| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Doublecortin, a stabilizer of microtubules
Introduction
Results
Intracellular Doublecortin
Doublecortin and microtubule interaction
Doublecortin and microtubule assembly
Mapping of Doublecortin and microtuble interaction
Discussion
Doublecortin is a MAP
Doublecortin stabilizes microtubules
Materials And Methods
Cell culture
Plasmid construction
Antibodies
Immunostaining
Live-cell microscopy
Detergent extraction assay
Microtubule assembly in vitro
Microtubules binding assay
Acknowledgements
Note Added In Proof
References
Doublecortin, a stabilizer of microtubules
Received May 7, 1999; Revised and Accepted June 21, 1999
X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an isoelectric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene (LIS1) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis.
INTRODUCTION
Cortical lamination in humans occurs over months of prenatal development. The final proper layer formation relies on an intricate balance between events of the cell cycle, proliferation, neuronal path finding and migration. The lissencephaly syndromes in humans involve abnormal cortical lamination and are medically categorized as neuronal migration defects (1,2). The various human lissencephalies are classified according to morphology or putative etiology (3,4). Two types of lissencephaly have been defined (5,6). In type I, also known as `classical lissencephaly', the cortex consists of four layers instead of the normal six, whereas in type II, also known as `cobblestone lissencephaly', the cortex is unlayered (4). The cortex in the case of lissencephaly type I consists of an outer layer, corresponding to the molecular layer. Inner to it is a second layer harboring neurons with morphology normally seen in layers III, V and VI. The third observed layer is cell sparse and the fourth layer contains neurons that are misplaced (heterotopic neurons) and seem to be arrested in migration (1,2,7). Understanding the molecular basis of lissencephaly may promote our understanding of events regarding normal brain patterning. During the recent years several lissencephaly disease genes have been identified. The first lissencephaly gene cloned was LIS1 (8; recently reviewed in ref. 9). Mutations in one allele of this gene (8,10) are sufficient to cause lissencephaly type I. LIS1 is a multifunctional protein, found to interact with tubulin and to influence microtubule dynamics (11). It has also been cloned independently as a non-catalytic subunit of platelet-activating factor acetylhydrolase (PAF-AH) (12). An additional interaction described is with the mammalian homolog of NudC (13), a gene involved in the nuclear migration pathway in Aspergillus nidulans (14). LIS1 expression during development as well as the brain phenotype in lissencephaly patients suggested a role in neuronal migration (15). Recent gene targeting experiments in the mouse demonstrate that reduction in LIS1 protein levels affect neuronal migration in a cell autonomous manner (16).
A second lissencephaly type I gene was identified and mapped to the X chromosome (17,18). This gene was recently cloned and dubbed doublecortin (19,20). It has been hypothesized that, due to random X-inactivation, affected females heterozygous for the X-linked mutation show a milder phenotype; so far this has not been conclusively shown and other mechanisms may be involved. The milder phenotype consists of a brain with bilateral plates or bands of gray matter located beneath the cortex and ventricle but separated from both, hence the descriptive term double cortex, also known as subcortical laminar heterotopia (21). Doublecortin is highly expressed during brain development; however, its function is as yet unknown (19,20). In situ hybridization in human fetal brain demonstrated strong labeling of the ventricular zone and the cortical plate (19). Recently it was also reported to be expressed in the adult brain, specifically in the frontal lobe (22). Expression of the doublecortin murine homolog was reported to be high, especially in the cortical plate (23). Various types of mutation were identified throughout the doublecortin gene, including nonsense, splice site and missense mutations (19,20,22,24,25). Therefore, it has been suggested that loss of function of Doublecortin is the major cause of subcortical laminar heterotopia.
In order to study the function of Doublecortin, we have used an ectopic expression approach in non-neuronal COS cells. In these large well-spread cells it is easier to study the cytoskeleton and as they do not express Doublecortin, there is no interference from the endogenous protein. Doublecortin is a basic protein with an isoelectric point of 10, typical of microtubule-binding proteins. However, the Doublecortin sequence contains no known microtubule-binding domain(s). In this paper, we have demonstrated that Doublecortin binds to microtubules and increases their bundling and stabilization.
RESULTS
Intracellular Doublecortin
One of the key questions in understanding the function of Doublecortin concerns its intracellular localization. First, we examined the expression of Doublecortin in primary neuronal culture. These stainings revealed an intracellular localization that resembled that of microtubules (Fig. 1a-c). However, such studies are difficult to conduct in neurons because of the highly asymmetric shape and high local concentration of microtubules in protruding processes. In order to define the intracellular localization of Doublecortin, we transiently transfected COS-7 epithelial cells with a FLAG-Doublecortin construct and immunostained them with anti-FLAG antibodies. The FLAG-Doublecortin was stained as a network of fibers reaching the cell periphery; in some cells the nuclei were stained as well (Fig. 1d). The same pattern was also observed by staining with anti-Doublecortin antibodies (Fig. 2b and e). This was the first indication that Doublecortin co-localizes with cytoskeletal proteins. Double staining of transfected cells with anti-Doublecortin antibodies and with anti-[alpha]-tubulin antibodies revealed significant overlaps (Fig. 2b and e, anti-Doublecortin antibodies with the secondary antibody labeled with FITC and rhodamine, respectively; Fig. 2a and d, the corresponding cells stained with anti-[alpha]-tubulin antibodies; Fig. 2c and f, overlaps in the staining). Furthermore, GFP-Doublecortin revealed a similar fibrillar pattern in both live and fixed cells (data not shown). This fibrillar structure persisted (Fig. 2g) even when the actin cytoskeleton was completely disrupted by cytochalasin B treatment (shown in Fig. 2h, by staining of the cell in Fig. 2g with fluorescein-phalloidin, which binds to actin). Co-localization with the microtubule cytoskeleton was further demonstrated by disruption of the microtubules with nocodazole. Subsequent staining using both Doublecortin [using anti-FLAG antibodies (Fig. 2i) as well as anti-Doublecortin antibodies (Fig. 2j)] and tubulin revealed that the fibrillar structured staining disappeared (Fig. 2k).
Figure 1. Doublecortin localizes to fibrous structures. (a) Rat primary neurons were co-stained with primary anti-Doublecortin antibodies in conjunction with secondary anti-rabbit FITC and (b) anti-tubulin antibodies with a Cy3 secondary anti-mouse antibody 72 h after plating. The neurons were visualized using confocal microscopy. Note staining of all neurites. (c) Computerized overlapping demonstrates a high degree of similarity in the staining pattern. (d) COS-7 cells were transfected with FLAG-Doublecortin and 48 h after transfections cells were fixed with cold methanol and stained with primary anti-FLAG antibodies and secondary anti-mouse rhodamine.
Figure 2. Doublecortin co-localizes with microtubules. COS-7 cells were transfected with FLAG-Doublecortin and 48 h after transfections cells were fixed with cold methanol and double stained with anti-[alpha]-tubulin antibodies and secondary anti-mouse rhodamine (a) and anti-Doublecortin primary antibodies and secondary anti-rabbit FITC (b) or anti-[alpha]-tubulin antibodies and secondary anti-mouse FITC (d) and with anti-Doublecortin primary antibodies and secondary anti-rabbit Cy3 anti-rabbit coupled with anti-[alpha]-tubulin antibodies (e). (c and f) Co-localization of fibers is shown. (g) Cytochalasin B did not disrupt the fibers (stained with anti-FLAG antibodies). (h) This treatment completely disrupted the actin cytoskeleton (fluorescein-phalloidin staining). (i) The fibrous structured staining of Doublecortin was completely disrupted by nocodazole treatment, depicted by anti-FLAG staining. (j) Doublecortin staining is disrupted by nocodazole. (k) The disruption in microtubule staining is visualized by anti-[alpha]-tubulin antibodies.
Our results of overexpression experiments of Doublecortin in COS-7 cells suggested that Doublecortin may act similarly to classical microtubule-associated proteins (MAPs) and may influence microtubule stability. We used several approaches to examine this hypothesis. Stable microtubules are known to be acetylated in cells (26-28); therefore, we double stained transfected cells with anti-Doublecortin antibodies and anti-acetylated tubulin antibodies. As seen in Figure 3a-f, cells that overexpress Doublecortin are positive for acetylated tubulin fibers that are organized in a typical `spring' fashion (compare Fig. 3a-c with d-f, respectively). Acetylated tubulin fibers are barely visible in non-transfected cells. Furthermore, microtubule bundles are observed in cells exhibiting high level overexpression of Doublecortin. This can be seen in the cell in Figure 1d, where some of the visualized fibers appear bundled.
Figure 3. Doublecortin stabilizes microtubules. COS-7 cells were transfected with FLAG-Doublecortin and 48 h after transfections cells were fixed with cold methanol and stained. (a-c) Cells stained with anti-Doublecortin primary antibodies and anti-rabbit FITC. (d-f) The corresponding cells were stained with anti-acetylated tubulin and anti-mouse rhodamine. Note bundles in the cells. (f) It is possible to see low level expression of acetylated tubulin in two non-transfected cells that are located above the transfected cells. (g-j) Transfected cells were treated with cytochalasin B and then fixed and stained. Note the delicate protrusions extending from the cells. (g and i) Cells stained with anti-[alpha]-tubulin and anti-mouse rhodamine. (h and j) The cell in (g) or (i) stained with anti-Doublecortin antibodies and anti-rabbit FITC. (k-m) Live cell microscopy of cells transfected with FLAG-Doublecortin and treated with cytochalasin B. Arrows denote the extended protrusions. (k) Cell before addition of cytochalasin B. (l) Cell 3 min after addition of cytochalasin B. Note the protrusions. (m) Cell 45 min after addition of cytochalasin B.
Stable tubulin fibers form a typical `spring' structure due to physical constraints imposed by the cortical actin network. The cortical actin polymers act as a physical barrier, preventing extension of the MAP-induced microtubule bundles beyond the cell periphery (29). When the cortical actin filaments are disrupted by cytochalasin B treatment, the MAP-induced microtubule bundles straighten and push out spike-like protrusions from the cell surface. This phenomenon was initially observed in cells overexpressing MAP2 (30). Indeed, when FLAG-Doublecortin or GFP-Doublecortin (data not shown) overexpressing cells were treated with cytochalasin B, typical protrusions appeared (Fig. 3g-j). It was possible to follow the development of these protrusions in living cells using time-lapse microscopy (Fig. 3k-m). The effect of cytochalasin B was already visible 3 min after treatment (Fig. 3l). The above experiments suggest that Doublecortin is a MAP. The precise mode of interaction between Doublecortin and microtubules was further investigated using biochemical methods.
Biochemical fractionation of exogenous expressed protein corroborated the cytoskeletal localization of Doublecortin which had been suggested by immunostaining. We used the well-elaborated protocol of gentle cell extraction with non-ionic detergent (0.5% Triton X-100) that removes lipids and soluble proteins, leaving intact the detergent-insoluble matrix composed of the nucleus, the cytoskeleton framework and cytoskeleton-associated proteins. In transfected COS-7 cells, the exogenous Doublecortin (recognized by the FLAG tag) appeared mainly in the detergent-insoluble fraction (90 ± 3%) (Fig. 4a), with 10% in the soluble fraction. In a similar fashion tubulin, which has a constant soluble pool, was found in both fractions (Fig. 4a and b). Moreover, it was clear from the overexpression experiments that the ratio of insoluble tubulin to soluble tubulin increased dramatically with Doublecortin transfection compared with mock-transfected cells (Fig. 4). While in untransfected cells 56% of tubulin was found in the insoluble fraction, in transfected cells 83% of the tubulin was insoluble. This result confirmed our previous results that Doublecortin might affect parameters of microtubule stability. In addition, pretreatment of the cells with nocodazole resulted in a shift of Doublecortin from the insoluble to the soluble fraction (Fig. 4a), suggesting that the insolubility of Doublecortin is due to its interaction with microtubules. In addition, acetylated tubulin, a marker for stabilized microtubules, was detected only in the insoluble fraction of Doublecortin-transfected COS cells (Fig. 4a).
Figure 4. Doublecortin-transfected cells and recombinant Doublecortin. (a) COS-7 cells were transfected with FLAG-Doublecortin and 48 h after transfection cells were subject to detergent extraction as described in Materials and Methods. Ten micrograms of total protein were separated on 10% SDS-PAGE gels, immunoblotted and reacted with the corresponding antibodies; (upper panel) anti-[alpha]-tubulin; (second panel) anti-Doublecortin; (third panel) anti-FLAG; (lower panel) anti-acetylated tubulin. Note that mock-transfected cells do not express Doublecortin, as demonstrated both by the anti-Doublecortin and anti-FLAG antibodies. Transfected cells were treated with nocodazole. In extracts from the treated cells there is a shift of tubulin and Doublecortin from the insoluble to the soluble fraction. (b) The distribution of tubulin in the insoluble and soluble fractions of mock and transfected cells (as described above) was quantified from the results of five repeats of the experiment. (c) Microtubules were pre-assembled from purified tubulin in PEM buffer. Polymerization was done in the presence of a microtubule-stabilizing drug (Taxol), a microtubule-destabilizing drug (Nocodazole, Noc) or without addition of drugs (-). GST-Doublecortin or GST alone were added after polymerization and incubated with the microtubules for 10 min at 37°C. Assembled microtubules and associated proteins (pellet, P) were sedimented by centrifugation. Pellets and supernatants (S) containing soluble proteins were separated on SDS-PAGE gels. Gels were stained with Coomassie blue.
Doublecortin and microtubule interaction
In order to examine whether the interaction between Doublecortin and microtubules is direct, we used tubulin purified from bovine brain and recombinant Doublecortin in an in vitro microtubule-binding assay. We used nocodazole as a microtubule-disrupting drug or taxol as a microtubule-stabilizing agent to change the solubility of tubulin. Doublecortin bound to pre-assembled taxol-stabilized microtubules. As can be seen in Figure 4c, the changes in the solubility of Doublecortin reflected the changes in solubility of tubulin. These results demonstrate a direct association between Doublecortin and the microtubule cytoskeleton.
Doublecortin and microtubule assembly
The next issue pursued was whether Doublecortin has an effect on microtubule assembly. To determine whether Doublecortin is involved in microtubule formation, we measured the assembly rate of tubulin using a light scattering assay (Fig. 5e). This assay is based on an increase in optical density absorbance as microtubules polymerize. Addition of recombinant GST-Doublecortin increased the rate of assembly and the total amount of assembled tubulin in a dose-dependent manner far beyond those obtained with tubulin itself (Fig. 5a) or by addition of GST control protein to tubulin (shown in a similar experiment in Fig. 7e). Addition of Doublecortin also reduced the critical concentration of tubulin required for polymerization, as tested by DIC microscopy. Tubulin will polymerize without the need for external nucleation sites at specific temperature-dependent concentrations (a higher temperature will require a lower concentration of tubulin), termed the `critical concentration' (31). We assayed whether the critical concentration of tubulin polymerization is altered by the addition of purified Doublecortin. Different tubulin concentrations were incubated at varying temperatures ranging from 26 to 38°C with GST-Doublecortin or GST alone as a control (data not shown). Addition of GST-Doublecortin (1:5 molar ratio) to tubulin (16 µM) caused a reduction of 6°C in the temperature at which initial polymerization was observed. Likewise, using 20 µM tubulin and GST-Doublecortin microtubules reduced the temperature required for initial polymerization by 4°C. These observations were supported by varying tubulin concentrations incubated at a fixed temperature of 26°C. The effect of overexpression of Doublecortin in COS-7 cells suggested that this protein may cause microtubule bundling. Similarly, addition of Doublecortin to tubulin resulted in microtubule bundling in vitro, clearly visualized by DIC microscopy (Fig. 5a-d). Increasing concentrations of Doublecortin were used while tubulin concentrations were held constant at 10 µM (Fig. 5b-d). While in the control there are a few visible microtubules (Fig. 5a), the number of filaments increased dramatically when Doublecortin was added (Fig. 5b-d). Clear bundles were visible when the Doublecortin:tubulin ratio was increased to 1:2 (Fig. 5c); the effect was even more striking when this ratio was increased to 1:1 (Fig. 5d).
Figure 5. Doublecortin affects microtubule assembly and bundling. (a-d) Microtubule bundling in vitro was visualized by DIC microscopy: (a) 10 µM tubulin at 26°C, few visible fibers; (b-d) increasing concentrations of Doublecortin; (b) Doublecortin:tubulin ratio 1:10; (c) Doublecortin:tubulin ratio 1:2; (d) Doublecortin:tubulin ratio 1:1. The assembly rate of tubulin was measured using a light scattering assay. (e) The Doublecortin:tubulin ratio was changed as indicated; the concentration of tubulin used was 16 µM. The optical absorbance was recorded every minute.
Mapping of Doublecortin and microtuble interaction
In order to map the cytoskeletal interaction we generated several truncated fragments of Doublecortin (Fig. 6a). As computer analysis did not reveal any putative functional domains and no crystal structure is yet available for this protein, we arbitrarily divided the coding region into three domains. Both in vivo experiments using a series of FLAG-Doublecortin constructs expressed in COS cells and in vitro assembly experiments using GST-tagged protein produced in bacteria (Fig. 6) were used to delineate the interacting domain(s). COS-7 cells were transfected with the different fragments and the pattern of staining as well as biochemical fractionation were determined as have been described. It was clear that FLAG-Doublecortin EcoRV (1-213) was associated with the microtubules while FLAG-Doublecortin EcoRV-end (213-361) was soluble (Fig. 6b and c). This was demonstrated by detergent extraction where essentially all of the FLAG-Doublecortin EcoRV (1-213) was found in the insoluble fraction (Fig. 6b). Furthermore, immunostaining of FLAG-Doublecortin EcoRV (1-213) depicted a clear fibrillar structure versus the FLAG-Doublecortin EcoRV-end (213-361), which stained the cell with no fibers (Fig. 6c versus d). FLAG-Doublecortin BamHI-end (110-361) was also expressed in COS-7 cells, but did not stain microtubules (Fig. 6e). In addition, in vitro analysis of GST-Doublecortin EcoRV (1-213) revealed direct binding to tubulin (Fig. 6g), while the fragment of GST-Doublecortin BamHI-end (110-361) demonstrated very weak binding (Fig. 6f). The GST-Doublecortin EcoRV (1-213) fragment was also tested for bundling activity. Samples with and without this Doublecortin fragment were processed for electron microscopy. Dense bundles were visible by negative staining (Fig. 7a and c). Most of the bundles appeared very dense and dark (Fig. 7a), while some were less dense (Fig. 7c). Without the addition of Doublecortin, only single microtubule filaments are visible (Fig. 7b and d). Two Doublecortin fragments, Doublecortin EcoRV (1-213) and Doublecortin BamHI-end (110-361) were tested in the light scattering assay. As clearly demonstrated in Figure 7e, while Doublecortin EcoRV (1-213) had activity very similar to the full-length Doublecortin. Doublecortin BamHI-end (110-361) activity was very low and the curve was quite similar to the measured activity of pure tubulin or to the activity of tubulin with GST control protein. This same fragment was tested for bundling activity using DIC microscopy; no bundles were apparent (data not shown). Our attempts to express shorter fragments were unsuccessful: the first third of the protein (1-110) was not expressed in mammalian cells nor in bacteria and the second third (110-213) was not expressed in mammalian cells (Fig. 6). The simplest interpretation of our results localizes the binding/bundling activity of Doublecortin to the first third of the protein. Future experiments combining structural data with mutagenesis of specific amino acids will allow us to define more accurately this interaction.
Figure 6. Microtubule-binding domain localization. (a) Different constructs of Doublecortin used in this study are schematically presented. For in vivo experiments FLAG tag was used while for in vitro experiments recombinant GST-tagged protein produced in bacteria was used; the expression of each construct is indicated. (b) The expression and solubility of three FLAG-tagged Doublecortin constructs were examined using western blot analysis. Note that FLAG-Doublecortin EcoRV (1-213) is detected only in the insoluble fraction, while Doublecortin EcoRV-end (213-361) is detected only in the soluble fraction. (c) COS-7 cells were transfected with FLAG-Doublecortin EcoRV (1-213) and 48 h after transfection cells were fixed with cold methanol and stained with primary anti-FLAG antibodies. Note the clear fibrillar structures. (d) COS-7 cells were transfected with Doublecortin EcoRV-end (213-361) and 48 h after transfection cells were fixed with cold methanol and stained with primary anti-FLAG antibodies. No fibrillar structures are visible. (e) COS-7 cells were transfected with Doublecortin BamHI-end (110-361) and 48 h after transfection cells were fixed with cold methanol and stained with primary anti-FLAG antibodies. No fibrillar structures are visible. (f) Microtubules were pre-assembled from purified tubulin in PEM buffer in the presence of a microtubule-stabilizing drug (Taxol). GST-Doublecortin BamHI-end (110-361) or GST alone were added after polymerization and incubated with the microtubules for 10 min at 37°C. Assembled microtubules and associated proteins (pellet, P) were sedimented by centrifugation. Pellets and supernatants containing soluble proteins were separated on SDS-PAGE gels. Gels were stained with Coomassie blue (right) or blotted and reacted with anti-Doublecortin antibodies (left). (g) Microtubules were pre-assembled from purified tubulin in PEM buffer. Polymerization was done in the presence of a microtubule-stabilizing drug (Taxol), a microtubule-destabilizing drug (Nocodazole, Noc) or without addition of drugs (-). GST-Doublecortin EcoRV (1-213) or GST alone were added after polymerization and incubated with the microtubules for 10 min at 37°C. Assembled microtubules and associated proteins (pellet, P) were sedimented by centrifugation. Pellets and supernatants containing soluble proteins were separated on SDS-PAGE gels. Gels were stained with Coomassie blue.
Figure 7. Doublecortin fragments affect microtubule assembly and bundling. (a-d) Tubulin (15 µM) with (a and c) or without (b and d) the addition of 9 µM of Doublecortin EcoRV (1-213) fragment were incubated as described for DIC microscopy, put on a grid stained with uranyl acetate and examined by electron microscopy. Most bundles were dense, as shown in (a); some bundles were less dense, as shown in (c). (b and d) Tubulin (15 µM) with no addition of Doublecortin. Only individual microtubules are visible. The magnification used is ×28 000. The black bar is 0.85 µm. The assembly rate of tubulin was measured using a light scattering assay. (e) The molar ratio recombinant proteins:tubulin was 1:10; the concentration of tubulin used was 16 µM. The optical absorbance was recorded every minute for 40 min. The recombinant proteins used were GST, GST-Doublecortin, GST-Doublecortin BamHI-end (110-361) and GST-Doublecortin EcoRV (1-213).
DISCUSSION
Doublecortin is a MAP
Our results suggest that Doublecortin may function as a MAP. The interaction between Doublecortin and microtubules is direct and does not require any additional mediators. This interaction results in stabilization and bundling of microtubules both in vivo and in vitro. Doublecortin is a basic protein with an isoelectric point of 10, typical of other microtubule-binding proteins. Sequence analysis did not detect any microtubule-binding domains; however, our deletion analysis localizes the region of binding to amino acids 1-110. We believe that this function is highly significant for the role of Doublecortin during brain development. Several events involved in the formation of the structure of the cerebral cortex require the presence of microtubules. These include neuronal migration, nuclear displacement and process formation. Cell migration is characterized by dynamic interactions between the substrate and the cytoskeleton-associated motile apparatus inside the cell (32). The role of microtubules in the formation of growth cones is also well documented (33,34). The active migration of neurons from their sites of origin to their final destination requires unidirectional translocation of the nuclei and somatic cytoplasm within the growing leading processes. It has been suggested that the dynamics of slow polymerization in combination with fast disintegration of oriented microtubules create forces that contribute to the piston-like salutatory displacement of the nucleus and cytoplasm within the membrane cylinder of the leading process of the migrating neuron (35). Indeed, microtubules are a predominant component of the neuronal cytoskeleton and comprise 15-20% of total cell protein in brain and they are essential for the development of neuronal morphology (36). The proposed role of microtubules in nuclear displacement during neuronal migration does not exclude the synergistic action of actin-like contractile proteins which may also participate in this event (37). However, it does fit with the finding that disruption of microtubule structure results in collapse of the migrating cell body and cessation of nuclear translocation (37).
The outgrowth of neuronal processes involves the interaction of two major components of the neuronal cytoskeleton, microtubules and actin filaments. These produce counteractive forces, tension generated by actin filaments of the cortical cytoskeleton (38,39) and compression produced by microtubule bundles in the cytoplasm (40,41). To be capable of supporting processes, the neuronal microtubules should have several special properties. They must be more stable than the microtubules of non-neuronal cells and they must be sufficiently stiff to support the elongate processes as they are formed. These special properties are thought to depend on a set of structural MAPs.
Doublecortin stabilizes microtubules
We have demonstrated here that Doublecortin stabilizes microtubules when ectopically expressed in non-neuronal cells. Microtubule stabilization was visible from the shift of tubulin to the insoluble fraction using a detergent extraction procedure, by the existence of acetylated tubulin fibers and protrusion of microtubule spikes after disruption of the cortical actin network. Furthermore, in vitro analysis demonstrated that Doublecortin reduced the critical concentration required for polymerization and caused bundling. Doublecortin is encoded by 361 amino acids that did not reveal any functional domains by computer searches. Several MAPs have been shown to bind to microtubules via short sequences with repeated amino acids motifs. However, we did not detect short repeated amino acids motifs or any clusters of charged amino acids that would imply microtubule binding. One mode of regulating Doublecortin microtubule binding may be through phosphorylation. In general, it has been shown that there is an inverse correlation between phosphorylation and microtubule binding; the more phosphorylated the protein, the less it binds to microtubules and stabilizes them (42-44). The phosphorylation state of MAPs changes during development (reviewed in refs. 34,45). This may be due to combined activities of kinases or phosphatases (46). This post-translational modification is believed to be the major mode of MAP function regulation. Within Doublecortin's first 100 amino acids there is a putative site for c-Abl phosphorylation (20). We have preliminary results indicating that Doublecortin is phosphorylated by c-Abl (O. Reiner, unpublished data) and we are investigating how this affects Doublecortin function. The most probable interpretation of our results localizes the region of Doublecortin-microtubule interaction to the first third of this protein. Bundling requires at least two binding sites that may reside within a single full-length Doublecortin sequence or result from homodimerization of two Doublecortin molecules. We favor the first option, as our preliminary data do not demonstrate homodimerization (T. Sapir, unpublished data).
We believe that the function of Doublecortin as a stabilizer of microtubules is highly relevant to the disease phenotype. We have previously characterized the interaction between LIS1 and microtubules and its influence on microtubule dynamics (11). As mutations in LIS1 or doublecortin result in lissencephaly type I it is likely that their interaction with the microtubule cytoskeletal component of neuronal cells is an important feature in the final obstruction of cortical layering. Lis1 gene targeting in the mouse demonstrated a direct neuronal migration defect (16; O. Reiner, unpublished data). We have previously proposed that nuclear migration may be important for the neuronal migration defect (13). This hypothesis was based on defects in nuclear migration due to mutations of the LIS1 homolog in A.nidulans, NudF (14). Furthermore, in higher eukaryotes LIS1 physically interacts with gene products that are involved in the nuclear migration pathway in A.nidulans. This includes LIS1 interactions with tubulin (11) and with the mammalian homolog of NudC (13). At present, we do not know whether LIS1 and Doublecortin are part of the same or different pathways. Not all lissencephaly-associated gene products will interact with microtubules. For example, a gene involved in Fukuyama-type congenital muscular dystrophy, which is associated with type II lissencephaly, was identified (47). Its protein product, Fukutin, is a secreted protein of unknown function (47); however, as a secreted protein it is not expected to interact with the cytoskeleton.
The stabilizing effect of Doublecortin on microtubules ascribes a function to this disease gene that may explain part of the pathophysiology of the disease phenotype. Furthermore, it expands our growing understanding of basic processes that are involved in cortical layer formation.
MATERIALS AND METHODS
Cell culture
COS-7 cells (48) were grown at 37°C with 5% CO2 and 95% air in DMEM nutrient medium supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cultures were split using standard trypsinization procedures. COS-7 cells were transfected using the DEAE transfection procedure (49) with 20 µg of DNA in the absence of serum in the medium. Four hours after transfection the medium was aspirated and 10% DMSO was added for 2 min. The cells were washed and fresh standard growth medium added. Transfected cells were then analyzed at various times for expression of exogenous protein. Efficiency of transfections was estimated using a GFP expression vector (Clontech, Palo Alto, CA).
Primary neuronal cultures were prepared from cortex of E15.5 mouse strain MF1. The neurons were dissociated, plated on poly(ornithine)-coated coverslips and grown in culture as described (50).
Plasmid construction
The constructs discussed in this paper are summarized in Figure 6a. The GST fusion proteins were made in pGEX4T-1 (Pharmacia, Piscataway, NJ). The full-length clone was constructed by preparation of a 1.2 kb HindIII-SmaI fragment, using the HindIII site 53 nt upstream of the start methionine and a SmaI site present in the cDNA vector. The HindIII site was blunt ended and the fragment was cloned in the pGEX4T-1 SmaI site. The construct was checked by sequencing of both ends. pGEX-BamHI-end is a subclone of a BamHI fragment in pGEX4T-1 digested with BamHI. The construct was checked by sequencing. pGEX-BamHI was prepared by digestion of a 370 bp HindIII-BamHI fragment blunt ended and cloned in the SmaI site of pGEX4T-1. The construct was checked by sequencing. GST fusion proteins were produced in the BL21 lysE strain of Escherichia coli and purified using glutathione-agarose (Sigma, Rehovot, Israel). Proteins that were not present in soluble form in bacteria were regarded as not expressed, as it was not possible to purify the proteins in the native conformation for biological experiments. The tagged proteins for expression in mammalian cells were made by subcloning the described fragments into pECE (51) with a FLAG epitope at the N-terminus or a GFP tag using pEGFP-N1 (Clontech). For the 5[prime] and 3[prime] ends, EcoRI sites derived from construct pGEX4T-1 were used. PCR of amino acids 110-213 was carried out using 5[prime] primer 5[prime]-CACGGTACCACGGATCCAGGAAGATCGG-3[prime], introducing a new KpnI site, and 3[prime] primer MYST2 5[prime]-CTGTTTTCCATCCAGAGTGTAGAG-3[prime]. The PCR product was digested with KpnI and EcoRV and the fragment was cloned into PECE-FLAG using KpnI and SmaI sites. Amino acids 110-end were introduced into PECE-FLAG using 5[prime] primer 5[prime]-CACGGTACCACGGATCCAGGAAGATCGG-3[prime], introducing a new KpnI site, and the 3[prime] primer from the pGEX vector. The PCR product was digested with KpnI and EcoRI and cloned into PECE-FLAG digested with the same restriction enzymes. Plasmids with inserts derived from PCR were completely sequenced. Expression of FLAG-tagged proteins was examined by immunohistochemistry and western blot analysis.
Antibodies
Anti-[alpha]-tubulin (monoclonal, clone DM1A) or anti-acetylated tubulin (monoclonal, clone no. 6-11B-1) were purchased from Sigma. Anti-doublecortin polyclonal antibody J74 was produced against a mixture of peptides (52). Anti-FLAG M2 monoclonal antibody was obtained from Kodak (New Haven, CT). All secondary antibodies were from Jackson Immunoresearch (West Grove, PA); peroxidase-conjugated affinipure goat anti-mouse IgG (H+L); lissamine/rhodamine-conjugated affinipure goat anti-mouse IgG (H+L); Cy3-conjugated affinipure goat anti-rabbit IgG (H+L); FICT-conjugated affinipure goat anti-mouse or anti-rabbit IgG (H+L).
Fluorescein-phalloidin (Molecular Probes, Eugene, OR) was used at 2 U/ml.
Immunostaining
Briefly, transfected cells were plated on glass coverslips. After 48 h they were washed twice with phosphate-buffered saline (PBS), then fixed and permeabilized simultaneously in cold methanol for 10 min. After fixation the cells were incubated with 30 µl of the first antibody for 60 min at room temperature, then washed three times with PBS and incubated for 30 min with 30 µl of fluorescent-conjugated secondary antibodies. The coverslips were washed three times with PBS, drained and mounted. The immunostaining was visualized using an Olympus microscope (IX50 model; Hamburg, Germany) using the appropriate filters. Photography was with Kodak 160T film. For microtubule disruption, cells were treated with 5 µM nocodazole (Sigma) overnight. For actin disruption cells were treated with 20 µM cytochalasin B for 2 h (Sigma) as described before (30). Neurons (Fig. 1) were observed under a Bio-Rad (Hercules, CA) confocal microscope.
Live-cell microscopy
For live microscopy, COS cells were plated on coverslips glued with parafilm across holes in the bottom of Petri dishes. Transfection was done with Doublecortin and GFP plasmids in a 9:1 ratio as described. Approximately 48 h after transfection the dishes were moved to the microscope. Prewarmed mineral oil was spread across the surface of the medium to prevent evaporation and loss of CO2. Transfected cells were located using the GFP marker. They often had an atypical morphology and in some cases bundles of microtubules could be seen directly.
Observations were made in differential interference contrast on an inverted microscope (Zeiss Axiovert 35, Fluar 100×/1.3 objective) with red light illumination. The objective was heated electrically to 37°C (Bioptechs, Butler, PA). Temperature of the cells was maintained by thermal conduction through the immersion oil. Video images were recorded by a digital CCD camera (iSight; Tirat haCarmel, Israel) onto S-VHS cassettes.
After choosing a cell, 1 ml of new medium containing cytochalasin B was introduced by syringe underneath the mineral oil. The process shown in Figure 3k-m took 50 min, following which the medium was exchanged three times for fresh medium without cytochalasin. The prominent branches bent and retracted, some partially and others completely (data not shown).
Detergent extraction assay
The detergent extraction assay was performed essentially as described by Cohen et al. (53). Sub-confluent cultures of transfected COS-7 cells, grown on 9 cm plates, were washed once with PBS and then with MES buffer (50 mM MES pH 6.8, 2.5 mM EGTA, 2.5 mM MgCl2). The cells were extracted for 3 min with 0.5 ml of 0.5% Triton X-100 in MES buffer supplemented with protease inhibitors (Sigma). The supernatant (the soluble fraction, termed Sol) was collected, centrifuged for 2 min at 16 000 g at 4°C and the clear supernatant was then transferred to new tubes. Two volumes of cold ethanol were added and the tubes were incubated at -20°C overnight, centrifuged for 10 min at 16 000 g at 4°C and resuspended in 200 µl of 2× protein sample buffer without dye. The detergent-insoluble matrix (InSol) remaining on the plate was extracted in 200 µl of 2× protein sample buffer, scraped from the plate with a rubber policeman and collected into tubes. Treatment with nocodazole was at 10 µM for 4 h prior to extraction. The samples were loaded on 10% SDS-PAGE gels; equal volumes of Sol and InSol were loaded according to the volume that contained 100 µg of protein in the soluble fraction. Proteins were detected by western blot analysis using primary anti-FLAG antibodies (1:300 dilution), antibodies against Doublecortin (1:2000 dilution), anti-tubulin antibodies (1:2000 dilution) and peroxidase-conjugated secondary antibodies (1:10 000 dilution). The chemiluminescence reaction was developed using SuperSignal substrate (Pierce, Rockford, IL).
Microtubule assembly in vitro
Rate measurements.
Tubulin was purified as described (11). The assembly rate of tubulin to form polymers was monitored using a light scattering assay (54,55). Purified tubulin was diluted in PEM buffer (100 mM PIPES pH 6.9, 1 mM MgSO4, 1 mM EGTA) supplemented with 1 mM GTP to a final concentration of 16 µM. Recombinant proteins were dialyzed against PEM buffer prior to addition to the tubulin solution. Absorbance was measured at 350 nm at 1 min intervals in a Uvicon spectrophotometer equipped with temperature controlled cells. Switching the temperature to 37°C induced assembly.
Microscopy of microtubules.
The effect of recombinant proteins on microtubule bundling was visualized by video-enhanced DIC microscopy. A solution (10 µl) containing phosphocellulose-purified tubulin and recombinant proteins, in PEM buffer supplemented with 1 mM GTP at the indicated molar ratio, was allowed to polymerize by incubation in an Eppendorf tube for 30 min at 26°C. A sample of the tube contents was transferred to microscope slides with coverslips mounted over parafilm spacers and sealed with wax. For presentation, all pictures in the figures were recorded in thin cells made by covering a 4 µl droplet of sample with a coverslip and sealing with wax. (Adsorption of protein to the glass surface made thin cells unreliable for concentration measurements, though the confinement of microtubules to a plane improved their visibility.) DIC microscopy was carried out at 26-37°C as indicated, using the same optics as employed for live cell microscopy.
Measurements of the effect of recombinant proteins on the critical concentration needed for spontaneous tubulin assembly were done in a similar fashion. Samples of increasing tubulin concentration were prepared while the molar ratio of tubulin to recombinant protein was kept constant. Solutions were incubated at 26°C for 30 min before transferring to slides.
Microtubules binding assay
Binding of recombinant proteins to stabilized microtubules.
Phosphocellulose-purified tubulin was assembled into microtubules by incubation of 10 µl of 30 µM tubulin in PEM buffer supplemented with 1 mM GTP. Taxol (final concentration 30 µM) or nocodazole (final amount 10 µg/ml) was added to the tubulin. The tubulin was incubated for 10 min at 37°C before addition of 10 µl of recombinant protein at the different indicated concentrations. Mixtures were incubated at 37°C for an additional 10 min and then layered on top of 25 µl of prewarmed 60% glycerol in PEM buffer in siliconized airfuge tubes. Microtubules were sedimented at 39 000 g for 30 min at room temperature. Supernatant fractions were collected and pellets were dissolved in electrophoresis sample buffer. Supernatants and pellets were analyzed on 10% SDS-PAGE gels.
Electron microscopy.
Samples were prepared as described for DIC microscopy using tubulin at a concentration of 15 µM with or without the addition of 9 µM EcoRV 1-213 fragment and incubated for 15 min at 37°C. Eight microliters of each sample were applied to a carbon-coated copper 400 mesh electron microscope grid, which had previously been glow-discharged to render the carbon surface hydrophilic. The grid was then rinsed with water, and 10 µl of uranyl acetate stain (1% in water) was applied. The grid was blotted after 30 s. Samples were examined with a Philips CM12 electron microscope operating at 100 kV.
ACKNOWLEDGEMENTS
The authors thank Dr Alexander Bershadsky for useful discussions and Harry Burgess for critical reading of this manuscript. We wish to thank the anonymous referees for their useful comments. This work was supported in part by the Minerva Foundation and the Minna James Heineman Foundation (to O.R.). O.R. is the incumbent of the Aser Rothstein Career Development Chair in Genetic Diseases. M.E. is the incumbent of the Delta Career Development Chair and acknowledges support from the Israel Academy of Sciences and the Gerhard M.J. Schmidt Minerva Center for Supramolecular Architecture.
NOTE ADDED IN PROOF
A paper was published after this manuscript was accepted which describes Doublecortin as a MAP: Gleeson, J.G., Lin, P.T., Flanagan, L.A. and Walsh, C.A. (1999) Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron, 23, 257-271.
REFERENCES
+These authors contributed equally to this work
§To whom correspondence should be addressed. Tel: +972 8 9342319; Fax: +972 8 9344108; Email: lvreiner{at}wicc.weizmann.ac.il
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|
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