation of peripheral actin structures in response to NGF (Fig 7 A). These findings are consistent with the view that PI(4,5)P2 microdomain modulation by GMC promotes the assembly and accumulation of plasmalemmal actin-based structures.
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To investigate more directly the possible involvement of PI(4,5)P2 in GMC-regulated actin dynamics, we carried out experiments in the presence of 10 mM neomycin. This specific PI(4,5)P2 sequestering agent (Fig 1 B) had three major effects. First, it mimicked the overall effects of GMC on the distribution of the actin cytoskeleton, dramatically potentiating the accumulation of peripheral actin-based structures, particularly filopodia, at the plasmalemma, and greatly reducing the pool of comparatively amorphous cytosolic and perinuclear filamentous actin (Fig 6A and Fig B, and Fig 7 B). Second, it partially counteracted the effects of the dominant negative ED mutants (Fig 7 B). Third, it counteracted the effects of GMC, with respect to the local accumulation of larger actin structures, cell polarization (Fig 6 and Fig 7 B), and neurite outgrowth (Fig 8). As a result, cells treated with neomycin spread in radially symmetrical, round shapes, with dense accumulations of evenly distributed actin-based filopodia and lamellae at their edges. The effects of neomycin were mimicked most closely by the GAP43(Ser42Asp) mutant that does not bind calmodulin (CaM) and cannot be phosphorylated by PKC (Fig 6 B and 7 A), suggesting that regulation of GMC proteins by calcium/CaM and PKC may promote the local accumulation of larger actin structures (see Fig 10). The neomycin effects did not depend on the presence of NGF, which, like GMC proteins, potentiated the accumulation of larger peripheral actin structures, particularly spikes (Fig 6 A). Interestingly, GMC and NGF had similar effects on neomycin-treated PC12 cells, and their combination partially rescued actin accumulation polarity at the plasmalemma of these cells (Fig 6 A), suggesting that with respect to actin regulation, GMC proteins and NGF may control synergistic pathways. Because it prevents the access of this enzyme to its substrate, neomycin is frequently used as an inhibitor of phospholipase C (PLC). However, direct inhibition of PLC with U-73122 had an effect opposite to that of neomycin: it reduced spreading and suppressed dynamic actin structures at the plasmalemma (Fig 6A and Fig B, and Fig 7 B). As discussed below (Fig 6 A, schematic), these findings are consistent with the interpretation that, under local resting conditions, unmasked plasmalemmal PI(4,5)P2 sequesters actin-regulating proteins that promote actin structure dynamics, thus, indirectly stabilizing the actin-based cortical cytoskeleton, and preventing the formation of dynamic actin structures involved in protrusive activity and spreading.
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Critical Role of Plasmalemmal GMC for Neurite Outgrowth in PC12 Cells
To investigate possible biological implications of microdomain and actin regulation by GMC proteins, we analyzed NGF-induced neurite outgrowth in stable PC12B clones overexpressing wild-type and ED-deficient constructs. As expected (ED) or pMARCKS(
ED) suppressed NGF- induced neurite outgrowth (Fig 8 A). These findings are entirely consistent with the effects of GMC constructs on PI(4,5)P2-containing microdomains and actin dynamics, and provide evidence for a critical role of GMC protein-mediated regulation in neurite outgrowth.
To determine whether GMC can induce process outgrowth in these cells in the absence of NGF, we monitored attachment and spreading of PC12 clones on a collagen substratum. Unlike wild-type cells, GMC-overexpressing cells formed neuritelike processes during spreading in the presence of high serum concentrations and no NGF (Fig 8 B). Under these experimental conditions, process formation was transient: it peaked at ~3 h after plating, and processes disappeared after 15 h (not shown). In contrast to those formed in the presence of NGF, processes were short, not exceeding 50 µm in length, and were not affected by the presence of the PI3-kinase inhibitor LY294002 (not shown). The specific PI(4,5)P2 sequestering agent neomycin promoted symmetrical cell spreading, and prevented spontaneous process formation (Fig 8 B) and NGF-induced neurite outgrowth (not shown, but see Fig 7 B) in GMC-overexpressing cells. Comparable effects were obtained with 10 mM LiCl (Fig 8 B), which inhibits the metabolism of phosphoinositides, and greatly reduces the levels of plasmalemmal PI(4,5)P2 (Fig 1 B). In contrast to neomycin, U-73122, which directly inhibits PLC, effectively inhibited cell spreading (see also Fig 6 A).
NGF-induced neurite outgrowth involves the activity of PI3-kinases (ED mutants affected the accumulation of the 3-phosphoinositides PIP3 (Fig 8 D) or PI(3,4)P2 (not shown) in response to NGF, which is consistent with the notion that the regulation of neurite outgrowth by GMC proteins specifically involves PI(4,5)P2.
Role of GMC-mediated Regulation in Peripheral Nerve Regeneration and Stimulus-induced Nerve Sprouting at the Neuromuscular Junction in the Adult
Although GAP43 has been implicated in axonal growth, GAP43-deficient mice only exhibit restricted axonal pathfinding defects (ED) specifically in neurons. We used a mouse Thy1.2 expression cassette, thus, achieving high expression levels restricted to postnatal neurons, including motoneurons and their neuromuscular synapses (Fig 9 A;
In control wild-type mice, nerve terminal branches at the neuromuscular junction exhibit a regular decrease in diameter as they elongate towards the periphery of the synapse (Fig 9 B). This characteristic pattern was detected at most adult gastrocnemius muscle synapses that we examined (at least 465/500 synapses from three mice). In contrast, in GAP43(ED)-overexpressing mice, nerve terminal branches were irregular in shape, with frequent enlargements at their tips (Fig 9 B). In addition, the nerve frequently exhibited a strikingly circular course (Fig 9 B). The circular course was reminiscent of CAP23-/- neurons, where the phenotype could be phenocopied by cytochalasin D, supporting the notion that it was due to a defect in the actin cytoskeleton (
GAP43 and CAP23 promote nerve sprouting at the neuromuscular junction (ED) on stimulus-induced nerve sprouting, we paralyzed lower hindlimb muscles of transgenic mice with botulinum toxin A. 7 d after toxin treatment, >95% of the synapses in the soleus muscle of wild-type mice exhibit robust ultraterminal sprouting, whereas sprouting in the medial gastrocnemius is much less pronounced, and is restricted to slow-type synapses (
ED) (Fig 9 D). Sprouting in the absence of toxin treatment was minimal (Fig 9 D), suggesting that the presence of GAP43(
ED) interfered with negative control of stimulus-induced sprouting at these synapses. When compared with those induced in wild-type mice, sprouts were strikingly curved, with frequent side branches and local expansions (Fig 9 C). Again, these features were reminiscent of the abnormal neurite outgrowth patterns in CAP23-/- or cytochalasin Dtreated neurons (
Finally, to explore the role of GMC proteins in axonal regeneration, we crushed the sciatic nerve of Thy1-GAP43 (ED) mice and monitored peripheral nerve regeneration. As shown in Fig 9 (E, F), GAP43(
ED) inhibited regeneration and reinnervation of skeletal muscle in a dose-dependent manner. Ultrastructural analysis of myelinated axonal profiles proximal to the crush site revealed normal numbers in the mutant mice (not shown). In addition, although with a substantial delay, skeletal muscle was apparently fully reinnervated also in the transgenic line expressing high levels of GAP43(
ED). Therefore, the presence of excess GAP43(
ED) greatly delayed, but did not completely prevent peripheral nerve regeneration. These results are consistent with the in vitro data with PC12 cells, and indicate that regulation by GMC proteins plays a critical role in axonal outgrowth in vivo.
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Discussion |
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We have provided evidence that GAP43, MARCKS, and CAP23 regulate plasmalemmal microdomain PI(4,5)P2, cell cortex actin recruitment, and morphogenic processes such as neurite outgrowth. The effects of GMC constructs on the actin cytoskeleton and morphogenic processes correlated closely with those on plasmalemmal PI(4,5)P2, and were mimicked by pharmacological agents that act on PI(4,5)P2, which is consistent with the notion that a main function of these proteins is to locally modulate PI(4,5)P2, upstream of actin cytoskeleton and cell cortex regulation. In the following sections, we discuss the regulation of PI(4,5)P2 microdomains by GMC-like proteins and their function in actin dynamics, neurite outgrowth, and anatomical plasticity.
A Lipid Microdomain that Brings Together PI(4,5)P2 and the Plasmalemma-associated Proteins GAP43, MARCKS, and CAP23
The results of this study suggest that PI(4,5)P2 and GMC proteins accumulate together at a subtype of cholesterol-rich plasmalemmal microdomains found in many, and possibly all types of cells. Thus, endogenous and transgenic GMC accumulated in cyclodextrin-sensitive raft fractions (Fig 1 D;
Definitive information about the properties and the regulation of GMC-PI(4,5)P2 microdomains will require further studies, including their biochemical isolation, and molecular identification of their lipid and protein components, as well as a characterization of their dynamic properties. However, some conclusions can already be drawn from this study. First, like previously described rafts, they are sensitive to cholesterol-depleting drugs, implying that significant domain promoting forces possibly related to those operating in sphingolipid rafts reside within the lipid environment. Second, direct interactions between PI(4,5)P2 and GMC proteins appear to promote PI(4,5)P2 retention at the domains. Thus, overexpression of GMC, induced larger macrodomains, and partially counteracted cyclodextrin-induced loss and dispersion of plasmalemmal PI(4,5)P2. In contrast, effector domain mutant accumulation may reduce the net binding capacity of the domain for PI(4,5)P2, thus, reducing masking and retention of this lipid second messenger. Along similar lines, the higher total contents of PI(4,5)P2 in MARCKS, but not in GAP43-overexpressing cells may be due to the fact that the ED of MARCKS has a significantly higher density of basic residues (13 lysine and arginine out of a total of 25 residues in MARCKS, versus 8/23 in GAP43), thus, binding PI(4,5)P2 with higher avidity. These combined findings suggest that direct electrostatic interactions between the ED-containing proteins and acidic phospholipids, including PI(4,5)P2, may be involved in domain stabilization. However, additional, presently unidentified, components are likely to be involved in microdomain nucleation and recruitment. Thus, for example, since mutant constructs of MARCKS or GAP43 lacking the ED codistribute with the wild-type proteins and PI(4,5)P2, protein interactions not involving the ED must be involved in GMC targeting to these domains.
With respect to their regulation, an important difference to previously described rafts is that while sphingolipid/GPI-linked protein complexes occupy the outer leaflet of the plasmalemma, where they can be regulated by signals from the extracellular environment, GMC-PI(4,5)P2 complexes are located at the inner leaflet. The interaction of the basic domains of GMC with acidic phospholipids, including PI(4,5)P2, is subject to regulation by several signaling pathways (
Functions of GMC Proteins in Actin Regulation, Neurite Outgrowth, and Anatomical Plasticity
The question of whether GMC are signaling or structural proteins, and what, if any, are their downstream targets has been difficult to address experimentally in the past. Because they can be such abundant proteins, and because their EDs interact with several molecules involved in signaling, including calcium/CaM, PKC, PI(4,5)P2, and actin filaments, it has been suggested that MARCKS and GAP43 may regulate the pools of any of these interacting molecules. However, we now find that GMC can modulate PI(4,5)P2 independent of binding to CaM or actin cytoskeleton integrity, and that all effects of GMC on the actin cytoskeleton, cell spreading and neurite outgrowth correlate with their effects on microdomain PI(4,5)P2. The global effects on cell spreading and peripheral actin recruitment are mimicked by drugs that reduce the availability of PI(4,5)P2, whereas the effects on cell polarization and process formation are, in part, prevented by these same drugs. This may reflect the regulatory cycle outlined above, i.e., masking of PI(4,5)P2 under resting conditions, which is mimicked by neomycin, and local unmasking of clustered PI(4,5)P2 in response to calcium/CaM and/or PKC activation, which is not mimicked by neomycin (Fig 10). This interpretation is also consistent with the observation that GAP43(Ser42Asp), which does not bind CaM, cannot be regulated by PKC, but still binds PI(4,5)P2, affected the actin cytoskeleton in a manner that was particularly reminiscent of that of neomycin (Fig 6 B and 7 A). Therefore, we propose that PI(4,5)P2 is a main effector of GMC, and that, rather than being regulated by GMC, calcium/CaM and PKC regulate the interactions of GMC with PI(4,5)P2. Accordingly, a main function of GMC would be to act as PI(4,5)P2 modulatory pipmodulins, to retain and mask PI(4,5)P2 at plasmalemmal lipid microdomain platforms, where they would couple its availability for actin cytoskeleton and cell cortex regulation to signal transduction pathways involving calcium/CaM and PKC (Fig 10). In addition to modulating PI(4,5)P2, MARCKS and GAP43 can also interact directly with actin filaments (
How does GMC-modulated PI(4,5)P2 affect the actin cytoskeleton and cell-surface activity? Based on the results of this study, the fact that GMC expression correlates with cell cortex dynamics, and the known effects of PI(4,5)P2 on the actin cytoskeleton and the cell cortex, we suggest that GMC levels affect local PI(4,5)P2 availability, that in turn directly controls the activity of key actin regulating proteins. This model is consistent with recent evidence that PI(4,5)P2 promotes membranecortical cytoskeleton interactions (
The PI(4,5)P2 modulatory mechanism suggested by our results is likely to be operating throughout the cell surface. Thus, although some local accumulation can be detected at sites of cellsubstratum attachment and ruffling activity, the immunocytochemistry data suggest the presence of substantial amounts of evenly distributed PI(4,5)P2 and GMC throughout the cell cortex. However, because it should be affected by the intrinsic, cell-specific properties of the cortical cytoskeleton, and by the local accumulation of structural, regulatory, and signaling components that initiate and regulate actin structure formation, its outcome is likely to exhibit highly cell specific and local features. Taking all these factors into account, actin regulation by GMC and PI(4,5)P2 may involve the following mechanisms (see also Fig 10). First, under local resting conditions, with low contents of components such as Arp2/3, CapZ, and activated Rho-type GTPases that promote the formation of dynamic structures, and with low levels of GMC proteins, accessible PI(4,5)P2 would favor cortical actin cytoskeleton stability, suppressing local dynamics and inhibiting membrane fusion. Second, under the same conditions, but with higher levels of GMC proteins, PI(4,5)P2 masking would lower the threshold for cell-surface dynamics, promoting processes such as cell spreading and membrane fusion. Third, in the presence of signals and components that promote the formation of actin structures, for example, at a forming axonal growth cone or phagosome, stimulus-induced release of PI(4,5)P2 from masking by GMC would support local actin filament assembly, coupling this process to regulation by calcium/CaM and PKC signals. Overall, according to this model, the expression of proteins such as GAP43, MARCKS, or CAP23 would promote and regulate cell-surface dynamics, phagocytosis, cell attachment, and regulated morphogenic processes such as neurite outgrowth (Fig 10). These predictions are in good agreement with a wealth of data from cultured cells and genetically modified mice.
The effects of GMC constructs on neurite outgrowth in PC12 cells, peripheral nerve regeneration, and stimulus-induced nerve sprouting at the neuromuscular junction of soleus muscle provide strong experimental evidence for a critical role of GMC proteins in promoting process outgrowth. Together with those of the accompanying paper (ED) in neurons not only impaired proper axonal regeneration and synaptic sprouting, but also paradoxically allowed paralysis-induced sprouting at neuromuscular synapses that do not sprout in wild-type mice. Such sprouts were abnormal in shape and exhibited several features reminiscent of cytochalasin Dtreated or CAP23-/- neurons. It is well established that disrupting the actin cytoskeleton of pioneer neurons in situ abolishes growth cone guidance and induces extensive twisted growth of neurites (
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Footnotes |
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1 Abbreviations used in this paper: CaM, calmodulin; GMC, GAP23, MARCKS, and CAP23; MARCKS, myristoylated alanine-rich C kinase substrate; ED, effector domain; PKC, protein kinase C; PLC, phospholipase C.
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Acknowledgements |
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We thank S. Arber, J. Hagmann, W. Krek, and U. Mueller (Friedrich Miescher Institute), and E. Stoeckli (University of Basel) for valuable comments on the manuscript.
Submitted: 27 October 1999
Revised: 23 May 2000
Accepted: 24 May 2000
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References |
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