Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality

Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.     

Global developmental gene expression and pathway analysis of normal brain development and mouse models of human neuronal migration defects

Heterozygous LIS1 mutations are the most common cause of human lissencephaly, a human neuronal migration defect, and DCX mutations are the most common cause of X-linked lissencephaly. LIS1 is part of a protein complex including NDEL1 and 14-3-3ε that regulates dynein motor function and microtubule dynamics, while DCX stabilizes microtubules and cooperates with LIS1 during neuronal migration and neurogenesis. Targeted gene mutations of Lis1, Dcx, Ywhae (coding for 14-3-3ε), and Ndel1 lead to neuronal migration defects in mouse and provide models of human lissencephaly, as well as aid the study of related neuro-developmental diseases. Here we investigated the developing brain of these four mutants and wild-type mice using expression microarrays, bioinformatic analyses, and in vivo/in vitro experiments to address whether mutations in different members of the LIS1 neuronal migration complex lead to similar and/or distinct global gene expression alterations. Consistent with the overall successful development of the mutant brains, unsupervised clustering and co-expression analysis suggested that cell cycle and synaptogenesis genes are similarly expressed and co-regulated in WT and mutant brains in a time-dependent fashion. By contrast, focused co-expression analysis in the Lis1 and Ndel1 mutants uncovered substantial differences in the correlation among pathways. Differential expression analysis revealed that cell cycle, cell adhesion, and cytoskeleton organization pathways are commonly altered in all mutants, while synaptogenesis, cell morphology, and inflammation/immune response are specifically altered in one or more mutants. We found several commonly dysregulated genes located within pathogenic deletion/duplication regions, which represent novel candidates of human mental retardation and neurocognitive disabilities. Our analysis suggests that gene expression and pathway analysis in mouse models of a similar disorder or within a common pathway can be used to define novel candidates for related human diseases.     

Lis1 and Ndel1 influence the timing of nuclear envelope breakdown in neural stem cells

Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1^+/− mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1–Ndel1 disruption.     

The essential role of LIS1, NDEL1 and Aurora-A in polarity formation and microtubule organization during neurogensis

Lissencephaly is a devastating neurological disorder due to defective neuronal migration. LIS1 (or PAFAH1B1), the gene mutated in lissencephaly patients and its binding protein NDEL1 were found to regulate cytoplasmic dynein function and localization. LIS1 and NDEL1 also play a pivotal role on a microtubule regulation and determination of cell polarity. For example, LIS1 is required for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. On the other hand, NDEL1 is essential for mitotic entry as an effector molecule of Aurora-A kinase. In addition, an atypical protein kinase C (aPKC)-Aurora-A-NDEL1 pathway is critical for the regulation of microtubule organization during neurite extension. These findings suggest that physiological functions of LIS1 and NDEL1 in neurons have been ascribed for proteins fundamentally required for cell cycle progression and control. In turn, cell cycle regulators may exert other functions during neurogenesis in a direct or an indirect fashion. Thus far, only a handful of cell cycle regulators have been shown to play physiological cell-cycle-independent roles in neurons. Further identification of such proteins and elucidation of their underlying mechanisms of action will likely reveal novel concepts and/or patterns that provide a clear link between their seemingly distinct cell cycle and neuronal functions.     

The structure of the coiled-coil domain of Ndel1 and the basis of its interaction with Lis1, the causal protein of Miller-Dieker lissencephaly

Ndel1 and Nde1 are homologous and evolutionarily conserved proteins, with critical roles in cell division, neuronal migration, and other physiological phenomena. These functions are dependent on their interactions with the retrograde microtubule motor dynein and with its regulator Lis1--a product of the causal gene for isolated lissencephaly sequence (ILS) and Miller-Dieker lissencephaly. The molecular basis of the interactions of Ndel1 and Nde1 with Lis1 is not known. Here, we present a crystallographic study of two fragments of the coiled-coil domain of Ndel1, one of which reveals contiguous high-quality electron density for residues 10-166, the longest such structure reported by X-ray diffraction at high resolution. Together with complementary solution studies, our structures reveal how the Ndel1 coiled coil forms a stable parallel homodimer and suggest mechanisms by which the Lis1-interacting domain can be regulated to maintain a conformation in which two supercoiled alpha helices cooperatively bind to a Lis1 homodimer.     

Ndel1 controls the dynein-mediated transport of vimentin during neurite outgrowth

Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein alphaCOP. Ndel1 promotes the interaction between Lis1, alphaCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/alphaCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.     

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.     

The essential role of LIS1, NDEL1 and Aurora-A in polarity formation and microtubule organization during neurogensis

Lissencephaly is a devastating neurological disorder caused by to defective neuronal migration. LIS1 (or PAFAH1B1), the gene mutated in lissencephaly patients and its binding protein NDEL1 were found to regulate cytoplasmic dynein function and localization. LIS1 and NDEL1 also play a pivotal role on a microtubule regulation and determination of cell polarity. For example, LIS1 is required for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. On the other hand, NDEL1 is essential for mitotic entry as an effector molecule of Aurora-A kinase. In addition, an atypical protein kinase C (aPKC)-Aurora-A-NDEL1 pathway is critical for the regulation of microtubule organization during neurite extension. These findings suggest that physiological functions of LIS1 and NDEL1 in neurons have been ascribed for proteins fundamentally required for cell cycle progression and control. In turn, cell cycle regulators may exert other functions during neurogenesis in a direct or an indirect fashion. Thus far, only a handful of cell cycle regulators have been shown to play physiological cell cycle-independent roles in neurons. Further identification of such proteins and elucidation of their underlying mechanisms of action will likely reveal novel concepts and/or patterns that provide a clear link between their seemingly distinct cell cycle and neuronal functions.Key words: microtubule, mitotic kinase, neurite, cell polarity, migrationDuring the development of the mammalian central nervous system, the self-renewal of neural stem cells can occur either by symmetric cell divisions, which generate two daughter cells with the same fate, or by asymmetric cell divisions, which generate one daughter cell that is identical to the mother cell and a second, different non-stem-cell progenitor (reviewed in refs. ¹ and ²). Neural non-stem-cell progenitors typically undergo symmetric, differentiating divisions, each of which generates two neurons, which are terminally differentiated, post-mitotic cells. These post-mitotic neural progenitors migrate from their birth place at the ventricular zone to their final destinations in cortical plate (reviewed in ref. ³). Coinciding with the proper positioning of post-mitotic neurons, neurons project neurite and dendrites to targets with the assistant of molecular guidance cues in the local environment. Proper navigations of neurite and dendrite processes ensure synapse formations, which are the basis of brain function. In the series of developmental steps, the determination of neuronal polarity is critically important (reviewed in refs. ⁴ and ⁵). A polarity complex of Par3, Par6, and atypical protein kinase C (aPKC) functions in various cell-polarization events including axon formation.^6,7 GTPases that regulate actin cytoskeletal dynamics have been implicated in cell polarization. Recent findings provide insights into polarization mechanisms and show intriguing crosstalk between small GTPases and members of polarity complexes in regulating cell polarization (reviewed in ref. ⁸). Thus, determination of neuronal polarity and regulation of cytoskeletal organization are intimately related.LIS1 was identified as the first gene mutated in isolated lisssencephaly sequence (ILS), a human neuronal migration defect.^9,10 LIS1 and its binding protein, NDEL1 regulate the function and localization of cytoplasmic dynein^11,13 as part of an evolutionarily conserved pathway.^14,15 Genetic analysis of fungi displaying defective nuclei migration led to the identification of a number of genes and their protein products involved in this process. For example, mutations of nudA (coding for cytoplasmic dynein heavy chain) and genes coding for other subunits of the dynein complex inhibit nuclear migration, including nudC (mammalian NudC, mNudC), nudE (Ndel1 and Nde1) and nudF (Lis1). We recently demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules in vitro (Suppl. movies 1 and 2), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein.¹⁶ We demonstrated anterograde co-migration of cytoplasmic dynein and LIS1 (Suppl. movies 3 and 4). When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Lis1 KO cells exhibited biased distribution around the centrosome and aberrant distribution of organelles. Our favorite model is that LIS1 fixes cytoplasmic dynein on soluble microtubules in an “idling” state, thereby creating a microtubule-LIS1-dynein complex, which could be transported by kinesin to the plus-end of microtubules.Lis1 is also essential for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells.¹⁷ 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. We believe the role of LIS1 in promoting the anterograde transport of cytoplasmic dynein on kinesin as part of a microtubule-LIS1-dynein complex, as described in the previous paragraph, is responsible for controlling spindle orientation, since when LIS1 is reduced, cortical dynein fixed on the surface of the cell is also reduced. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells.¹⁷ Thus, Lis1 is intimiately involved in the determination of cell polarity as an effector molecule, which regulates dynein localization and/or function as well as microtubule organization.Interestingly, more than half of LIS1 protein is degraded at the cell cortex after transport to the plus-end of MTs via calpain-dependent proteolysis. We recently demonstrated that inhibition or knockdown of calpain protects LIS1 from proteolysis resulting in the augmentation of LIS1 levels in Lis1^+/− mouse embryonic fibroblast (MEF) cells, which leads to rescue of the aberrant distribution of cytoplasmic dynein and intracellular components including mitochondria and β-COP positive vesicles.¹⁸ We also showed that presence of calpain inhibitors improves neuronal migration of Lis1^+/− cerebellar granular neurons.¹⁸ This study demonstrates that stabilization of proteins in disorders caused by haploinsufficiency is a potential therapeutic strategy and provides a proof-of-principle for this notion.NDEL1, a binding partner of LIS1 is also essential for the regulation of cytoplasmic dynein and microtubule organization.^12,13 In particular, NDEL1 is phosphorylated by cyclin dependent kinase1 (CDK1) in mitotic cells, or CDK5 in post-mitotic neurons, and this phosphorylation is essential for proper targeting of NDEL1 binding proteins to the centrosome.¹⁹ NDEL1 is also a substrate of the mitotic kinase Aurora-A, by which NDEL1 connects Aurora-A to other target molecules for the regulation of microtubule organization.²⁰ Interestingly, NDEL1 is differentially phosphorylated by Aurora-A and CDK1. It is possible that distinct pools of NDEL1 may be targeted by each kinase, or conversely the affects of each kinase may counteract each other within the same pool of NDEL1.Aurora-A is a one of representative mitotic kinase, whose homologues have been reported in various organisms including yeast, nematodes, fruit flies and vertebrates (reviewed in ref. ²¹). The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. In particular, Aurora-A plays a pivotal role on microtubule reorganization during remodeling from interphase microtubules to mitotic microtubules, i.e., the mitotic spindle. We recently reported molecular and cell biological data that support a unique role of aPKC-Aurora-A-NDEL1 pathway on microtubule dynamics at the neurite hillock during neurite extension.²² PKCζ phosphorylates Aurora-A at T287 and activates it, which augments interaction with TPX2 and facilitates activation of Aurora-A at the neurite hillock, followed by S251 phosphorylation of NDEL1 and recruitment. Inhibition of PKCζ/λ, depletion of Aurora-A and disruption of Ndel1 severely affected neurite extension and microtubule dynamics, suggesting that the aPKC-Aurora-A-NDEL1 pathway is an important regulatory system of microtubule oranization within neurite processes (Fig. 1A).Open in a separate windowFigure 1Models of microtubule remodeling. (A) Neurite extension: an unknown upstream cue polarity activates aPKC followed by T287 phosphorylation of Aurora-A. T287 phosphorylation of Aurora-A facilitates binding of the Aurora-A activator, TPX2 resulting in activation of Aurora-A at the neurite hillock, which leads to phosphorylation of NDEL1, one of effector molecules of Aurora-A. Finally, phosphorylation of NDEL1 triggers remodeling microtubules during neurite extension. (B) Spindle formation: NDEL1 is differentially phosphorylated at T219 and Ser251 by CDK1 and Aurora-A, respectively at the beginning of mitotic entry. NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. (C) Neuronal migration: during neuronal migration, NDEL1 may be differentially phosphorylated at T219 and Ser251 by CDK5 and Aurora-A, respectively. 14-3-3ɛ might negatively regulate Aurora-A kinase.Our preliminary data suggest that Aurora-A is also activated by neurons during migration, and may further link signaling components already implicated in neuronal migration. Mice deficient in Ywhae that encondes 14-3-3ɛ have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Lis1.²³ Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. Heterozygous deletions of 17p13.3 in human result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mice carrying double heterozygous mutations of Ywhae and Lis1 are therefore thought to be a mouse model of MDS. Intriguingly, 14-3-3ɛ binds to NDEL1 after phosphorylation by CDK1/CDK5, protecting phospho-NDEL1 from phosphatase attack.14-3-3 proteins mediate multiple cellular events, including scaffolding of signaling molecules, regulation of enzyme catalysis, and subcellular targeting. In the C. elegans, 14-3-3 homolog, Par5 is required for correct anterior-posterior zygote polarization.²⁴ In addition, phosphorylation-dependent interactions between 14-3-3ɛ, and the tight junction-associated protein Par3 had been reported.²⁵ Intriguingly, 14-3-3ɛ is a centrosomal protein,²⁶ suggesting that 14-3-3ɛ, Aurora-A and NDEL1 might create a complex at the centrosome, which may then be involved in the determination of polarity and neuronal migration. These findings might be the result of the known role of Aurora-A as a regulator of microtubule network. Microtubules are emanated from MTOC, and are extended into the chromosome, nucleus or the cell periphery (Fig. 1). These microtubule flows associated with the dynamic remodeling will provide enough force to maintain a neurite process, a spindle body or a leading process.Post-mitotic neurons, however, lose their mitotic competence permanently. Intuitively, once a neural progenitor differentiates into a neuron, the post-mitotic neurons have severed all ties with the cell cycle, in which the expression of cell cycle proteins are assumed to be not expressed. Emerging evidence reveals that this holds true for a handful of core cell cycle regulators, which facilitate the differentiation and maturation of neurons, suggesting that “core“ cell cycle regulators serve diverse postmitotic functions that span various developmental stages of a neuron, including neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity (reviewed in ref. ²⁷). Among the essential kinases that function in mitosis are Aurora kinases, evolutionarily conserved serine-threonine kinases that maintain genomic stability and are required for mitotic progression. Although they share conserved regions, each member (Aurora A, B and C) contributes distinctly to cell cycle progression. Aurora-A is essential for mitotic entry, centrosome maturation during late G2 and prophase, centrosome separation during bipolar spindle assembly and mitotic spindle organization (reviewed in refs. ²¹ and ²⁸). During mitotic progression, Aurora-A loss of function prevents centrosomal separation prior to mitotic spindle formation and results in monopolar spindles.²⁹ We reported an essential role of Aurora-A during neurite extension. Wirtz-Peitz et al. reported that Aurora-A phosphorylates Par-6.³⁰ This phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis, which provides further evidence for crosstalk of PAR proteins and Aurora-A.³⁰ Apart from neurons, the interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora-A kinase at the basal body of cilia had been reported.³¹ This pathway is both necessary and sufficient for ciliary resorption and constitutes an unexpected non-mitotic activity of Aurora-A in vertebrates.Aurora-A kinase, Plk1 or CDK1 has been recognized as a mitotic kinase, which regulates mitotic entry. Cells in which these genes are mutated display defective mitotic entry. Individual proteins however, have multiple functions within specific cellular context. For example, Aurora-A may participate remodeling microtubule in mitotic spindle formation and in remodeling of microtubule organization during neurite extension or neuronal migration. Apart from existing concept, elucidation of multiple functions of cell cycle regulators will provide us with a better understanding of the extent to which they exert physiological cell cycle-independent neuronal functions.     

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.     

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.     

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

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 (RGPC) 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.     

Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase

Mutations in the LIS1 gene cause lissencephaly, a human neuronal migration disorder. LIS1 binds dynein and the dynein-associated proteins Nde1 (formerly known as NudE), Ndel1 (formerly known as NUDEL), and CLIP-170, as well as the catalytic alpha dimers of brain cytosolic platelet activating factor acetylhydrolase (PAF-AH). The mechanism coupling the two diverse regulatory pathways remains unknown. We report the structure of LIS1 in complex with the alpha2/alpha2 PAF-AH homodimer. One LIS1 homodimer binds symmetrically to one alpha2/alpha2 homodimer via the highly conserved top faces of the LIS1 beta propellers. The same surface of LIS1 contains sites of mutations causing lissencephaly and overlaps with a putative dynein binding surface. Ndel1 competes with the alpha2/alpha2 homodimer for LIS1, but the interaction is complex and requires both the N- and C-terminal domains of LIS1. Our data suggest that the LIS1 molecule undergoes major conformational rearrangement when switching from a complex with the acetylhydrolase to the one with Ndel1.     

Lis1, the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation.

Lissencephaly is a severe congenital brain malformation resulting from incomplete neuronal migration. One causal gene, LIS1, is homologous to nudF, a gene required for nuclear migration in A. nidulans. We have characterized the Drosophila homolog of LIS1 (Lis1) and show that Lis1 is essential for fly development. Analysis of ovarian Lis1 mutant clones demonstrates that Lis1 is required in the germline for synchronized germline cell division, fusome integrity and oocyte differentiation. Abnormal packaging of the cysts was observed in Lis1 mutant clones. Our results indicate that LIS1 is important for cell division and differentiation and the function of the membrane cytoskeleton. They support the notion that LIS1 functions with the dynein complex to regulate nuclear migration or cell migration.     

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development

Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.     

Distinct Functions of Nuclear Distribution Proteins LIS1, Ndel1 and NudCL in Regulating Axonal Mitochondrial Transport

Neurons critically depend on the long‐distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein‐LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.      

Cenp-F links kinetochores to Ndel1/Nde1/Lis1/dynein microtubule motor complexes

Cenp-F is a nuclear matrix component that localizes to kinetochores during mitosis and is then rapidly degraded after mitosis [1]. Unusually, both the localization and degradation of Cenp-F require it to be farnesylated [2]. Five studies recently demonstrated that Cenp-F is required for kinetochore-microtubule interactions and spindle checkpoint function [3-7]; however, the underlying molecular mechanisms have yet to be defined. Here, we show that Cenp-F interacts with Ndel1 and Nde1, two human NudE-related proteins implicated in regulating Lis1/Dynein motor complexes (reviewed in [8]). We show that Ndel1, Nde1, and Lis1 localize to kinetochores in a Cenp-F-dependent manner. In addition, Nde1, but not Ndel1, is required for kinetochore localization of Dynein. Accordingly, suppression of Nde1 inhibits metaphase chromosome alignment and activates the spindle checkpoint. By contrast, inhibition of Ndel1 results in malorientations that are not detected by the spindle checkpoint; Ndel1-deficient cells consequently enter anaphase in a timely manner but lagging chromosomes then manifest. A major function of Cenp-F, therefore, is to link the Ndel1/Nde1/Lis1/Dynein pathway to kinetochores. Furthermore, our data demonstrate that Ndel1 and Nde1 play distinct roles to ensure chromosome alignment and segregation.     

Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine‐tuning the activity of the retrograde motor cytoplasmic dynein.     

Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/- neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear "cage" to the centrosome. Lis1+/- neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.     

Functional dissection of LIS1 and NDEL1 towards understanding the molecular mechanisms of cytoplasmic dynein regulation

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected the regulatory effects of LIS1 and NDEL1 on dynein motility using full-length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1, and the mechanisms of cytoplasmic dynein regulation.