Article |
Address correspondence to Joseph G. Gleeson, University of California, San Diego, MTF 312, 9500 Gilman Drive, La Jolla, CA 92093-0624. Tel.: (858) 822-3535. Fax: (858) 534-1437. email: jogleeson{at}ucsd.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: migration; Lis1; doublecortin; centrosome; nucleus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LIS1, located on chromosome 17p13.3, encodes the LIS1 protein. LIS1 functions on an evolutionarily conserved pathway regulating microtubule (MT) function and dynein motor activity. In Aspergillus nidulans, the LIS1 homologue nudF is required for proper nuclear transport into and within hyphal processes (Xiang et al., 1995), and other nud (nuclear distribution defect) genes such as nudA, nudG, and nudK encode for dynein components (Xiang et al., 1994, 1999). The mammalian orthologues of NUD proteins bind to LIS1 (Faulkner et al., 2000; Feng et al., 2000; Niethammer et al., 2000; Sasaki et al., 2000; Smith et al., 2000), suggesting that the fungal nuclear migration pathway may be conserved in mammals to mediate nuclear translocation. The fact that the main defect observed in cultured Lis1+/ mouse neurons is in nuclear movement within the limits of the cell membrane, but not in neurite length (Hirotsune et al., 1998; unpublished data), also supports the recapitulation of LIS1 in mammalian nuclear translocation.
DCX, located on Xq22.3-q23, encodes for an MT-associated protein, doublecortin (DCX). DCX directly polymerizes purified tubulin into MTs, and misexpression in heterologous cells leads to the formation of depolymerization-resistant MTs (Francis et al., 1999; Gleeson et al., 1999a; Horesh et al., 1999). Similarly, DCX has evolutionarily conserved orthologues, where in Caenorhabditis elegans mutations in zyg-8 result in defects in MT stability and karyokinesis during asymmetric division at the one-cell stage (Gönczy et al., 2001), suggesting that DCX also functions in migration on an evolutionarily conserved pathway. Although Dcx knockout mice do not display a major disruption in migration, acute inactivation in rodents produces significant migration defects (Corbo et al., 2002; Bai et al., 2003).
Recent data suggest that Lis1 and Dcx display overlapping localization and may interact. In fixed cells of various types, Lis1 localizes to the centrosome (Feng et al., 2000; Sasaki et al., 2000; Smith et al., 2000), the perinuclear region (Coquelle et al., 2002), kinetochores (Faulkner et al., 2000), the plus end of MTs (Coquelle et al., 2002; Lee et al., 2003; Xiang, 2003), and the leading cell cortex (Swan et al., 1999; Dujardin et al., 2003). Evidence suggests Dcx localizes to MT structures in both the leading process (Friocourt et al., 2003) and the perinuclear region (Gleeson et al., 1999a). Dcx and Lis1 coimmunoprecipitate from brain lysate, and purified Dcx and Lis1 bind cooperatively to MTs (Caspi et al., 2000). These data suggest that they may share functionality during migration. However, their roles have not thus far been tested in mammalian migrating neurons, the cells directly affected in lissencephaly. Here, we use mouse cerebellar granule neurons and demonstrate that Lis1 and Dcx function with dynein to mediate nucleuscentrosome (N-C) coupling in neuronal migration. We propose that proper N-C coupling may be critical in neuronal migration.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cerebellar granule neurons were dissociated from postnatal d 5 mice and cultured with retrovirus, resulting in spherical cellular reaggregates that were transferred to poly-D-lysine and laminin-coated slides (Liang and Crutcher, 1992). A fraction of neurons migrated radially from each. After 12 h of migration, the distance between transduced cell bodies and the edge of the reaggregate was measured, allowing for an integrated estimate of the migration rate (Fig. 1).
|
To quantitate the amount of retrovirally transduced proteins, lysates were collected from infected cells and analyzed by SDS-PAGE/Western blotting for the presence of endogenous and epitope-tagged Dcx or Lis1 (Fig. 2 A). Quantitation of band intensity and correction for percentage of transduced cells indicated the epitope-tagged Dcx and Lis1 proteins were produced at 85% of the endogenous level (Fig. 2 A, see table), meaning transduction increased protein levels a little less than twofold.
|
Patient mutations abrogate the effect of overexpression on migration
Intragenic LIS1 or DCX mutations in patients with lissencephaly include missense amino acid changes and truncations (Cardoso et al., 2000, 2002; Gleeson et al., 2000). The mutant proteins are either incorrectly folded (Sapir et al., 1999; Caspi et al., 2003) and fail to associate with binding partners (Feng et al., 2000) or fail to bind MTs (Taylor et al., 2000), but no data exist on the effect of these mutations on migration. To assess the function of the mutant proteins quantitatively, patient-related mutant Dcx (R89G, T203R, 303stop; Gleeson et al., 1998) or Lis1 (H149R, S169R, D317H; Lo Nigro et al., 1997; Pilz et al., 1999) was overexpressed. Neurons transduced with single amino acid substitution mutations in either Dcx or Lis1 displayed a mean migration slightly above GFP control, but were not statistically different (Fig. 3). There were slight differences in the bin distribution for each mutation, but the overall migration distances were not significantly different from one another. Truncated Dcx showed no increase in migration over the control level. Thus, patient mutations abrogate the effect of overexpression of Dcx and Lis1 on migration and appear to function as null or hypomorphic alleles.
|
|
Lis1 is localized at the centrosome in migrating neurons
In fixed cells of various types, Lis1 has been reported to overlap in localization with several structures. To test the localization in live migrating neurons, RFP-tagged Lis1 (Lis1-RFP) was transduced in granule neurons. Localization of Lis1-RFP was nearly identical with native Lis1 visualized by immunostaining (Fig. 5 A), suggesting that it reports the localization of the native protein.
|
Lis1 localizes prominently to the nuclear membrane after MT disruption
A previous report has found that treatment with the MT-depolymerizing drug nocodazole led to some Lis1 localized to the nuclear membrane in fixed nonneuronal cells (Smith et al., 2000), suggesting that some fraction of Lis1 may be localized to the nuclear membrane and that an intact MT cytoskeleton is required for Lis1 localization to the centrosome. We tested this in migrating neurons using nocodazole. In approximately half of the cells analyzed at 5 and 50 µg/ml nocodazole concentration, there was redistribution of Lis1 to the perinuclear region (Fig. 5 D). There continued to be some localization of Lis1 to the region of the presumed centrosome, although it was less intense than in untreated cells. The half of the cells not showing perinuclear distribution also displayed less intense centrosomal Lis1 localization. Therefore, a fraction of Lis1 is redistributed to the nuclear membrane after MT disruption.
Because there is very little perinuclear cytoplasm in neurons, the previous experiment could not easily distinguish between perinuclear versus cytoplasmic localization of Lis1. Therefore, nuclei were isolated from granule neurons in the presence of nocodazole and immunostained for Lis1. Nucleoporin, a constitutive nuclear pore complex protein (Davis and Blobel, 1986), outlined the nuclear membrane, whereas Lis1 was detected as punctate signals (Fig. 5 E). Together, these data suggest that some Lis1 is associated with the nuclear membrane and that intact MT structures may be required in localizing Lis1 to the centrosome.
Dcx outlines MTs extending from the perinuclear "cage" to the centrosome
To study the localization of Dcx during migration, RFP-tagged Dcx (Dcx-RFP) was transduced in granule neurons. The localization of Dcx-RFP was nearly identical to endogenous Dcx (Fig. 5 F). Dual transduction with Dcx-RFP and GFP-CETN2 displayed a bright fibrillar bundle-like signal of Dcx encapsulating the nucleus and converging to the centrosome ahead of the nucleus throughout migration (Fig. 5 G). Cotransduction with Dcx-RFP and GFP-tubulin indicated that the fibrillar signal represented MTs (Fig. 5 H), thus Dcx outlined the perinuclear cage-like MT structures previously described in migrating neurons (Rivas and Hatten, 1995). Together, these data suggest that Lis1 and Dcx display distinct but adjacent localization during migration.
Coupling of the nucleus to the centrosome is defective in Lis1-deficient neurons
Our time-lapse recording of migrating neurons using GFP-CETN2 demonstrated that in essentially every cell that was observed moving, the centrosome precedes the nucleus in the direction of migration. According to the reported MT organization in neurons (Aumais et al., 2001; Hatten, 2002), MTs would couple (1) the leading process to the centrosome and (2) the centrosome to the nucleus, in order to translocate the nucleus (Fig. 6 A). Our data on subcellular localization in migrating neurons, together with the known role of Lis1 in mediating dynein motor function, suggests that Lis1 and Dcx might be involved in coupling the nucleus to the preceding centrosome (Fig. 6 B).
|
|
Dynein inhibition results in defects in N-C coupling and neuronal migration
Previous works demonstrated the direct and functional interaction between Lis1 and cytoplasmic dynein (Faulkner et al., 2000; Sasaki et al., 2000; Smith et al., 2000), so we reasoned that inhibiting dynein function may recapitulate the defects due to Lis1 deficiency. To test this, dynamitin overexpression, which disassembles the cytoplasmic dynein component dynactin (Echeverri et al., 1996), was used to disrupt dynein function in migrating neurons.
WT neurons were transduced with RFP-tagged CETN2 (CETN2-RFP) and GFP-dynamitin, nuclei were labeled with Hoechst stain, and the N-C distance was measured in each dual-transduced neuron (Fig. 7 D). Dynamitin overexpression led to a 63% increase in the mean N-C distance with greater variability (Fig. 7 E; mean 1.09 µm, SD 0.77 µm, n = 46, control vs. mean 1.78 µm, SD 1.33 µm, n = 21, GFP-dynamitin; P < 0.01). Thus, N-C coupling in migrating neurons is defective with disruption of dynein function.
To test the effect of dynein inhibition on neuronal migration, the migration assay was performed on granule neurons overexpressing GFP-dynamitin. This resulted in a shift in a migration bin distribution similar to Lis1+/ neurons (Fig. 7 F), and led to a 30% decrease in mean migration distance (47 µm, n = 356, GFP-dynamitin vs. 65 µm, n = 704, GFP control; P < 0.01, t test). Thus, dynein motor function is required for proper N-C coupling and neuronal migration. These data indicate that recapitulation of the N-C coupling defect using a different method also leads to defective migration.
Dcx rescues the impaired N-C coupling caused by dynein inhibition
As Dcx overexpression rescued the N-C coupling defect in Lis1+/ neurons, we examined whether Dcx overexpression rescued the coupling defect seen with dynamitin overexpression. WT neurons were transduced with CETN2-RFP and GFP-dynamitin, and some were additionally transduced with Dcx-RFP (Fig. 7 G). Although both CETN2 and Dcx are labeled with the same fluorophore, localization of these two is distinct, and dual-transduced cells were easily identifiable. Dcx overexpression corrected the N-C coupling defect caused by dynamitin overexpression back to a WT level (Fig. 7 H; mean 1.48 µm, SD 1.34 µm, n = 60, GFP-dynamitin vs. mean 0.84 µm, SD 0.77 µm, n = 36, GFP-dynamitin with Dcx-RFP; P < 0.01). These data suggest that the mechanism by which Dcx overexpression rescues Lis1 deficiency is through restoration of dynein function. Together, our data suggest that Lis1 and Dcx function with dynein to couple the nucleus to the centrosome in neuronal migration.
Disruption of MTs leads to N-C coupling defects
The N-C coupling model suggests that intact MT structures are required. To test this, MTs were disrupted by nocodazole in WT neurons transduced with GFP-CETN2, and N-C distances during migration were analyzed. Mean N-C distances were increased significantly by treatment with nocodazole (Fig. 7 I; mean 0.92 µm, SD 0.70 µm, n = 60, control vs. mean 1.54 µm, SD 1.43 µm, n = 52, 100 nM nocodazole, P < 0.01; mean 2.41 µm, SD 1.42 µm, n = 35, 1 µM nocodazole, P < 0.01, SNK test). These data indicate correct N-C coupling requires MT integrity, and is consistent with the dynein-based coupling mechanism.
Dcx is part of the cytoplasmic dynein complex
The data mentioned in the previous paragraph suggest that Dcx may interact with dynein. We tested whether Dcx, like Lis1 (Faulkner et al., 2000; Sasaki et al., 2000; Smith et al., 2000), is part of the dynein motor complex. Protein complexes were immunoprecipitated using specific antisera. Before immunoprecipitation, whole brain lysates from postnatal d 5 mice were incubated at 4°C in order to depolymerize MTs, and were then centrifuged to remove MT-bound proteins. In this way, proteins complexed through MT bridges alone should be largely removed. Dcx immunoprecipitated with both dynein heavy chain (DHC) and dynein intermediate chain (DIC) antibodies in a fashion similar to Lis1 (Fig. 8). This suggests that Dcx is part of a complex with dynein.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous reports showed that LIS1 overexpression in COS-7 or MDCK cells resulted in altered organization and distribution of MTs, cytoplasmic dynein, dynactin, and the Golgi complex (Smith et al., 2000), increase in mitotic index, a variety of spindle defects, unattached chromosomes, and irregular cell shapes (Faulkner et al., 2000). These analyses were performed in nonneuronal cells in which the physiological LIS1 levels were lower than neuronal cells (Smith et al., 2000), and used adenoviral or cytomegalovirus promoters in which expression levels are much higher than with the retroviral 5' long terminal repeats promoter used here. The resultant nonphysiological changes in LIS1 dosage might be responsible for the toxic effect on these cells. In our work, the level of Lis1 or Dcx after retroviral transduction was a little less than twice the WT level. In this range, overexpression of Dcx or Lis1 appears to be capable of increasing neuronal migration without obvious detrimental or toxic effects. It remains to be seen if even higher levels of expression would produce yet further enhancement in migration.
Patient-related mutations of Dcx or Lis1 abrogate the effects on migration
Overexpression of patient-related mutant proteins had little to no obvious effects on migration. This is the first demonstration of a system that can be used to test the effect of patient mutations on neuronal migration. Previous reports have demonstrated that Lis1 with introduced patient mutations is either unstable (Sapir et al., 1999), displays abnormal folding (Caspi et al., 2003), or cannot properly bind to ligands such as mNudE (Feng et al., 2000). Dcx with introduced patient mutations leads to impaired MT polymerizing ability (Sapir et al., 2000; Taylor et al., 2000) or cannot bind to the FIGQY-phosphotyrosine motif in neurofascin (Kizhatil et al., 2002). Our data clearly indicate these patient-related mutations abrogate their ability to positively regulate migration rate, suggesting the mutant proteins are non- or hypo-functional, and lead to the migration defects that result in the lissencephaly phenotype in humans.
N-C coupling requires normal levels of Lis1 and is regulated by Dcx and dynein
We confirmed that in live migrating neurons the centrosome is nearly uniformly positioned ahead of the nucleus, as previously reported in fixed cells (Rakic, 1971; Gregory et al., 1988; Rivas and Hatten, 1995). In migrating neurons, Lis1 localized at the centrosome and after MT disruption redistributed to the perinuclear region, suggesting that Lis1 translocates from the perinuclear region toward the centrosome via MT structures in physiological conditions, or that at least its localization elsewhere requires MTs. Dcx localized to a perinuclear cage-like MT structure intersecting the centrosome. In Lis1-deficient neurons, we identified defective N-C coupling. Based on our evidence, the known effects of Dcx on MT polymerization, the minus end of MTs being located at the centrosome, and association of Lis1 with the dynein motor complex, we speculate that Dcx is required for proper formation of the perinuclear MT structure, and that the Lis1dynein complex may move in an MT minus end direction, attached to the nuclear membrane, to displace the nucleus toward the centrosome (Fig. 6 B). Our preliminary data suggest a possible defect in perinuclear MT structure in Dcx null migrating neurons, but more work is needed.
There is almost nothing known about N-C coupling during neuronal migration. We demonstrated that in WT migrating neurons, the N-C distance measured 0.81.1 µm on average, and that manipulation of Lis1, Dcx, dynein, or MTs significantly changed this distance. We measured the distance projected in a single horizontal plane in which migration occurred. Any differences in the z axis should have been randomly distributed and should not interfere with these analyses. All our analyses were done on deconvolved images that had clear contours, assuring accuracy in measurement. Our findings are consistent: (1) the increase in N-C distance by Lis1 deficiency was rescued by Lis1-forced expression or Dcx overexpression, which also rescued migration defects; (2) N-C distance was increased by disruption of MTs as well as dynein inhibition; and (3) the differences detected between experimental conditions were highly reproducible, and although this distance was dynamic as neurons move, measurements were collected from several hundred neurons and so these internal variations due to the phases of migration should be captured in our dataset. Therefore, we conclude that the N-C distances collectively measured in live neurons reflect the N-C coupling states and pertain to migration.
Dcx, Lis1, and dynein pathways in nuclear translocation
Previous data indicated Lis1-deficient cells display a defect of dynein function (Sasaki et al., 2000; Smith et al., 2000). Our data show that Lis1 deficiency and dynein inhibition display the same effect on N-C coupling and neuronal migration, suggesting that Lis1 deficiency affects N-C coupling by impairment of dynein function. Dcx overexpression rescued the coupling defects caused by Lis1 deficiency and dynein inhibition. Additionally, we showed coimmunoprecipitation between Dcx and dynein subunits, which may be mediated by Lis1. These data suggest that the mechanism by which Dcx overexpression rescues Lis1 deficiency might be through restoration of dynein function. However, other possible mechanisms of rescue exist, including a modulation of the MT cytoskeleton by Dcx, which might compensate for less efficient Lis1-mediated N-C coupling. Further genetic and cell biological evidence for this rescue will be necessary.
How these proteins function to mediate N-C coupling remains to be determined. There is a growing body of evidence that the dyneindynactin complex associates with nuclear membranes in many cell types, and these complexes coupled to the nucleus may move in a minus enddirected fashion toward the centrosome. Synthetic nuclei assembled from Xenopus egg extracts move along MTs toward their minus ends, the movement required both dynein and the nuclear membrane, and female pronuclear movement is suggested to use this mechanism (Reinsch and Karsenti, 1997). Dynein is required for positioning or nuclear attachment of centrosomes in C. elegans (Gönczy et al., 1999) and Drosophila (Robinson et al., 1999), and localizes to the nuclear envelope of mammalian cells and C. elegans embryos before nuclear envelope breakdown (Busson et al., 1998; Gönczy et al., 1999; Salina et al., 2002). Recently, compelling evidence of a dynein-dependent N-C coupling mechanism has emerged from the study of the Hook protein ZYG-12 in C. elegans (Malone et al., 2003), which localizes to both the nuclear envelope and the centrosome, binds dynein light intermediate chain, and is proposed to move the centrosome toward the nucleus.
Together, these data suggest an evolutionarily conserved pathway using dynein in centrosomenuclear interactions. Mammalian neurons may use this mechanism to translocate the nucleus during migration, where we propose the centrosome is fixed in location by MT structures so that the nucleus moves toward the centrosome. Molecular mechanisms of capture and translocation of the nucleus toward the centrosome and the role of dynein should be further investigated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of recombinant retroviruses
Recombinant ecotropic replication-incompetent Moloney murine leukemia retrovirus (Naviaux et al., 1996; Tomoda et al., 1999) or pseudotyped retrovirus (Yee et al., 1994) was produced according to standard protocols.
Reaggregate neuronal migration assay
Cerebellar granule cells were isolated as described previously (Hatten, 1985) and cultured at 106 cells/ml with retrovirus for 12 h, resulting in uniform-sized reaggregates (100150 µm in diameter), which were then transferred to poly-D-lysine (Sigma-Aldrich) and laminin (Sigma-Aldrich)-treated slides (Bix and Clark, 1998). Imaging was commenced after an additional 12 h using a 20x objective lens and images were analyzed using MetaMorph® v.4.5 (Universal Imaging Corp.).
Isolation of nuclei from cerebellar granule neurons
Nuclei were isolated from cerebellar granule neurons according to the protocols for isolation of mammalian cerebellar nuclei with modifications (Blobel and Potter, 1966; Thompson, 1973; Thomas and Thompson, 1977). Neurons were treated with 10 µg/ml nocodazole and 10 µg/ml latrunculin B for 2 h, centrifuged at 2,000 rpm for 10 min, and the pellet was resuspended in 2.0 M sucrose in TKM (50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM PMSF, 1 mM DTT) and homogenized in an Aldrich-type homogenizer. The homogenate diluted with 2.0 M sucrose/TKM was centrifuged at 64,000 g for 30 min. The pellet was resuspended in 2.4 M sucrose/TKM, overlaid with 1.8 M sucrose/TKM, and recentrifuged at 85,000 g for 30 min. The pellet at the interface of two sucrose layers was collected, resuspended in 0.25 M sucrose/TKM, and centrifuged for 10 min at 1,000 g to remove remaining contaminants.
Immunofluorescence
Neurons were fixed in 4% PFA for 10 min, blocked in 4% normal donkey serum in PBS, and immunostained with anti-Lis1 (provided by L.-H. Tsai, Harvard Medical School, Boston, MA, and D. Smith, University of South Carolina, Columbia, SC; 1:250; Smith et al., 2000), anti-Dcx (1:300; Gleeson et al., 1999a), and anti-nucleoporin (1:100, Mab414; Covance) antibodies controlled by omitting primary antibodies. Cells were washed and stained with Alexa Fluor® secondary antibodies (1:600; Molecular Probes, Inc.).
Image acquisition
Images in Fig. 5 (AF and H) and Fig. 7 were collected using a DeltaVision® deconvolution imaging system (Applied Precision) on a microscope (model IX-70; Olympus) with an oil immersion 100x (NA 1.4) or 60x (NA 1.4) lens at 37°C in a 5% CO2 chamber, using 0.280.35-µm Z-steps, with 10 cycles of deconvolution by softWoRx v.3.2.3. Images in Fig. 5 G were collected by a DeltaVision® system on a microscope (model TE200; Nikon) with a 100x lens (NA 1.4) at 37°C, using 0.3-µm Z-steps, with 10 cycles of deconvolution by softWoRx v.2.5.
Western analysis and immunoprecipitation
Cells were lysed with modified RIPA buffer with protease inhibitors (PMSF, aprotinin, leupeptin), incubated at 4°C for 30 min, calf intestinal alkaline phosphatase was added, and cells were further incubated at 37°C for 1 h. For immunoprecipitation, whole brains from postnatal d 5 mice were homogenized in 1 ml modified RIPA buffer, incubated at 4°C for 30 min, centrifuged at 14,000 rpm, and the supernatant was collected. To 250 µl brain lysate, 10 µg antibody, anti-Dcx (Gleeson et al., 1999a), anti-DIC (CHEMICON International), anti-DHC (CHEMICON International), or control antibody was added and incubated for 1 h, followed by addition of 30 µl protein ASepharose beads (Amersham Biosciences) for 30 min. Beads were washed with PBS four times, boiled in sample buffer, analyzed by Western blotting as previously described (Taylor et al., 2000) with anti-Dcx (1:2,000) or anti-Lis1 (1:200, N-19; Santa Cruz Biotechnology, Inc.), and were developed using chemiluminescence. Band intensities were quantitated in ImageQuant v.1.1 (Molecular Dynamics).
Statistical analysis
For single pairwise comparisons, a two-tailed t test was used. For multiple pairwise comparisons, the SNK test was used, which corrects the P value for multiple comparisons (Glantz, 1996).
Animal experiments
All animal work was performed at the animal facility at the University of California, San Diego School of Medicine under approved animal protocols and in accordance with institutional guidelines.
![]() |
Acknowledgments |
---|
This work was supported by a Post-doctoral Research Training Fellowship (T. Tanaka) and the Junior Investigator Research Grant from the Epilepsy Foundation of America, by the National Institute of Neurological Disorders and Stroke (K12NS01701, R01 NS41537), the University of California, San Diego Neuroscience Microscopy Shared Facility (NS047101), the John Merck Award in the Developmental Disabilities in Childhood, the Searle Scholars Program, and the Klingenstein Foundation.
Submitted: 4 September 2003
Accepted: 26 April 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akagi, T., T. Shishido, K. Murata, and H. Hanafusa. 2000. v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc. Natl. Acad. Sci. USA. 97:72907295.
Aumais, J.P., J.R. Tunstead, R.S. McNeil, B.T. Schaar, S.K. McConnell, S.H. Lin, G.D. Clark, and L.Y. Yu-Lee. 2001. NudC associates with Lis1 and the dynein motor at the leading pole of neurons. J. Neurosci. 21:RC187.
Bai, J., R.L. Ramos, J.B. Ackman, A.M. Thomas, R.V. Lee, and J.J. LoTurco. 2003. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat. Neurosci. 6:12771283.[CrossRef][Medline]
Berg, M.J., G. Schifitto, J.M. Powers, C. Martinez-Capolino, C.T. Fong, G.J. Myers, L.G. Epstein, and C.A. Walsh. 1998. X-linked female band heterotopia-male lissencephaly syndrome. Neurology. 50:11431146.[Abstract]
Bix, G.J., and G.D. Clark. 1998. Platelet-activating factor receptor stimulation disrupts neuronal migration In vitro. J. Neurosci. 18:307318.
Blobel, G., and V.R. Potter. 1966. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 154:16621665.[Medline]
Busson, S., D. Dujardin, A. Moreau, J. Dompierre, and J.R. De Mey. 1998. Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells. Curr. Biol. 8:541544.[Medline]
Cardoso, C., R.J. Leventer, N. Matsumoto, J.A. Kuc, M.B. Ramocki, S.K. Mewborn, L.L. Dudlicek, L.F. May, P.L. Mills, S. Das, et al. 2000. The location and type of mutation predict malformation severity in isolated lissencephaly caused by abnormalities within the LIS1 gene. Hum. Mol. Genet. 9:30193028.
Cardoso, C., R.J. Leventer, J.J. Dowling, H.L. Ward, J. Chung, K.S. Petras, J.A. Roseberry, A.M. Weiss, S. Das, C.L. Martin, et al. 2002. Clinical and molecular basis of classical lissencephaly: Mutations in the LIS1 gene (PAFAH1B1). Hum. Mutat. 19:415.[CrossRef][Medline]
Caspi, M., R. Atlas, A. Kantor, T. Sapir, and O. Reiner. 2000. Interaction between LIS1 and doublecortin, two lissencephaly gene products. Hum. Mol. Genet. 9:22052213.
Caspi, M., F.M. Coquelle, C. Koifman, T. Levy, H. Arai, J. Aoki, J.R. De Mey, and O. Reiner. 2003. LIS1 missense mutations: variable phenotypes result from unpredictable alterations in biochemical and cellular properties. J. Biol. Chem. 278:3874038748.
Coquelle, F.M., M. Caspi, F.P. Cordelieres, J.P. Dompierre, D.L. Dujardin, C. Koifman, P. Martin, C.C. Hoogenraad, A. Akhmanova, N. Galjart, et al. 2002. LIS1, CLIP-170's key to the dynein/dynactin pathway. Mol. Cell. Biol. 22:30893102.
Corbo, J.C., T.A. Deuel, J.M. Long, P. LaPorte, E. Tsai, A. Wynshaw-Boris, and C.A. Walsh. 2002. Doublecortin is required in mice for lamination of the hippocampus but not the neocortex. J. Neurosci. 22:75487557.
Davis, L.I., and G. Blobel. 1986. Identification and characterization of a nuclear pore complex protein. Cell. 45:699709.[Medline]
Dobyns, W.B., and C.L. Truwit. 1995. Lissencephaly and other malformations of cortical development: 1995 update. Neuropediatrics. 26:132147.[Medline]
Dobyns, W.B., E. Andermann, F. Andermann, D. Czapansky-Beilman, F. Dubeau, O. Dulac, R. Guerrini, B. Hirsch, D.H. Ledbetter, N.S. Lee, et al. 1996. X-linked malformations of neuronal migration. Neurology. 47:331339.[Abstract]
Dobyns, W.B., C.L. Truwit, M.E. Ross, N. Matsumoto, D.T. Pilz, D.H. Ledbetter, J.G. Gleeson, C.A. Walsh, and A.J. Barkovich. 1999. Differences in the gyral pattern distinguish chromosome 17-linked and X-linked lissencephaly. Neurology. 53:270277.
Dujardin, D.L., L.E. Barnhart, S.A. Stehman, E.R. Gomes, G.G. Gundersen, and R.B. Vallee. 2003. A role for cytoplasmic dynein and LIS1 in directed cell movement. J. Cell Biol. 163:12051211.
Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132:617633.[Abstract]
Faulkner, N.E., D.L. Dujardin, C.Y. Tai, K.T. Vaughan, C.B. O'Connell, Y. Wang, and R.B. Vallee. 2000. A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function. Nat Cell Biol. 2:784791.[CrossRef][Medline]
Feng, Y., E.C. Olson, P.T. Stukenberg, L.A. Flanagan, M.W. Kirschner, and C.A. Walsh. 2000. LIS1 regulates CNS lamination by interacting with mNudE, a central component of the centrosome. Neuron. 28:665679.[Medline]
Francis, F., A. Koulakoff, D. Boucher, P. Chafey, B. Schaar, M.C. Vinet, G. Friocourt, N. McDonnell, O. Reiner, A. Kahn, et al. 1999. Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron. 23:247256.[Medline]
Friocourt, G., A. Koulakoff, P. Chafey, D. Boucher, F. Fauchereau, J. Chelly, and F. Francis. 2003. Doublecortin functions at the extremities of growing neuronal processes. Cereb. Cortex. 13:620626.
Gambello, M.J., D.L. Darling, J. Yingling, T. Tanaka, J.G. Gleeson, and A. Wynshaw-Boris. 2003. Multiple dose-dependent effects of Lis1 on cerebral cortical development. J. Neurosci. 23:17191729.
Glantz, S.A. 1996. Primer of Biostatistics. 4th ed. McGraw-Hill Inc., New York. 473 pp.
Gleeson, J.G., K.M. Allen, J.W. Fox, E.D. Lamperti, S. Berkovic, I. Scheffer, E.C. Cooper, W.B. Dobyns, S.R. Minnerath, M.E. Ross, and C.A. Walsh. 1998. doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell. 92:6372.[Medline]
Gleeson, J.G., P.T. Lin, L.A. Flanagan, and C.A. Walsh. 1999a. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron. 23:257271.[Medline]
Gleeson, J.G., S.R. Minnerath, J.W. Fox, K.M. Allen, R.F. Luo, S.E. Hong, M.J. Berg, R. Kuzniecky, P.J. Reitnauer, R. Borgatti, et al. 1999b. Characterization of mutations in the gene doublecortin in patients with double cortex syndrome. Ann. Neurol. 45:146153.[CrossRef][Medline]
Gleeson, J.G., S. Minnerath, R.I. Kuzniecky, W.B. Dobyns, I.D. Young, M.E. Ross, and C.A. Walsh. 2000. Somatic and germline mosaic mutations in the doublecortin gene are associated with variable phenotypes. Am. J. Hum. Genet. 67:574581.[CrossRef][Medline]
Gönczy, P., S. Pichler, M. Kirkham, and A.A. Hyman. 1999. Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J. Cell Biol. 147:135150.
Gönczy, P., J.-M. Bellanger, M. Kirkham, A. Pozniakowski, K. Baumer, J.B. Phillips, and A.A. Hyman. 2001. zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-like kinase. Dev. Cell. 1:363375.[Medline]
Gregory, W.A., J.C. Edmondson, M.E. Hatten, and C.A. Mason. 1988. Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro. J. Neurosci. 8:17281738.[Abstract]
Hatten, M.E. 1985. Neuronal regulation of astroglial morphology and proliferation in vitro. J. Cell Biol. 100:384396.[Abstract]
Hatten, M.E. 2002. New directions in neuronal migration. Science. 297:16601663.
Hirotsune, S., M.W. Fleck, M.J. Gambello, G.J. Bix, A. Chen, G.D. Clark, D.H. Ledbetter, C.J. McBain, and A. Wynshaw-Boris. 1998. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat. Genet. 19:333339.[CrossRef][Medline]
Horesh, D., T. Sapir, F. Francis, S.G. Wolf, M. Caspi, M. Elbaum, J. Chelly, and O. Reiner. 1999. Doublecortin, a stabilizer of microtubules. Hum. Mol. Genet. 8:15991610.
Kizhatil, K., Y.X. Wu, A. Sen, and V. Bennett. 2002. A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin. J. Neurosci. 22:79487958.
Lee, W.L., J.R. Oberle, and J.A. Cooper. 2003. The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast. J. Cell Biol. 160:355364.
Liang, S., and K.A. Crutcher. 1992. Neuronal migration on laminin in vitro. Brain Res. Dev. Brain Res. 66:127132.[Medline]
Lo Nigro, C., C.S. Chong, A.C. Smith, W.B. Dobyns, R. Carrozzo, and D.H. Ledbetter. 1997. Point mutations and an intragenic deletion in LIS1, the lissencephaly causative gene in isolated lissencephaly sequence and Miller-Dieker syndrome. Hum. Mol. Genet. 6:157164.
Lois, C., J.M. Garcia-Verdugo, and A. Alvarez-Buylla. 1996. Chain migration of neuronal precursors. Science. 271:978981.[Abstract]
Malone, C.J., L. Misner, N. Le Bot, M.C. Tsai, J.M. Campbell, J. Ahringer, and J.G. White. 2003. The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus. Cell. 115:825836.[Medline]
Matsumoto, N., R.J. Leventer, J.A. Kuc, S.K. Mewborn, L.L. Dudlicek, M.B. Ramocki, D.T. Pilz, P.L. Mills, S. Das, M.E. Ross, et al. 2001. Mutation analysis of the DCX gene and genotype/phenotype correlation in subcortical band heterotopia. Eur. J. Hum. Genet. 9:512.[CrossRef][Medline]
Naviaux, R.K., E. Costanzi, M. Haas, and I.M. Verma. 1996. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70:57015705.[Abstract]
Niethammer, M., D.S. Smith, R. Ayala, J. Peng, J. Ko, M.S. Lee, M. Morabito, and L.H. Tsai. 2000. NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein. Neuron. 28:697711.[Medline]
Pilz, D.T., J. Kuc, N. Matsumoto, J. Bodurtha, B. Bernadi, C.A. Tassinari, W.B. Dobyns, and D.H. Ledbetter. 1999. Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1. Hum. Mol. Genet. 8:17571760.
Rakic, P. 1971. Guidance of neurons migrating to the fetal monkey neocortex. Brain Res. 33:471476.[CrossRef][Medline]
Reinsch, S., and E. Karsenti. 1997. Movement of nuclei along microtubules in Xenopus egg extracts. Curr. Biol. 7:211214.[Medline]
Rivas, R.J., and M.E. Hatten. 1995. Motility and cytoskeletal organization of migrating cerebellar granule neurons. J. Neurosci. 15:981989.[Abstract]
Robinson, J.T., E.J. Wojcik, M.A. Sanders, M. McGrail, and T.S. Hays. 1999. Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila. J. Cell Biol. 146:597608.
Ross, M.E., K. Swanson, and W.B. Dobyns. 2001. Lissencephaly with cerebellar hypoplasia (LCH): a heterogeneous group of cortical malformations. Neuropediatrics. 32:256263.[CrossRef][Medline]
Salina, D., K. Bodoor, D.M. Eckley, T.A. Schroer, J.B. Rattner, and B. Burke. 2002. Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell. 108:97107.[Medline]
Sapir, T., M. Eisenstein, H.A. Burgess, D. Horesh, A. Cahana, J. Aoki, M. Hattori, H. Arai, K. Inoue, and O. Reiner. 1999. Analysis of lissencephaly-causing LIS1 mutations. Eur. J. Biochem. 266:10111020.
Sapir, T., D. Horesh, M. Caspi, R. Atlas, H.A. Burgess, S.G. Wolf, F. Francis, J. Chelly, M. Elbaum, S. Pietrokovski, and O. Reiner. 2000. Doublecortin mutations cluster in evolutionarily conserved functional domains. Hum. Mol. Genet. 9:703712.
Sasaki, S., A. Shionoya, M. Ishida, M.J. Gambello, J. Yingling, A. Wynshaw-Boris, and S. Hirotsue. 2000. A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system. Neuron. 28:681696.[Medline]
Smith, D.S., M. Niethammer, R. Ayala, Y. Zhou, M.J. Gambello, A. Wynshaw-Boris, and L.H. Tsai. 2000. Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1. Nat. Cell Biol. 2:767775.[CrossRef][Medline]
Swan, A., T. Nguyen, and B. Suter. 1999. Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning. Nat. Cell Biol. 1:444449.[CrossRef][Medline]
Taylor, K.R., A.K. Holzer, J.F. Bazan, C.A. Walsh, and J.G. Gleeson. 2000. Patient mutations in doublecortin define a repeated tubulin-binding domain. J. Biol. Chem. 275:3444234450.
Thomas, J.O., and R.J. Thompson. 1977. Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons. Cell. 10:633640.[Medline]
Thompson, R.J. 1973. Studies on RNA synthesis in two populations of nuclei from the mammalian cerebral cortex. J. Neurochem. 21:1940.[Medline]
Tomoda, T., R.S. Bhatt, H. Kuroyanagi, T. Shirasawa, and M.E. Hatten. 1999. A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons. Neuron. 24:833846.[Medline]
White, R.A., Z. Pan, and J.L. Salisbury. 2000. GFP-centrin as a marker for centriole dynamics in living cells. Microsc. Res. Tech. 49:451457.[CrossRef][Medline]
Xiang, X. 2003. LIS1 at the microtubule plus end and its role in dynein-mediated nuclear migration. J. Cell Biol. 160:289290.
Xiang, X., S.M. Beckwith, and N.R. Morris. 1994. Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA. 91:21002104.[Abstract]
Xiang, X., A.H. Osmani, S.A. Osmani, M. Xin, and N.R. Morris. 1995. NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol. Biol. Cell. 6:297310.[Abstract]
Xiang, X., W. Zuo, V.P. Efimov, and N.R. Morris. 1999. Isolation of a new set of Aspergillus nidulans mutants defective in nuclear migration. Curr. Genet. 35:626630.[CrossRef][Medline]
Yee, J.K., T. Friedmann, and J.C. Burns. 1994. Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol. 43:99112.[Medline]