Correspondence to Brett P. Lauring: bl320{at}columbia.edu
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Introduction |
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The function of Spastin is unclear. Neuronal suppression of Drosophila melanogaster Spastin using RNAi results in undergrowth of the neuromuscular junction and increased synaptic currents, whereas overexpression results in decreased currents (Trotta et al., 2004).
AAA proteins often function as oligomers, with each protomer usually containing one or two AAA ATPase modules. These domains share several key structural motifs and are homologous to some degree among all AAA proteins. NH2-terminal domains of AAA family members that bind adaptors or directly to target proteins are usually highly divergent and, therefore, are determinant of specificity of cellular function. This portion of Spastin contains an MIT domaina domain of unknown function (Ciccarelli et al., 2003) found in several proteins that function in the endosome, including VPS4/SKD1 (an AAA ATPase involved in endosomal trafficking), and in sorting nexin 15. Interestingly, another hereditary spastic paraplegia gene, Spartin, contains an MIT domain.
Although the MIT domain mediated interaction of Spastin with the endosomal protein CHMP1B (Reid et al., 2005) raises the possibility an endosome-related function, Spastin's AAA ATPase domain (Frohlich, 2001; Frickey and Lupas, 2004) is most homologous to those found in VPS4, Fidegtin, and Katanin, a microtubule-severing AAA ATPase (McNally and Vale, 1993). First purified from mitotic sea urchin extracts, Katanin has been shown to play a role in microtubule reorganization at the onset of mitosis (McNally and Vale, 1993; McNally and Thomas, 1998). In neurons, Katanin appears to be a source of noncentrosomal microtubules (Ahmad et al., 1999). Katanin overexpression in cultured mammalian neurons or in transgenic D. melanogaster alters process outgrowth (Karabay et al., 2004) and results in developmental abnormalities of the mushroom body (Nicolai et al., 2003), respectively.
Spastin overexpression in cultured cells or in D. melanogaster tissues results in decreased microtubule content (Errico et al., 2002; Sherwood et al., 2004; Trotta et al., 2004) by an unknown mechanism. Based on the homology of Spastin's ATPase domain to that found in Katanin, Spastin has been hypothesized to have microtubule-severing activity. However, overexpression of many types of proteins including tubulin isoforms (Bhattacharya and Cabral, 2004), tubulin folding cofactors (Martin et al., 2000), and others (Antonsson et al., 1998) results in decreased microtubule content. To determine the precise mechanism by which Spastin alters microtubule content, we used video microscopic and biochemical assays to demonstrate that recombinant Spastin makes internal breaks along the lengths of microtubules both with purified in vitro assembled microtubules and when added to permeabilized cytosol extracted fibroblasts stably expressing GFP-tubulin. We show that Spastin is sufficient for severing, and that several disease-associated mutations in Spastin abolish both ATPase and severing activities.
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Results |
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To determine whether the enzymatic activity of Spastin is altered by disease-associated missense mutations, many of which change conserved residues in the AAA domain (Fonknechten et al., 2000; Lindsey et al., 2000), we produced and purified recombinant GST-Spastin (Fig. 1 A). Recombinant GST-Spastin hydrolyzes ATP with a Km and Vmax in the range for those reported for other AAA ATPases (Fig. 1, BD). None of the three disease-associated mutants tested had significant enzymatic activity (Fig. 1 E). Two of these mutations are in the P-loop (K388R and N386K), but the third involves the arginine finger motif (R499C). Removal of the NH2-terminal GST tag by site-specific proteolysis did not change ATPase activity (not depicted). Q347K Spastin lacked activity (not depicted, see also Fig. 2 h). Neither Spastin, with a canonical Walker A mutation (K388A) designed to prevent ATP binding, nor Spastin harboring a Walker B (E442Q) mutation designed to allow ATP binding but prevent hydrolysis, showed any significant activity. This suggests that the measured ATPase activity with WT enzyme results from Spastin's activity and not from contaminating ATPases.
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E442Q Spastin, which is predicted to bind but not hydrolyze ATP and thus remain kinetically trapped on target proteins, shows a filamentous pattern (Fig. 2 b). These filaments represent a subset of microtubules and are even observed in SW cells that lack cytosolic intermediate filament proteins (Sarria et al., 1994), suggesting that the filaments are indeed microtubules (unpublished data). Furthermore, nocodazole treatment abolished this pattern of Spastin fluorescence (Errico et al., 2002; unpublished data). These results suggest that, upon ATP binding, Spastin targets to microtubules. As a negative control, we engineered a canonical Walker A motif P-loop mutation involving an invariant Lys (position 388 in Spastin). Typically, mutation of this Lys to Ala results in an enzyme that does not bind ATP (Babst et al., 1998). In contrast to E442Q Spastin, K388A Spastin does not decorate microtubules (Fig. 2 f).
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Consistent with the idea that microtubules are a Spastin target, overexpression of WT Spastin results in a decreased steady-state number of both dynamic and stable microtubules in transfected cells (Fig. 2 a and Fig. 3, ad) as assessed by staining with antibodies specific for either Tyr or detyrosinated Glu-tubulin (Gundersen et al., 1984). We also examined the consequences of overexpression of representative Spastin mutants, including one that shows a primarily filamentous pattern and one that shows the punctate pattern. Compared with neighboring untransfected cells, the content and arrangement of both stable and dynamic microtubules appear relatively normal in the K388A expressing cell (Fig. 3, il). As seen in Fig. 2, the Spastin mutants that showed the filamentous pattern only decorated a subset of microtubules in cells, often centrally and surrounding the nucleus. To determine whether this decorated subset represents stable microtubules, we analyzed E442Q YFP-Spastin expressing by immunofluorescence microscopy using tubulin antibodies specific for Tyr and Glu tubulin. The latter antibody stains stable microtubules. In the majority of cells examined, overexpression of E442Q Spastin results in an increased content of stable microtubules (Fig. 3 f, compare nontransfected cells with transfected cell). Often, but not in every cell, the extent of Spastin colocalization with microtubules enriched in Glu-tubulin is greater than those enriched in Tyr-tubulin. Overexpression of Spastin mutants showing the filamentous pattern also causes bundling of decorated microtubules. At least in these overexpression experiments, filamentous Spastin mutants appear to act like a MAP, in that they stabilize and bundle microtubules. Similar results were obtained when we stained for acetylated rather than Glu tubulin and with other filamentous Spastin mutants (unpublished data).
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Discussion |
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It is currently unclear whether the two severing enzymes subserve different functions. Nevertheless, our demonstration that a second AAA ATPase is capable of microtubule severing suggests that cells use this means of regulating microtubule dynamics in a broad array of circumstances. Both Katanin and Spastin are expressed in a wide variety of neurons and nonneuronal tissues (Hazan et al., 1999; Charvin et al., 2003; Wharton et al., 2003; Karabay et al., 2004). Though Katanin and Spastin share the ability to make internal breaks in microtubules, they only share homology in the AAA region and their NH2-terminal regions are quite distinct. This might indicate use of distinct adaptor proteins or may indicate different biochemical mechanisms of action.
Whereas the precise role of Spastin in unclear, the disease phenotype where axons of some long neurons degenerate after apparently normal central nervous system development suggests that the microtubule-severing activity may be important in axonal maintenance and that Katanin is not able to compensate for Spastin deficiency in this circumstance. The recent finding that loss of D. melanogaster Spastin paradoxically results in decreased microtubule content at the neuromuscular junction with resultant decreases in transmitter release suggests that Spastin may be required for maintenance of axon terminals (Sherwood et al., 2004).
A growing number of neurodegenerative disorders are linked to microtubule function. Mutations in microtubule motors including KIF5a (Reid, 2003) and dynein complex components (Puls et al., 2003) cause hereditary spastic paraplegia and motor neuron degeneration, respectively. Tau, a microtubule-associated protein, is mutated in chromosome 17linked fronto-temporal dementia and accumulates in neurofibrillary tangles in Alzheimer's disease (Lee et al., 2001). Abnormalities of microtubule destabilizing enzymes can now be considered capable of contributing to neurodegeneration.
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Materials and methods |
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Malachite green ATPase assay
ATPase activity was measured using the malachite green colorimetric assay using sodium phosphate as a standard. Reactions (typically 20 µl) containing the concentrations of enzyme and ATP indicated in the figures were incubated at 37°C for the times indicated in the figures. Next, the absorbance (OD650nm) was recorded. Finally, the absorbances of control samples containing the same amount of ATP, but lacking the enzyme, were subtracted from all values.
Transfection, cell culture, and immunofluorescence
TC7 or NIH 3T3 cells stably transfected with GFP-tubulin were maintained in DME supplemented with 10% FCS, penicillin, and streptomycin at 37°C with 5% CO2. For transient transfections, cells were grown on coverslips in 12-well dishes and transfected with YFP-Spastin constructs using Transfectin (Bio-Rad Laboratories). After 2448 h, cells were rinsed and fixed with methanol for 5 min at 20°C. Cells were blocked in TBS containing 10% normal donkey serum and 0.2% Triton X-100 for 1 h at room temperature. Primary antitubulin antibody (YL1/2 [Chemicon] or antiGlu-tubulin [Gundersen et al., 1984] 1:500 dilution) was diluted in blocking buffer and applied to cells for 1 h at 25°C. After three 5-min washes, secondary antibody (Cy-3 or Cy-5labeled donkey antirat; Jackson ImmunoResearch Laboratories) was applied for 1 h at 25°C. Still images were obtained using an Optiphot (Nikon) with a 60x objective. Photographs were obtained using a cooled CCD camera (Princeton Instruments) and processed using Metamorph software.
Microtubule-severing assay in permeabilized cells
TC 7 or NIH 3T3 cells were rinsed three times in prewarmed PEM buffer at 37°C on a heating block, and then were permeabilized by addition of prewarmed PEM containing 0.2% Triton X-100 for 60 s. After aspiration, the microtubules in the permeabilized cells were stabilized by addition of PEM containing 10 µM taxol. Cells were rinsed in PEM/taxol, and all further manipulations were performed at 37°C.
For severing reactions, recombinant Spastin in enzyme buffer was diluted to 80 nM in PEM buffer. This mixture was added to cells and reactions were initiated by adding ATP to 0.25 mM or the concentration indicated in the figures. Reactions were stopped by removal of the enzyme and fixation in cold methanol. Microtubule content was assessed either by immunofluorescence or real-time imaging of GFP-tubulin.
Microtubule severing with fluorescently labeled microtubules
We used similar conditions to those described in Hartman et al. (1998). Flow cells were constructed by taping coverslips together with strips of double-sided tape placed 5 mm apart. Chambers hold 1015 µl. The chambers were first perfused with 10 µl of 15 µM of mutant rigor kinesin (E164A) provided by Susan Gilbert (University of Pittsburgh, Pittsburgh, PA). After 10 min, chambers were blocked with 10 µl of 1 mg/ml casein. Next, microtubules were assembled by incubating tubulin and rhodamine-tubulin at a ratio of 9:1 (Cytoskeleton, Inc.) at a final concentration of 2 mg/ml in PEM buffer (Pipes, pH 6.95, 1 mM EDTA, and 2 mM MgCl2) with 1 mM GTP and 20 µM taxol for at least 1 h at 37°C. These were diluted 10-fold before perfusion. Rhodamine-labeled microtubules (10 µl of 0.2 mg/ml stock) were perfused into chambers. After 3 min, chambers were washed with 50 µl PEM buffer containing 1 mM ATP, and with an oxygen-scavenging cocktail to prevent photodamage (220 µg/ml glucose oxidase, 22.5 mM glucose, 36 µg/ml catalase, and 71.5 mM ß-mercaptoethanol). After selecting a field to image, chambers were perfused with 80 nM GST-Spastin in PEM buffer containing 10 µM taxol, the oxygen-scavenging cocktail, and 1 mM ATP.
Imaging E442Q Spastin bound to microtubules
Unlabeled microtubules were immobilized on kinesin-coated coverslips as described in the preceding paragraph and perfused with 100 nM GST-E442Q Spastin or 100 nM GST alone. After 5 min of binding, chambers were washed three times and fixed for 3 min at 25°C with 1% glutaraldehyde. Samples were stained for tubulin (Tyr-tubulinspecific antibody) and for GST (Amersham Biosciences).
Time-lapse video microscopy
Cells or perfusion chambers were transferred to a microscope (model TE300; Nikon), equipped with a heated (34°C) chamber and a 60x (1.4 NA) plan Apo lens. Epifluorescence images were collected with a camera (model CoolSnap HQ; Roper Scientific). The exposure time was controlled with a Lambda 102 filter changer (Sutter Instruments) driven by Metamorph (Universal Imaging Corporation). Images were processed with a low-pass filter.
Microtubule binding assay
Microtubules were assembled by incubating tubulin (Cytoskeleton, Inc.) at 0.2 mg/ml in PEM buffer with 1 mM GTP and 20 µM taxol for at least 1 h at 37°C. 5 µl of microtubules was added to each reaction containing recombinant 1 µg WT or mutant Spastin and ATP as indicated in the figures. Reactions were performed in PEM buffer, and the final volume was 50 µl. After 10 min at 37°C, microtubules were separated from soluble tubulin by ultracentrifugation (TL100 rotor [Beckman Coulter]; run at 50,000 rpm for 10 min, 37°C, 100,000 gav). Equal portions of pellet and supernatant fractions were analyzed by SDS-PAGE.
Online supplemental material
Fig. S1 shows Spastin-mediated severing in permeabilized TC7 cells. Fig. S2 shows that recombinant E442Q Spastin binds along the length of purified microtubules. Five videos are available online (Videos 15). In Videos 13 microtubules in permeabilized NIH 3T3 cells stably expressing GFP-tubulin were imaged in real time. Video 1 shows a cell incubated with WT Spastin; Video 2 shows a region of detail from the cell imaged in Video1; and Video 3 shows a cell incubated with mutant Spastin. The complete videos for the data in Fig. 5 are shown in Videos 4 and 5. Video 4 shows incubations with WT enzyme, whereas Video 5 shows an incubation with mutant E442Q Spastin. Legends and methods for the videos are available online at http://www.jcb.org/cgi/content/full/jcb.200409058/DC1.
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Acknowledgments |
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This work was supported by the Paul Beeson Physician Faculty Scholars Program (to B.P. Lauring) and by grants from the National Institutes of Health (to B.P. Lauring and G.G. Gundersen) and the Dystonia Medical Research Foundation (to B.P. Lauring). E.R. Gomes was supported by Fundação para a Ciencia e Tecnologia of Portugal.
Submitted: 10 September 2004
Accepted: 22 December 2004
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