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. Author manuscript; available in PMC: 2009 Feb 8.
Published in final edited form as: Cell. 2008 Feb 8;132(3):474–486. doi: 10.1016/j.cell.2008.01.026

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division

Jessica Yingling 1,2,3, Yong Ha Youn 1,3,5, Dawn Darling 1, Kazuhito Toyo-oka 1,4,5, Tiziano Pramparo 1,5, Shinji Hirotsune 4, Anthony Wynshaw-Boris 1,5,6
PMCID: PMC2265303  NIHMSID: NIHMS40368  PMID: 18267077

Abstract

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1 deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.

INTRODUCTION

The neural tube at embryonic day 8–8.5 (E8–8.5) in the mouse consists of a single pseudostratified epithelial layer of neuroepithelial stem cells (NESC) that undergo rapid symmetrical proliferative divisions as the neural tube grows and the progenitor pool is expanded. Neurogenesis begins at about E12 as the NESCs first undergo asymmetric divisions to generate one radial glial progenitor cell (RGPC) and one migratory post-mitotic daughter neuron. The neocortex partitions into the proliferative ventricular zone (VZ), intermediate zone (IZ), cortical plate (CP) and marginal zone (MZ). Several embryonic lamina are established between E13–19 in the mouse in an “inside-out” pattern as neurons generated from the RGPCs in the VZ migrate through the IZ toward the CP. Similar events occur during the development of other areas of the brain such as the midbrain and hindbrain (reviewed in Gupta et al. 2002).

The maintenance of proliferative NESCs and their transition to RGPC at the onset of neurogenesis is fundamental to the formation of the brain (reviewed in Gotz and Huttner 2005). NESCs and RGPCs span the neural tube and/or brain, attached at the apical (ventricular) and basal (pial) surfaces. Both types of progenitors display interkinetic nuclear migration (Takahashi et al. 1993, reviewed in Caviness et al. 2003). Nuclei migrate from apical to basal surfaces in NESCs, while interkinetic nuclear migration in RGPCs is restricted to the VZ. Both types of progenitors display apical-basal polarity, with apical localization of the centrosome, PAR3, PAR6/atypical PKC (aPKC) and junctional proteins (reviewed in Gotz and Huttner 2005). NESCs are generated from symmetrical proliferative divisions perpendicular to the apical-basal axis in the single layer of the neuroepithelium (Chenn and McConnell 1995; Haydar et al. 2003). In the RGPCs that act as neuronal progenitors during neurogenesis as well as radial guides for migration of daughter post-mitotic cells (Noctor et al. 2001; Miyata et al. 2001), both symmetric and asymmetric divisions occur (Chenn and McConnell 1995; Haydar et al. 2003; Kosodo et al. 2004). Symmetrical divisions in NESCs likely result in the symmetrical distribution of polarized apical components while asymmetric divisions in RGPCs distribute components asymmetrically to produce one progenitor and one neuron or glial cell (reviewed in Gotz and Huttner 2005). The importance of mitotic cleavage plane during asymmetric cell division in RGPCs was demonstrated for NDE1 (Feng and Walsh 2004), DCLK (Shu et al. 2006), Gβγ and AGS3 (Sanada and Tsai 2005), but little is known about differences in the regulation of symmetric and asymmetric divisions in NESCs and RGPCs. Importantly, no genetic mutants disrupting symmetrical divisions in NESCs or distinguishing mechanisms of symmetric from asymmetric divisions have yet been identified.

Here, in the course of studying its in vivo role, we identify Lis1 (also known as Pafah1b1) as a critical component in NESCs. Heterozygous loss or mutation of LIS1 is sufficient to cause the human neuronal migration defect lissencephaly (“smooth brain”), characterized by a smooth cortical surface, abnormal cortical layering and enlarged ventricles (reviewed in Gupta et al. 2002). We produced an allelic series of mice with varying doses of Lis1 generated from different combinations of two mutant alleles of Lis1 in mice (see Supplemental Figure 1), a null knock-out (Lis1ko) and a conditional knock-out/hypomorphic allele (Lis1hc), and demonstrated in vivo Lis1-dosage dependent neuronal migration defects (Hirotsune et al. 1998; Gambello et al. 2003; Tanaka et al. 2004). RNAi evidence also supports the role of LIS1 in neuronal migration in vivo (Shu et al. 2004; Tsai et al. 2005). LIS1 also has a broader role in mammalian development (reviewed in Vallee and Tsai 2006). Lis1 null mice are peri-implantation lethal (Hirotsune et al. 1998; Cahana et al. 2001). Lis1+/ko and Lis1hc/ko mice exhibited defects in neurogenesis and interkinetic nuclear migration at the ventricular zone of the cortex, implicating LIS1 in neurogenesis (Gambello et al. 2003). LIS1 overexpression or disruption by antibody injection in cell culture support a role for LIS1 in a variety of dynein-dependent mitotic functions (Faulkner et al. 2000; Tai et al. 2002). Finally, siRNA knock-down of Lis1 in rat cortical slice cultures resulted in defects in migration, interkinetic nuclear migration, and ventricular zone/intermediate zone defects in cell division (Tsai et al. 2005). These and other studies suggest an important role for LIS1 in cell division and neurogenesis, but its precise role is not well-defined.

We directly demonstrate an essential and surprising role for Lis1 in the neuroepithelium at times prior to the onset of radial glial neurogenesis and neuronal migration as well as in mitotic spindle orientation and symmetric division at the apical surface of cells. This effect appears to result from a requirement for LIS1 in dynein-mediated cortical microtubule capture. This novel function for LIS1 during mitosis and neurogenesis reveals a unique and critical role for the control of vertical spindle orientation and symmetric cleavage during neuroepithelial expansion of NESCs, and supports the established role of spindle orientation in RGPC neurogenesis.

RESULTS

LIS1 is essential in the neuroepithelium prior to neuronal migration

To examine the role of Lis1 during progenitor expansion and neurogenesis, we ablated Lis1 in restricted spatial and/or temporal patterns in the developing brain using a Lis1 conditional knock-out and compared the phenotype of littermates heterozygous or homozygous for the Lis1 hypomorphic-conditional allele (Lis1hc) with various Cre transgenes (Supplemental Figure 1). The Rosa26-lacZ Cre reporter (Soriano 1999) marked Cre-exposed cells.

Pax2-Cre transgenic mice express Cre predominantly at the midbrain-hindbrain junction starting at E8.0 in the neuroepithelium prior to the onset of neuronal migration (Lewis et al. 2004). Lis1hc/hc; Pax2-Cre; Rosa26 pups did not survive past P0. Strikingly, the midbrain, midbrain-hindbrain junction and the cerebellum were absent in nearly all (n=10) Lis1hc/hc; Pax2-Cre; Rosa26 mice, resulting in the separation of the forebrain from the hindbrain at birth (data not shown). In whole embryos at E9.5, there was a small but noticeable reduction in X-gal staining in the branchial arches (Figure 1A, B and E, F, arrows) and the midbrain-hindbrain region (Figure 1A, B to E, F, arrowheads) of Lis1hc/hc; Pax2-Cre; Rosa26 compared with Lis1+/hc; Pax2-Cre; Rosa26 embryos. The majority of the X-gal positive cells in the midbrain-hindbrain, but not the X-gal negative cells in the telencephalon, underwent apoptotic cell death in the Lis1hc/hc; Pax2-Cre; Rosa26 embryos (Figure 1G, H) whereas control embryos displayed negligible numbers of apoptotic cells (Figure 1C, D).

Figure 1.

Figure 1

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(A–D) Heterozygous Lis1+/hc; Pax2-Cre; Rosa26 and (E–H) homozygous Lis1hc/hc; Pax2-Cre; Rosa26 embryos stained with X-gal at E9.5. Lateral (A, E) and top (B, F) views of whole mounts with blue stained pharyngeal arches (arrows) and midbrain (arrowheads) indicative of Cre expression. Mid-sagittal sections demonstrate the midbrain-hindbrain expression of Cre (C, G). An adjacent section (D, H) stained with DAPI (blue) and TUNEL (bright spots) demonstrates massive apoptosis in the Xgal-positive regions in homozygotes compared with heterozygous controls. (I–L) Heterozygous Lis1+/hc; Wnt1-Cre; Rosa26 and (M–P) homozygous Lis1hc/hc; Wnt1-Cre; Rosa26 embryos at E10.5 (I, J, M, N) and E12.5 (K, L, O, P), stained with X-gal. Arrow (N, P) denotes drastic loss of blue cells in the midbrain in the homozygote. (Q–T) Wild-type Lis1+/+; hGFAP-Cre; Rosa26 and (U–X) homozygous Lis1hc/hc; hGFAP-Cre; Rosa26 neonates (P3). (Q, U) Cresyl violet staining. (Q–T, V–X) Immunohistochemistry with anti-testis-1 (to mark CA1 of the hippocampus and layers 2 and 5 of the cortex). Note absence of the hippocampus in the homozygote (arrows in V, W) with severe disruption and underpopulation of cortical layers 2 and 5 of the cortex (arrow in X).

Wnt1-Cre (Chai et al. 2000) is expressed at the midbrain-hindbrain junction and in the telencephalon at about E8–8.5 (see Figure 1I, J). The broader expression pattern of Wnt1-Cre resulted in severe neuronal loss in the telencephalon as well as in the midbrain-hindbrain junction of Lis1hc/hc; Wnt1-Cre; Rosa26 embryos and death at about E13.5. At E10.5, the telencephalon of Lis1hc/hc; Wnt1-Cre; Rosa26 embryos was significantly smaller and degenerated compared to control (compare Figure 1I, J with M, N), and degeneration of the telencephalon and midbrain was observed at E11.5 (compare Figure 1K, L with O, P). These acute phenotypes were observed in neuronal progenitor cells in the neuroepithelium prior to the onset of RGPC neurogenesis and neuronal migration, demonstrating that LIS1 is essential during neuroepithelial expansion of NESCs in the midbrain-hindbrain junction as well as the telencephalon.

LIS1 is essential for neuroepithelial expansion but not radial glial neurogenesis

To determine whether LIS1 plays an important role during RGPC neurogenesis as well as NESC expansion in the neuroepithelium, we used the hGFAP-Cre transgenic mouse to inactivate Lis1 (Zhuo et al. 2001). hGFAP-Cre expression begins at E12.5–13.5 in distinct developmental stages in neuronal precursor cells of the neocortex and hippocampus (Malatesta et al. 2003). hGFAP-Cre is active in the RGPCs of virtually all radially migrating cells in the neocortex, but not in the neocortical neuroepithelium prior to E12.5. By contrast, hGFAP-Cre is expressed in the neuroepithelium in the hippocampal region at E12.5, a time when only immature mitotic NESCs are present. If Lis1 is critical in NESCs and RGPC, then the phenotype should be similarly severe in both the hippocampus and cortex, but if Lis1 is more important in NESCs than RGPCs, then the phenotype should be more severe in the hippocampus than the cortex. In wild-type Lis1+/+; hGFAP-Cre; Rosa26 (Figure 1Q–T) neonates, the cortex and hippocampus were well developed. Lis1hc/hc; hGFAP-Cre; Rosa26 neonates survived to P5, but were completely missing the hippocampus (Figure 1U–X, see arrows in Figure 1V, W). The cortex of these neonates was present, although thinner than control animals. Nearly all cells present in the cortex had some blue staining, indicating that Cre was present in these cells (data not shown). A few apoptotic cells were observed in the cortex of Lis1hc/hc; hGFAP-Cre; Rosa26 embryos (data not shown) in contrast to the massive and acute apoptosis observed when Lis1 was deleted during NESC expansion. These studies demonstrate an immediate and essential role for Lis1 in dividing neural progenitor cells of the neuroepithelium, while the loss of Lis1 in radial glial progenitors undergoing neurogenesis and neuronal migration have less immediate consequences for survival.

LIS1 is essential for apical spindle orientation in the ventricular zone

To identify factors that differentiate NESCs and RGPCs (reviewed in Gotz and Huttner 2005), we determined whether LIS1 affects apical-basal polarity and/or mitotic spindle orientation during neuroepithelial expansion and/or radial glial neurogenesis. Apical determinants such as centrosomes (marked by pericentrin), β-catenin, aPKC, Numb and the cadherin hole (Kosodo et al. 2004) were apically located in wild-type or Lis1+/ko or Lis1hc/ko mutant embryos with 50% or 35% of wild-type LIS1 protein respectively (see Gambello et al. 2003) in the neuroepithelium (E9.5, Figure 2A, B, Supplemental Figure 2, 3) and radial glia (E14.5, Figure 2C, D, Supplemental Figure 2, 3), although not as tightly localized to the apical surface of mutant embryos in a Lis1 dosage-dependent fashion. This may be due to the less organized cellular structure from long-term deficiencies of LIS1 as indicated by nestin (Figure 2A, B, Supplemental Figure 2). Of note, the cadherin hole was present at the apical surface of wild-type and all mutant embryos (Figure 2A–D, Supplemental Figures 2, 3) consistent with the maintenance of apical polarity. Nuclei undergoing interkinetic nuclear migration are elongated and distributed throughout the apical-basal axis, as seen in wild-types (Figure 2A, C), but in Lis1 mutants, the nuclei were round and accumulated near the apical surface (Figure 2B, D), consistent with previously described interkinetic nuclear migration defects (Gambello et al. 2003; Shu et al. 2004; Tsai et al. 2005).

Figure 2.

Figure 2

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Left top panels: Single optical confocal sections of neural tubes from wild-type (A, C) and Lis1hc/ko (B, D) embryos at E9.5 (A, B) and E14.5 (C, D) were stained with nestin (neural stem cell marker, green) and pericentrin (centrosomal marker, red) in the left panels, and pancadherin (apical marker, red) in the right panels to detect the cadherin hole (arrows). All sections were counterstained with DAPI. Dotted lines are examples of spindle cleavage plane measurements relative to the ventricular zone surface, and arrows the apical location of the cadherin hole. Right top panels: Single optical confocal sections of neural tubes from Lis1hc/hc littermates either without (E, G) or with (F, H) Cre-ERTM, a tamoxifen inducible Cre, after tamoxifen injection into pregnant females at E8.5, and embryo isolation 12 or 24 hr later. Neural tubes were stained with nestin (neural stem cell marker, green) and pericentrin (centrosomal marker, red) in the left panels, aPKCζ (apical marker, red) in the central panels and pan-cadherin (apical marker, red) in the right panels to detect the cadherin hole (arrows). All sections were counterstained with DAPI. (I) Mean (box) and standard deviation (lines) of angles of spindle cleavage of NE (E9.5) progenitors in WT (blue, 80.72° ± 9.56), Lis1+/ko (red, 64.73° ± 24.5) and Lis1hc/ko (green, 48.40° ± 22.0) embryos, as well as RG (E14.5) progenitors in WT (blue, 70.15° ± 18.3), Lis1+/ko (red, 65.27° ± 24.5) and Lis1hc/ko (green, 40.49° ± 25.9) embryos are shown. (J) Mean (box) and standard deviation (lines) of angles of spindle cleavage of littermate Lis1hc/ko (−CRE, blue) or Lis1hc/ko; Cre-ERTM (+CRE, orange) embryos 12, 24 and 36 hr after pregnant females were injected intraperitoneally at E8.5 with tamoxifen. Average angles at 12, 24 and 36 hr of −CRE embryos were 80.32° ± 8.33, 78.52° ± 9.93 and 79.75° ± 8.19, respectively, while for +CRE embryos the angles were 49.58° ± 26.5, 44.36° ± 25.2 and 50.34° ± 16.4 at 12, 24 and 36 hrs, respectively. (K) Mean (bars) and standard deviation (lines) of percentage of apoptotic cells of littermate Lis1hc/ko (−CRE, blue) or Lis1hc/ko; Cre-ERTM (+CRE, orange) embryos 12, 24 and 36 hr after pregnant females were injected intraperitoneally at E8.5 with tamoxifen. (L) Mean (bars) and standard deviation (lines) of angles of the number of mitotic cells measured by phopho-H3 histone of littermate Lis1hc/ko (−CRE, blue) or Lis1hc/ko; Cre-ERTM (+CRE, orange) embryos 12, 24 and 36 hr after pregnant females were injected intraperitoneally at E8.5 with tamoxifen. Green areas at the top of each bar represents the portion of phospho-H3 postitive cells away (non-apical) from their normal position at the apical surface (brackets). Asterisks in I-L indicate * p <0.05, ** p <0.02, *** p <0.0001 by unpaired ANOVA with Bonferroni post-hoc test.

Symmetrical apical mitotic cell divisions display vertically oriented cleavage planes (90° relative to the plane of the apical surface) to generate two proliferative daughter progenitor cells attached at the apical and basal surfaces, while asymmetric divisions generate one proliferative daughter cell and a postmitotic neuron or glial cell. At E9.5, all wild-type anaphase neuroepithelial cells in the neural tube displayed nearly vertical (symmetric) cleavage (Figure 2A, I, average angle: 80.72° ± 9.56), while the majority of anaphase cells in wild-type cortical slices at radial glial stages (E14.5) displayed less perpendicular spindle cleavage planes with more variability (Figure 2C, I, average angle: 70.15° ± 18.3, p < 0.016). In neural tubes at E9.5 and in cortex at E14.5 from Lis1+/ko or Lis1hc/hc embryos, spindle cleavage planes were more randomized and more horizontal to the ventricular surface than WT, with the degree of randomization proportional to the reduction of LIS1 (Figure 2B, D, I). This suggests that disruption of the orientation of apical spindle cleavage planes seen with reduction in LIS1 levels results in a severe and catastrophic disruption in the neuroepithelium during NESC expansion when cleavage plane is tightly controlled. During radial glial neurogenesis, spindle orientation defects result in a change of progenitor cell fate, reducing the progenitor pool population early in cortical development and decreasing the total number of cortical neurons. Consistent with this, there are reduced numbers of Tst-1-positive layer 2 cortical neurons (Figure 1X, arrow) and neurons marked with a series of layer specific markers (data not shown) in Lis1hc/hc; hGFAP-Cre; Rosa26 neonates.

To delineate the temporal sequence of events that occurred after acute loss of Lis1, we used a tamoxifen-inducible Cre (Cre-ERTM, Hayashi and McMahon 2002), and the Tg(ACTB-bgeo/DsRed.MST) Cre reporter (Vintersten et al. 2005), which switches from beta-geo to DsRed.MST expression after Cre recombination. We injected pregnant dams with tamoxifen (2 mg/40 g mouse) to activate Cre at E8.5, and examined the phenotypes of Lis1hc/hc; Cre-ERTM experimental and Lis1hc/hc control embryos without Cre from the same litter 12, 24 and 36 hours later. Within 12 hours of Cre activation, vertical spindle orientation was disrupted (Figure 2E, F, J). This immediate disruption of spindle orientation was associated with an increase in apoptosis (Figure 2K) and metaphase arrest with mitotic cells located away from the apical surface (phospho-H3 histone, Figure 2L), without disruption of either apical polarity or epithelial integrity (Figure 2E, F, Supplemental Figures 2 and 4). At later time points, 24 and 36 hours after Cre activation, the spindle, apoptosis and metaphase arrest phenotypes progressed further, with only minimal disruption of apical polarity and epithelial integrity (Figure 2G,H, J–L and Supplemental Figures 2, 4). These findings indicate that the immediate effect of the loss of Lis1 is the disruption of the orientation of apical spindle cleavage planes and proliferation resulting in a severe and catastrophic disruption in the neuroepithelium during NESC expansion when cleavage plane is tightly controlled.

LIS1 is essential for spindle microtubule organization and cell cycle progression

To examine cell cycle progression and spindle organization in vitro, mouse embryonic fibroblasts (MEFs) with decreasing doses of LIS1 were generated from mice with combinations of the Lis1ko and Lis1hc hypomorphic alleles (see Supplemental Figure 1), including Lis1hc/hc; Cre-ERTM MEFs to analyze the effects of complete loss of LIS1. Lis1+/ko and Lis1hc/ko MEFs displayed 50% (data not shown), 35% of wild-type LIS1, while Lis1hc/hc; Cre-ERTM MEFs 0h, 24h, 48h and 72h after tamoxifen treatment (hereafter termed Lis124h, Lis148h and Lis172h) displayed 80%, 52%, 25% and <10%, respectively, of wild-type levels by Western blot (Figure 3A). The doubling time of Lis1hc/ko, Lis1+/ko and wild-type MEFs was 38h, 26h and 18h, respectively (Figure 3B). There was a significant decrease in growth of Lis124h cells, a nearly normal increase Lis148h cells, and a plateau thereafter (Figure 3C). No effect of tamoxifen treatment on cell growth was seen with treatment of Lis1hc/hc MEFs without the Cre-ERTM transgene (data not shown). Cell death did not significantly increase in tamoxifen-treated versus untreated cells (data not shown). Mitotic index was 4.0±0.14%, 3.6±0.10%, 2.2±0.12% for wild-type, Lis1+/ko and Lis1cko/ko cells, respectively, while in Lis24h and Lis48h cells it was 13.1%±0.09% and 1.9±0.08%. Doubling time and mitotic index were significantly different among groups (p < 0.05). There was a block in mitosis from a mitotic delay in prometaphase at 24h (10.0%±0.09% vs. 4.0%±0.11% in control cells), which disappeared at 48 and 72h (Figure 3D, data not shown), similar to findings from LIS1 depletion experiments (Faulkner et al. 2000). Tamoxifen-treated Cre-ERTM; Lis1hc/hc MEFs underwent one cell division and then senesced or moved more slowly through all phases of the cell cycle, since there was little increase in prometaphase cells at 48h and 72h.

Figure 3.

Figure 3

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(A) Western Blot of LIS1 and actin levels in wild-type (wt) and Lis1hc/ko MEFs, as well as in Lis1hc/hc; Cre-ERTM; Rosa26 MEFs after 0-72h tamoxifen treatment. (B) Growth curve of wt, Lis1+/ko and Lis1hc/ko MEFS. (C) Growth curve of Lis1cko/cko; Cre-ERTM without (control) and with 1 µm tamoxifen treatment (tamoxifen). (D) Quantitation of mitotic index using antiphospho H3 Histone. Doubling time and mitotic index were significantly different among groups (p < 0.05) by one-way ANOVA with a Bonferroni post-hoc test. Astral MTs concentration measured by EB1 (green) and β-tubulin (red) display in wt (E), Lis1hc/ko (F) and Lis148h (G) MEFs. White lines indicate the edge of the cell, and blue is Hoechst. Prometaphase wt (H), Lis1hc/ko (I) and Lis148h (J) MEFs after immunohistochemistry with pericentrin (red) and α-tubulin (green). (K–N) MG-132 metaphase-arrested wt (K, L) and Lis1hc/ko (M, N) MEFs were untreated (K, M) or cold-treated on ice (L, N) for an hour to depolymerize unattached MTs within the spindle, and stained for α-tubulin (green) and Hoechst (blue). Spindles from wt (O), Lis1hc/ko (P), Lis124h (Q) and Lis148h (R) MG132-arrested MEFs stained with α-tubulin (green), SH-CREST (red) and Hoechst (blue). Lagging chromosomes (arrows) were observed in Lis148h MEFs. Anaphase structures of wt (S, T) and Lis124h (U, V) MEFs stained with α-tubulin (green) and Hoechst (blue). Arrows point to lagging chromosomes in Lis148h MEFs. Scale bars: H–J 5 µm, K–N 4 µm and O–R 5 µm.

MEFs with decreased LIS1 levels displayed severely shortened and sparse astral MTs, using an EB1 antibody to label growing MT plus ends (Figure 3E–G), an effect on either MT growth or stability proportional to the amount of LIS1. LIS1 reduction had no affect on the formation of bipolar spindles, spindle pole maintenance or number (Figure 3H–J). Prometaphase spindles of LIS1 mutant MEFs were smaller and slightly disorganized with fewer MTs (Figure 3I, J) and a few cells in anaphase and telophase were found. Metaphase spindles of Lis1hc/ko (Figure 3M), Lis124h and Lis148h (data not shown) MEFs treated with MG-132 (to provoke metaphase arrest) looked similar to bipolar spindles of wild-type MEFs (Figure 3K) but were smaller (Figure 3Q, R). Wild-type and Lis1hc/ko MEFs were treated with MG-132 for 3h and then placed on ice for an hour to depolymerize unattached MTs. Lis1hc/ko MEFs did not display a significant decrease in stabilized spindle MTs (Figure 3N) compared with wild-type (Figure 3L). Lis148h spindles had less tightly aligned chromosomes compared with wild-type and other Lis1 mutants (Figure 3O–R), suggesting impaired chromosome congression. No lagging chromosomes or chromosomal bridges during anaphase were found in wild-type (Figure 3S, T) but in Lis124h and Lis148h MEFs, many lagging chromosomes at anaphase were seen (10/24, Figure 3U, V) with a corresponding increase in micronuclei in the general population (72h, 51±3.3% compared to wild-type 4.5±1.2%, data not shown). Similar results were found in proliferation and mitosis in undifferentiated and proliferating neural progenitor cells (NPCs) from Lis1hc/hc; Cre-ERTM; Rosa26 (Lis1hc/hc NPCs) mice after tamoxifen treatment to induce inactivation of Lis1 (Supplemental Figure 5) but the scant cytoplasm of NPCs precluded examination of astral MTs.

LIS is required for dynein, NDEL1 and CLIP-170 localization at the cell cortex

The spindle orientation defects displayed by NESCs and RGPCs likely resulted from shortened and thinned astral MTs (Figure 3E–G). We examined the localization of known LIS1 binding partners and the interphase MT array in wild type and Lis1hc/ko MEFs with a uniform (35%) reduced level of LIS1. MEFs with reduced LIS1 doses displayed MTs that did not fully extend to the cell cortex and occupy the cell margin and lamellipod (Figure 4A, B). The area and density of stabilized acetylated MTs were decreased in Lis1hc/ko MEFs versus wild-type by immunocytochemistry (Figure 4C) and Western blot analysis (Figure 4E). The shortened length of interphase and astral MTs was not caused by decreased MT nucleation, since no differences were observed between wild-type and Lis1hc/ko MEFs treated with nocodazole for 1h and fixed the cells at 10 and 30 sec after drug removal (Figure 4D).

Figure 4.

Figure 4

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Dynein components were examined by immunocytochemistry in wild-type (wt, A) and Lis1hc/ko MEFs (hc/ko, B) with DIC (blue), LIS1 (red), and α-tubulin (TUB, green). White arrows and lines indicate areas at the edge of the cell. Yellow arrows in hc/ko cells indicate an area inside of the cell with increased LIS1 and DIC localization and MT stability. (C) Acetylated tubulin (ac-TUB, green,compare arrows and arrowheads in similar cells) and (E) western blot analysis. (D) α-tubulin (TUB, green) was depolymerized by nocodazole treatment in wild-type and Lis1hc/ko MEFs. MEFs were then washed and allowed to recover for 10s. White lines outline the area of MT recovery. (F) NDEL1 (aqua) was mislocalized to the perinuclear region in MEFs with complete loss of LIS1 (RED-CRE), compared with wild-type MEFs, which displayed diffuse NDEL1 localization out to the cortex. The edge of the cell with RED-CRE is outlined. DAPI (blue) and γ-tubulin (green) define the nucleus and centrosome. (G) Cells with abnormal NDEL1 (defined as away from the periphery and more perinuclear in localization) were quantified in CRE-negative (white bar) and CRE-positive (black bar). (H–I) Dynein and CLIP-170 were examined at the cell cortex with decreasing doses of LIS1. (H, J) wild-type; (I, K) Lis1hc/ko. All panels: LIS1, red; α-tubulin, blue; green: (K, L) DIC; (M, N) CLIP-170.

LIS1 and dynein intermediate chain (DIC) displayed strong co-localization and staining intensity at the cell cortex and throughout the cytoplasm in interphase wild-type MEFs (Figure 4A, H). DIC and LIS1 were significantly reduced at the cortex in Lis1hc/ko MEFs, like cortical MTs, and most of the DIC was distributed inward towards the nuclear region (Figure 4B, I). CLIP-170 was also reduced at the cell cortex (Figure 4J, K), although LIS1 and CLIP-170 staining did not show the same degree of overlap as LIS1 and DIC in wild-type MEFs (compare Figure 4H to Figure 4J). NDEL1, an evolutionarily conserved binding partner of both LIS1 and dynein (Niethammer et al. 2000; Sasaki et al; 2000), plays an important role in regulating dynein function (reviewed in Gupta et al. 2002). NDEL1 was normally present throughout the cytoplasm of MEFs, but when LIS1 was inactivated by Cre expression, NDEL was perinuclear (Figure 4F). Greater than 80% of Cre-positive cells displayed such a phenotype compared with less than 10% of Cre-negative cells (Figure 4G, p < 0.05). These findings suggest that LIS1 is important for localization of its binding partners NDEL1, dynein, and CLIP-170 and that this localization is important for MT stability and capture at the cell cortex.

LIS1 is essential for plus-end MT stability and cortical MT capture

EB1 labels growing plus-ends of MTs, and the apparent movement of the EB1-GFP provides a measure of MT growth rate (Mimori-Kiyosue et al. 2000; Piehl and Cassimeris 2003). We analyzed 100 MT ends (10 MT ends in 10 cells) of wild-type, Lis1hc/ko and Lis148h MEFs transfected with an EB1-GFP plasmid using live cell imaging. Average EB1 velocity was not significantly different in wild-type (25.4 ± 5.5 µm min−1), Lis1hc/ko (27.2 ± 7.1 µm min−1) and Lis148h MEFs (31.2 ± 5.0 µm min−1) (Movie 1–Movie 3; EB1-WT, EB1-CKO-KO, EB1-48h), suggesting that growth defects do not account for the inability of MTs to reach the cell cortex. There was no increase of labeled EB1, and APC localization was unaffected in fixed cells (Supplemental Figure 6), suggesting the EB1/APC pathway for end-on capture of MTs (Reilein and Nelson 2005) is unaffected.

Next, MT plus-end stability and capture at the cell cortex was analyzed by live cell imaging after transfection of β-tubulin-GFP into wild-type and Lis1hc/ko and Lis148h MEFs. In wild-type MEFs, MTs were dense and dynamic at the cell cortex, exhibiting sliding and curling consistent with stabilization and cortical capture (Movie 4; TUB-WT, Figure 5A–D). By contrast, the MTs of Lis1hc/ko MEFs were extended but rarely curled at the cortex and appeared to be in a probing transition state at the cell margin (Movie 5; TUB-CKO/KO). Some MTs destabilized and shrunk back to the centrosome, while others continued probing (Figure 5F, G, arrowhead and chevron), but the vast majority never displayed curling characteristic of cortical capture in wild-type MEFs. In Lis1hc/ko cells, some areas exhibited a few MTs with the curling, stabilized behavior at the cell cortex (Figure 5E–G, chevron), and were likely to be in pockets of high LIS1 concentration, since in fixed LIS1 mutant cells, areas of LIS1 localization at the cortex correlated with regions of microtubule stability and dynein (Supplemental Figure 7, arrows). In Lis148h MEFs (Movie 6: TUB-48h), there were small regions with a few stabilized MTs, as well as large empty regions that MTs would attempt to penetrate by the back and forth motion seen in Lis1hc/ko MEFs that then destabilized. The MT network was sparse, disorganized and decaying. No instances of MT cortical capture and curling were observed. Thus, LIS1 does not affect MT growth but is essential for MT plus-end stability and overall maintenance of the MT array, as well as for cortical capture of MTs, an effect that is likely mediated via cortical dynein (see Figure 4).

Figure 5.

Figure 5

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Representative (of 15 cells of each genotype) still images from live movies of tubulin-YFP from wild-type (A–D) and Lis1hc/ko (E–G) MEFs. (A, B) Low-magnification views of two wild-type images. (C, D) MTs in wild-type MEFs display constant curling behavior, with very dynamic activity. (E) Low-magnification view of a representative Lis1hc/ko MEF cell. (F) Regions of poor MT stability and (G) increased MT stability were seen in the same Lis1hc/ko cell. Arrowheads and chevrons track the movement of individual MTs over indicated periods of time in seconds. The arrowhead at 180s is not present because that MT was completely destabilized back to the centrosomes. (G) MTs in certain regions of Lis1hc/ko MEFs extend and bend at the cell cortex, exhibiting some curving movements and stabilization.

NDEL1 rescues MT capture and dynein localization defects in Lis1 mutant cells

We next tested whether NDEL1 or CLIP170, two highly conserved LIS1 interaction partners known to be important for LIS1 function, were involved in the MT phenotype (Sasaki et al. 2000; Shu et al. 2004; Coquelle et al. 2002). Increased expression of NDEL1 in Lis1hc/ko MEFs rescued the normal interphase MT array into the lamellipod with full extension of curled MTs at the cortical edge, whereas increased expression of CLIP-170 caused bundling of MTs without restoration of cortical MT capture (Supplemental Figure 8), suggesting that NDEL1 but not CLIP-170 drives enough remaining LIS1 to cortical sites to rescue cortical MT capture. NDEL1 is phosphorylated at three sites (Ser198, Thr219 and Ser231) by CDK5 or CDK1 in fibroblasts (Sasaki et al. 2000; Niethammer et al. 2000), and at a single distinct site (Ser251) by Aurora-A (Mori et al. 2006). To determine whether these phosphorylation events are necessary for the NDEL1 rescue effect, we transfected Lis1hc/hc MEFs with expression constructs for GFP, GFP-LIS1, GFP-NDEL1, GFP-NDEL1 (tripleS/T-A) with all three CDK1 sites mutated to alanine, and GFP-NDEL1 (S251A), with the Aurora-A kinase site mutated to alanine, in the presence or absence of dsRed-Cre recombinase. In all cases, GFP-LIS1 and GFP-NDEL1 rescued the abnormal localization of MTs (Figure 6A) and DIC (Figure 6B) in LIS1-deleted cells. Both GFP-NDEL1 (S251A) and GFP-NDEL1 (tripleS/T-A) rescued the localization abnormalities of DIC (Figure 6B), while neither of the mutant NDELs rescued the MT localization (Figure 6A). These results suggest that both CDK1 and Aurora-A phosphorylation of NDEL1 are required for rescue of MT but not dynein localization.

Figure 6.

Figure 6

Figure 6

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NDEL1 rescue of MEF defects in β-tubulin (A) and DIC (B) localization. Lis1hc/ko MEFs were transfected with no DNA (−), or with expression constructs for GFP, GFP-LIS1, GFP-NDEL1, GFP-NDEL1 (tripleS/T-A) with all three CDK1 sites mutated to alanine, and GFP-NDEL1 (S251A), with the Aurora-A kinase site mutated to alanine, in the presence or absence of dsRed-Cre recombinase (Toyo-oka et al. 2005). Three days after transfection, β-tubulin (A) and DIC (B) localization was determined. Upper panels, immunofluorescence staining with anti-β-tubulin (A) and anti-DIC. Bottom panels, statistical analysis of distribution of β-tubulin (A) and DIC (B) localization in Lis1hc/ko MEFs without (white) or with (black) Cre. Staining patterns of MEFs were classified as having peripheral MTs (A) or for perinuclear DIC (B). More than 100 cells were phenotyped for each measurement in three separate experiments. Arrows indicate centrosomal distribution of transfected proteins. Asterisks denote *** p <0.0001 by unpaired ANOVA with Bonferroni post-hoc test.

DISCUSSION

We demonstrate that NESCs and RGPCs can be distinguished by sensitivity to LIS1/dynein/NDEL1. LIS1 is absolutely required in vivo in the neuroepithelium but not during radial glial neurogenesis. We provide several lines of evidence that this phenotype results from the important role for LIS1 (via NDEL1 and dynein) in the control of spindle orientation. First, wild-type NESCs display spindle orientations perpendicular to the apical-basal axis with little variation, while spindle orientation is more variable in RGPCs. Second, tight control of spindle orientation requires LIS1 in both NESCs and RGPCs. Third, acute loss of LIS1 provoked disruption of vertical spindle orientation with metaphase arrest, increased apoptosis and proliferation defects, but without disruption of either apical polarity or epithelial integrity. Fourth, LIS1 loss results in reduced and weakened astral and spindle MTs. Finally, LIS1 is essential for cortical and MT capture via dynein components, an effect mediated through NDEL1.

Why are NESCs so sensitive to the precise regulation of spindle orientation? Apical spindle cleavage planes in the NESCs are tightly controlled to be more symmetric and vertical than in RGPCs, where both symmetric and asymmetric divisions occur to produce daughter cells that are progenitors (symmetric) or one progenitor and one neuron (or glial cell), respectively (Figure 7A). The apical and basal plasma membranes of NESCs are only a tiny fraction of the total cell membrane, so the orientation of cleavage must be precisely controlled during symmetrical proliferative divisions, such as those occurring in NESCs, to distribute apical and basal components equally to the daughter cells. We propose that this tiny cell membrane fraction that attaches each NESC to the apical or basal surface must be inherited by each daughter cell in order for the two cells to be attached at both apical and basal surfaces. The rapid rate of proliferation of NESCs probably does not allow time for cells without attachments at both surfaces to establish new attachments. In wild-type embryos, these events happen infrequently due to the strict control of spindle orientation in NESCs, but in Lis1 mutants, spindle orientation is not well controlled, resulting in massive loss of cells unattached at both surfaces. It is also possible that asymmetric cleavage in NESCs results in cells that lack either apical or basal proteins, and this may result in apoptosis. Symmetric and asymmetric divisions of NESCs and RGPCs are critical in order to distribute apical and basal intracellular contents such as proliferative and/or cell determining factors equally or differentially to the daughter cells. In any case, abnormalities of spindle orientation in NESCs appear to profoundly disrupt stem cell expansion, resulting in a catastrophic phenotype. In RGPCs, by contrast, dysregulation of plane of division results in depletion of progenitor pools and an eventual decrease in the production of neurons (Feng and Walsh 2004, Sanada and Tsai 2005; Shu et al. 2006), but resulting in small brain size without catastrophic consequences. This appears to be the case when Lis1 is lost in RGPCs (Figure 1, Figure 7A).

Figure 7.

Figure 7

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(A) Model for the role of LIS1 in neuroepithelial stem cells (NESCs) and radial glial progenitor cells (RGPCs or RGs) in developing cortex from wild-type and Lis1 mutants. M, G1, S, G2 refer to mitotic stages, and arrows next to soma reflect the direction of interkinetic nuclear migration. Spindle orientation in M-phase cells is indicated. Wild-type NESCs divide symmetrically to generate NESCs. Lis1 mutant NESCs divide asymmetrically, resulting in apoptosis. In RGPCs, interkinetic nuclear migration occurs only in the ventricular zone (VZ), but not in the intermediate zone (IZ) or cortical plate (CP), and cells divide symmetrically (Sym M) or asymmetrically (Asy M). In Lis1 mutants, the predominant asymmetric mode of division leads to progenitor depletion. (B) Model for the role of cortical dynein (o^o) in cortical MT capture. In wild-type cells, cellular contents can be moved by pulling MTs via cortical dynein, while in metaphase, the spindle can be rotated (arrows) by cortical dynein. In Lis1 mutant cells, cortical dynein is reduced, resulting in reduction in cortical MTs in interphase, as well as weakened spindle and astral MTs and an inability to rotate the mitotic spindle. See text for further details.

How does loss of LIS1 result in profound spindle orientation defects? LIS1 is essential for proliferation and the specification of mitotic spindle orientation via its critical role in cortical MT capture and stability (Figure 7B). In MEFs with reduced levels of LIS1, we observed shortened and sparse astral MTs while in interphase cells MTs do not fully extend to the cell cortex. LIS1 is required for dynein and dynactin localization to the cell cortex, suggesting that LIS1 stabilizes MTs by plus-end capture at the cell cortex via localization of dynein components, providing an explanation for the spindle orientation defects seen in NESCs and RGPCs. Live cell imaging in interphase MEFs transfected with GFP-tubulin directly demonstrated defective MT capture, sliding and stabilization at the cell cortex, with disorganization and degeneration of the MT array when LIS1 levels were greatly reduced, without affecting MT growth, as measured by GFP-EB1 velocity. In S. cerevisiae, cells lacking Pac1, the Lis1 homologue, dynein or dynactin do not exhibit MT capture and sliding at the cell cortex, supporting the hypothesis that LIS1/dynein/dynactin are part of a MT plus-end capture and stabilization mechanism (Lee et al. 2003). Pac1 is essential for the capture of MTs at the neck of the budding yeast, which then slides into the bud to help orient the spindle (reviewed in Gundersen 2002; Pearson and Bloom 2004). In Dictyostelium, the LIS1 homologue and XMAP215 are important for cortical MT capture via dynein (Rehberg et al. 2005). Similar to the function of Pac1 in yeast and LIS1 in Dictyostelium, we found defects in microtubule capture and sliding at the cell cortex when we reduce LIS1 dosage, suggesting evolutionary conservation of LIS1 function. We propose that LIS1/dynein/dynactin mediates MT plus-end capture, stabilization and sliding of astral MTs to facilitate spindle orientation and positioning, and that this is the mechanism for the spindle orientation defects seen in the NESCs of Lis1 mutants (Figure 7B). Consistent with this proposal, it was previously shown that LIS1/dynein/dynactin is necessary for astral MTs interaction with the cortex in mammalian cells (Busson et al. 1998; Faulkner et al. 2000). In further support of this model, dynein components were reduced at the kinetochore in Lis1 mutant cells (data not shown), suggesting that defects in kinetochore capture of MTs due to lack of dynein components provide an explanation for the chromosome congression defects in these cells.

NDEL1 but not CLIP-170 overexpression rescued the normal interphase MT array and cortical dynein localization. NDEL1 phosphorylation by both CDK1 and Aurora-A phosphorylation is required for rescue of MT but not dynein localization. Phosphorylation of NDEL1 by CDK1 facilitates Katanin p60 recruitment to the centrosome to trigger microtubule remodeling and shortening (Toyo-oka et al. 2005). NDEL1 phosphorylation by Aurora-A is essential for mitotic entry and centrosome separation by recruitment of TACC3 and XMAP215 at the centrosome (Mori et al. 2006), critical events for centrosome-mediated MT assembly in mitosis (Kinoshita et al. 2005). As noted above, in Dictyostelium, XMAP215 acts with LIS1 and dynein to capture MTs at the cortex (Rehberg et al. 2005). It appears that cortical dynein localization is independent of NDEL1 interaction with katanin and TACC3/XMAP215 that results from phosphorylation by CDK1 and Aurora A kinases, while cortical MT capture and stabilization requires interactions with phosphorylation-dependent NDEL1 partners.

We believe that the severe defects in NESC survival in Lis1 mutants result ultimately from defective cortical capture of MTs, producing weakened spindles that cannot rotate to orient properly in the apical-basal axis. We suggest that this unique LIS1 function in MT capture and stabilization may play a broader role throughout neural development in neuronal migration, neurite extension, and neuronal survival, besides its role neuroepithelial expansion and radial glial neurogenesis.

EXPERIMENTAL PROCEDURES

Mice

Hypomorphic conditional knock-out mice (Lis1hc/hc, also Pafah1b1-loxP, Hirotsune et al. 1998, deposited at Jackson Laboratory) were mated with Gt(ROSA)26 Cre-reporter (JAX #3474) or Tg(ACTB-bgeo/DsRed.MST) Cre-reporter (JAX #5441) and one of several Cre lines: Pax2-Cre (Lewis et al. 2004) and Wnt1-Cre (JAX #3829) kind gifts of A. McMahon, Harvard University, hGFAP-Cre (JAX #4600), a kind gift of Albee Messing, University of Wisconsin, and Cre-ERTM (JAX #4453).

Tissue preparation, histology and immunohistochemistry

Embryos were drop-fixed while adult mice were perfused with fixative. Xgal staining, cresyl violet (Nissl), hematoxylin/eosin (H&E) staining, immunohistochemistry with Tst-1 and apoptosis analysis by TUNEL assay (Apoptag Fluorescein Kit, Chemicon) were performed as described (Soriano 1999; Gambello et al. 2003).

Analysis of spindle orientation and apical-basal polarity

Embryonic brains were fixed in 4% paraformaldehyde (PFA) in PBS, soaked in 30% sucrose overnight, transferred into OCT compound (Sakura), frozen, and coronally sectioned (20 µm thickness). Frozen sections were postfixed with 4% PFA and immunostained with aPKCζ (Santa Cruz, rabbit, 1/100), nestin (Santa Cruz, mouse 1/100), γ-tubulin (Sigma, mouse 1/100) and pericentrin (Covance, rabbit, 1/1000), after dilution in 5% normal goat serum, 0.5% TritonX-100 in PBS. Secondary antibodies used were: rhodamine-conjugated anti-rabbit IgG 1/100 (Donkey, Jackson ImmunoResearch Lab); FITC-conjugated anti-mouse IgG 1/100 (Donkey, Jackson ImmunoResearch Lab). DAPI (Molecular Probes) was used for nuclear staining. Images were captured using confocal microscopy (FV1000, Olympus), which were arranged and labeled using Olympus Fluoview program. The angle of the mitotic cleavage plane of each labeled anaphase cell was measured relative to the surface plane of ventricular zone.

Cell culture

MEFs were generated from E14.5 embryos, and used below P5 to guard against culture-derived defects. For immunofluorescence, cells were grown on acid-washed, 0.01% poly-L-lysine (Sigma P4707) and 0.5% gelatin (Sigma G1393) coated coverslips. 4-OH tamoxifen (Sigma) was dissolved in ethanol at 1 mg/ml (1000x), and a 1x solution was made immediately before treatment. Growth Curve: 3 plates for each genotype were counted every 24h using a hemocytometer and trypan blue. Experiments were repeated twice with two different cell lines. One-way ANOVA with a Bonferroni post-hoc test was performed to examine differences among groups. Neural stem cells were isolated and cultured from adult mice using a modification of a previously described method (Palmer et al. 1999).

Western blot

Western blots were performed as described (Gambello et al. 2003) using 1:100 goat anti-LIS1 and 1:1000 rabbit actin (Santa Cruz Biotechnology, Inc) primary and HRP conjugated (Zymed/Invitrogen) secondary antibodies. Quantitation was performed on 4 separate samples run 3x each. A time versus intensity plot was made to ensure linearity of exposures.

Immunocytochemistry in mitotic and interphase cells

MEFs and NPCs were plated on 12 mm round coverslips, which were coated with poly-D-Lysine (Becton-Dickinson) and mouse Laminin (Sigma) for NPCs. Cells were metaphase arrested with 10 µM MG-132 for 2h, placed on ice for 1 h to depolymerize any unattached kinetochore MTs, washed with cold PBS and fixed as described below for MT stability. For MT stability staining, cells were washed with warm PBS, incubated at 37° C for 5 min in 0.5% Triton X-100 in PHEM buffer (2X: 120 mM PIPES, 50 mM HEPES, 20 mM EGTA 8 mM MgSO4, pH 7), fixed with cold methanol with 5 mM EGTA at −20° C for 5 min and then in 4% formaldehyde in PHEM for 10 minutes. Blocking and antibody incubation buffer was 0.2 M glycine, 2.5% fetal bovine serum, 0.1% Triton X-100 in PBS. Incubation was either for 2 h at RT or overnight at 4° C using goat anti-LIS1 1:200 (Abcam), rabbit anti-phospho-Histone H3 1:500 (Sigma), mouse anti-α-tubulin-FITC 1:200 (Sigma), rabbit anti-acetylated tubulin 1:200 (Sigma); rabbit anti-α-tubulin 1:200 (Abcam), mouse anti-EB1 1:200 (BD, Transduction Labs), mouse anti-p150glued 1:200 (BD, Transduction Labs), rabbit anti-CLIP-170 1:200 (Holly Goodson, Notre Dame), mouse anti-DIC 70.1 1:25 (Sigma or Abcam), human SH-CREST autoimmune serum 1:10,000 (kind gift of Brinkley Lab, Baylor), Mad2 1:200 (Cleveland Lab, UCSD), rabbit anti-Numb 1:00 (Abcam) and mouse anti-pan cadherin 1:100 (Sigma). Secondary antibodies (Jackson ImmunoResearch) were donkey anti-rabbit TRITC, donkey anti-goat CY5, donkey anti-mouse FITC, donkey anti-mouse CY3, donkey anti-human CY2, donkey anti-human CY3, and were incubated for 2 h at RT. Counterstains for DNA or actin were Hoechst 33432 1:10,000 (Molecular Probes) and Phalloidin (coumarin labeled). Images were obtained with a Deltavision Spectris (Applied Precision) and a CoolSnapHQ (Roper) CCD camera running Softworx acquisition and deconvolution software. Ten cycles of constrained iterative deconvolution and maximum intensity projections were made of the resultant 4D images (x,y,z).

Transfection, imaging and analysis

EB1-GFP (Yuko Mimori-Kiyosue, KAN Research Institute, Kyoto, Japan) and tubulin-GFP (Clontech) were transfected into wild-type, Lis1cko/ko and Lis148H MEFs using Effectene Transfection Reagent (Qiagen), and imaged 24 hours later using the Deltavision Spectris under full temperature control and CO2-incubation. EB1-GFP transfected cells were imaged every 2s for 100 frames (f) at 60X with 2×2 binning, 0.6s exposure, 32% neutral density (ND). Velocity was determined in Softworx Explorer v1.1 using line segment tool across timepoints. Tubulin-GFP was imaged every 2s, 100f, 150X, 3×3 bin, 0.6s exp, 32% ND. Movies were created with Softworx at 1f/s. Interphase cells were imaged using confocal microscopy on an Olympus FV1000 spectral deconvolution confocal run with Olympus Fluoview 1.4a software. 3D mage stacks were acquired at 1024×1024 pixels with a 60x 1.4 NA objective for Figures 4 and Supplemental Figure 3 and 150x 1.45 NA objective for Figure 5 and Supplemental Figure 4 at 0.25 micron z-steps with sequential scanning using 488, 543 and 653 nm laser light.

NDEL1 transfection and rescue

An RFP-tagged Cre expression vector was introduced into Lis1hc/hc MEFs using LIPOFECTAMINE 2000 reagents (Invitrogen). For rescue experiments, we similarly transfected Lis1hc/hc MEFs with expression constructs for GFP, GFP-LIS1, GFP-NDEL1, GFP-NDEL1 (tripleS/T-A) with all three CDC2 sites (Ser198, Thr219 and Ser231) mutated to alanine, and GFP-NDEL1 (S251A), with the Aurora-A kinase site mutated to alanine, with or without the RFP-Cre expression vector. Microtubule organization was detected by monoclonal anti-β-tubulin antibody (Clone TUB 2.1: Sigma), and dynein localization was determined by an antibody to DIC (Sigma).

Supplementary Material

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ACKNOWLEDGEMENTS

This work was supported in part by the NIH (NS041310 and HD047380 to A.W-B.) and an NSF Pre-Doctoral Fellowship (JY). We thank Donna Holland for animal handling, Jeff Long for statistical analysis, Albee Messing and Andrew McMahon for mice, as well as Holly Goodson and Yuko Mimori-Kiyosue for reagents. We especially thank Brendan Brinkman for technical expertise and support at the UCSD Neuroscience Microscopy Shared Facility (P30 NS047101), as well as Don Cleveland, Arshad Desai, Joe Gleeson and Geoff Rosenfeld for reagents, advice and comments on the manuscript.

Footnotes

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