LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co-migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin-1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co-precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein-LIS1 complex on transportable MTs, which is a possibility supported by our data.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

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.     

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.      

DISC1, PDE4B, and NDE1 at the centrosome and synapse

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.     

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.     

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.     

LIS1, CLIP-170's key to the dynein/dynactin pathway

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.     

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.     

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.     

LIS1 Clamps Dynein to the Microtubule

Cytoplasmic dynein is a motor essential for numerous mechanical processes in eukaryotic cells. How its activity is regulated is largely unknown. By using a combination of approaches including single-molecule biophysics and electron microscopy, Huang et?al. in this issue uncover the regulatory mechanism by which LIS1 controls the activity of cytoplasmic dynein.     

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.     

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.     

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.     

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.     

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.     

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.     

The L279P Mutation of Nuclear Distribution Gene C (NudC) Influences Its Chaperone Activity and Lissencephaly Protein 1 (LIS1) Stability

LIS1, a gene mutated in classical lissencephaly, plays essential roles in cytoplasmic dynein regulation, mitosis and cell migration. However, the regulation of LIS1 (lissencephaly protein 1) protein remains largely unknown. Genetic studies in Aspergillus nidulans have uncovered that the Nud (nuclear distribution) pathway is involved in the regulation of cytoplasmic dynein complex and a temperature-sensitive mutation in the nudC gene (L146P) greatly reduces the protein levels of NudF, an Aspergillus ortholog of LIS1. Here, we showed that L146 in Aspergillus NudC and its flanking region were highly conservative during evolution. The similar mutation in human NudC (L279P) obviously led to reduced LIS1 and cellular phenotypes similar to those of LIS1 down-regulation. To explore the underlying mechanism, we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90, which is also associated with LIS1. Inhibition of Hsp90 chaperone function by either geldanamycin or radicicol resulted in a decrease in LIS1 levels. Ectopic expression of Hsp90 partially reversed the degradation of LIS1 caused by overexpression of NudC-L279P. Furthermore, NudC was found to regulate the ATPase activity of Hsp90, which was repressed by the mutation of L279P. Interestingly, NudC itself was shown to possess a chaperone function, which also was suppressed by the L279P mutation. Together, these data suggest that NudC may be involved in the regulation of LIS1 stability by its chaperone function.     

A Highlights from MBoC Selection: Regulation of microtubule-based transport by MAP4

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.     

The LIS1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to specific subunits of dynein and dynactin and to alpha- and gamma-tubulin

The NUDF protein of Aspergillus nidulans, which is required for nuclear migration through the fungal mycelium, closely resembles the LIS1 protein required for migration of neurons to the cerebral cortex in humans. Genetic experiments suggested that NUDF influences nuclear migration by affecting cytoplasmic dynein. NUDF interacts with another protein, NUDE, which also affects nuclear migration in A. nidulans. Interactions among LIS1, NUDE, dynein, and gamma-tubulin have been demonstrated in animal cells. In this paper we examine the interactions of the A. nidulans NUDF and NUDE proteins with components of dynein, dynactin, and with alpha- and gamma-tubulin. We show that NUDF binds directly to alpha- and gamma-tubulin and to the first P-loop of the cytoplasmic dynein heavy chain, whereas NUDE binds directly to alpha- and gamma-tubulin, to NUDK (actin-related protein 1), and to the NUDG dynein LC8 light chain. The data suggest a direct role for NUDF in regulation of the dynein heavy chain and an effect on other dynein/dynactin subunits via NUDE. The interactions between NUDE, NUDF, and gamma-tubulin suggest that this protein may also be involved in the regulation of dynein function. Additive interactions between NUDE and dynein and dynactin subunits suggest that NUDE acts as a scaffolding factor between components.