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.     

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.     

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.     

Three-dimensional regulation of radial glial functions by Lis1-Nde1 and dystrophin glycoprotein complexes

Radial glial cells (RGCs) are distinctive neural stem cells with an extraordinary slender bipolar morphology and dual functions as precursors and migration scaffolds for cortical neurons. Here we show a novel mechanism by which the Lis1-Nde1 complex maintains RGC functions through stabilizing the dystrophin/dystroglycan glycoprotein complex (DGC). A direct interaction between Nde1 and utrophin/dystrophin allows for the assembly of a multi-protein complex that links the cytoskeleton to the extracellular matrix of RGCs to stabilize their lateral membrane, cell-cell adhesion, and radial morphology. Lis1-Nde1 mutations destabilized the DGC and resulted in deformed, disjointed RGCs and disrupted basal lamina. Besides impaired RGC self-renewal and neuronal migration arrests, Lis1-Nde1 deficiencies also led to neuronal over-migration. Additional to phenotypic resemblances of Lis1-Nde1 with DGC, strong synergistic interactions were found between Nde1 and dystroglycan in RGCs. As functional insufficiencies of LIS1, NDE1, and dystroglycan all cause lissencephaly syndromes, our data demonstrated that a three-dimensional regulation of RGC's cytoarchitecture by the Lis1-Nde1-DGC complex determines the number and spatial organization of cortical neurons as well as the size and shape of the cerebral cortex.     

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.     

Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.     

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.     

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.     

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.     

Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration

Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where Lis1, Ndel1, and 14-3-3? (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes. Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of Lis1 and Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.     

The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.     

Cortical development deNUDEd

The development of the cerebral cortex is a highly orchestrated process of cell division and migration. In this issue of Neuron, Feng and Walsh and Shu et al. examine the roles of two related proteins, Nde1 (mNudE) and Ndel1 (NUDEL), in cortical development. These proteins play a crucial role in centrosome positioning, with Nde1 functioning mainly during progenitor cell divisions and Ndel1 functioning in neuronal migration.     

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.     

Lis1 Acts as a "Clutch" between the ATPase and Microtubule-Binding Domains of the Dynein Motor

The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dynein's AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a "clutch" that prevents dynein's ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments.     

The structure of the N-terminal domain of the product of the lissencephaly gene Lis1 and its functional implications

Mutations in the Lis1 gene result in lissencephaly (smooth brain), a debilitating developmental syndrome caused by the impaired ability of postmitotic neurons to migrate to their correct destination in the cerebral cortex. Sequence similarities suggest that the LIS1 protein contains a C-terminal seven-blade beta-propeller domain, while the structure of the N-terminal fragment includes the LisH (Lis-homology) motif, a pattern found in over 100 eukaryotic proteins with a hitherto unknown function. We present the 1.75 A resolution crystal structure of the N-terminal domain of mouse LIS1, and we show that the LisH motif is a novel, thermodynamically very stable dimerization domain. The structure explains the molecular basis of a low severity form of lissencephaly.     

Roles for Ndel1 in keratin organization and desmosome function

Keratin intermediate filaments form dynamic polymer networks that organize in specific ways dependent on the cell type, the stage of the cell cycle, and the state of the cell. In differentiated cells of the epidermis, they are organized by desmosomes, cell–cell adhesion complexes that provide essential mechanical integrity to this tissue. Despite this, we know little about how keratin organization is controlled and whether desmosomes locally regulate keratin dynamics in addition to binding preassembled filaments. Ndel1 is a desmosome-associated protein in the differentiated epidermis, though its function at these structures has not been examined. Here, we show that Ndel1 binds directly to keratin subunits through a motif conserved in all intermediate filament proteins. Further, Ndel1 was necessary for robust desmosome–keratin association and sufficient to reorganize keratins at distinct cellular sites. Lis1, a Ndel1 binding protein, was required for desmosomal localization of Ndel1, but not for its effects on keratin filaments. Finally, we use mouse genetics to demonstrate that loss of Ndel1 results in desmosome defects in the epidermis. Our data thus identify Ndel1 as a desmosome-associated protein that promotes local assembly/reorganization of keratin filaments and is essential for robust desmosome formation.     

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.     

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.     

Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport

Haplo-insufficiency of human Lis1 causes lissencephaly. Reduced Lis1 activity in both humans and mice results in a neuronal migration defect. Here we show that Drosophila Lis1 is highly expressed in the nervous system. Lis1 is essential for neuroblast proliferation and axonal transport, as shown by a mosaic analysis using a Lis1 null mutation. Moreover, it is cell-autonomously required for dendritic growth, branching and maturation. Analogous mosaic analysis shows that neurons containing a mutated cytoplasmic-dynein heavy chain (Dhc64C) exhibit phenotypes similar to Lis1 mutants. These results implicate Lis1 as a regulator of the microtubule cytoskeleton and show that it is important for diverse physiological functions in the nervous system.