Characterization of LIS1 homologs in non-vertebrate species

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, a screen was carried out for mutations that are synthetically lethal in combination with a bni1 mutation and two genes were isolated. These were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant results in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant shows a synthetic lethal phenotype while the cin8 bni1 mutant does not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibit a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant show temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region does not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function (Fujiwara, 2000).

A temperature-sensitive mutation in the nudC gene (nudC3) of Aspergillus nidulans specifically prevents the microtubule-based movement of nuclei in this organism at the restrictive temperature. The mutation does not affect short term growth, nuclear division, or the movement of other subcellular organelles. Immunofluorescence analysis of cells blocked at the restrictive temperature, using antitubulin antibodies, shows that the inability of nuclei to move under these conditions is not related to an inability of a particular class of microtubule to form. The inability to move nuclei in this mutant is also shown to be independent of both mitosis and the number of nuclei in the cell because a double mutant carrying both nudC3 and a cell cycle-specific mutation blocks cell cycle progression with a single immotile nucleus at the restrictive temperature. The molecular cloning of the nudC gene and sequence analysis reveal that nudC encodes a previously unidentified protein of 22 kd. Affinity-purified antisera reactive to the nudC protein cross react to a single protein of 22 kD in Aspergillus protein extracts. This purified sera fails to reveal a subcellular location for the nudC protein at the level of indirect immunofluorescence. The data presented suggest that the 22-kD nudC gene product functions by interacting between microtubules and nuclei and/or is involved in the generation of force used to move nuclei during interphase (Osmani, 1990).

During a study of the genetics of nuclear migration in the filamentous fungus Aspergillus nidulans, a gene, nudF, was cloned that is required for nuclear migration during vegetative growth as well as development. The NUDF protein level is controlled by another protein, NUDC, and extra copies of the nudF gene can suppress the nudC3 mutation. nudF encodes a protein with 42% sequence identity to the human LIS-1 (Miller-Dieker lissencephaly-1) gene, which is required for proper neuronal migration during brain development. This strong similarity suggests that the LIS-1 gene product may have a function similar to that of NUDF and supports previous findings to suggest that nuclear migration may play a role in neuronal migration (Xiang, 1995).

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. Twenty genes have been identified whose products are specifically required by cells deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. Evidence is provided that the products of these genes function in concert with dynein. Among the dynein pathway gene products, homologs have been found of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family (Geiser, 1997).

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, a screen was initiated for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA-snfE, cause improved growth and nuclear migration at high temperatures, as compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dyncin heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicates that NUDA and NUDF act in the same nuclear migration pathway. Taken together, these data suggests that NUDF affects nuclear migration by acting on the dynein motor system (Willins, 1997).

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. A screen was carried out for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homolog of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homolog of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related (Efimov, 2000).

During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). This study reports the native-level localization of the dynein heavy chain and characterizes the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) results in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein results in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also results in selective accumulation of dynein on the plus ends of anaphase aMTs. It is proposed that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, it is suggested that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors (Sheeman, 2003).

A significant concentration of dynein is found at the plus ends of both polymerizing and depolymerizing aMTs. These results are consistent with observations in animal cells and Aspergillus. These experiments shed light on the mechanism by which dynein is recruited to the plus ends of MTs. Bik1p is a functional component of the dynein mechanism for spindle positioning and Bik1p, specifically its cargo binding domain, has an important role in recruiting dynein to aMT plus ends. These findings are in agreement with recent studies in mammalian cells reporting that the cargo binding domain of human CLIP-170 binds LIS1 and that overexpression of CLIP-170 can recruit LIS1, and potentially other dynein components, onto MTs. Evidence was also obtained for binding of Bik1p to Pac1p by two-hybrid experiments. Thus, the complex between CLIP-170- and LIS1-related proteins appears to be a highly conserved element of the mechanism for targeting dynein to MTs. Recruitment to MTs by a plus-end tracking protein (CLIP-170/Bik1p) may explain how a minus end-directed motor protein can be returned to the plus end (Sheeman, 2003).

p150Glued-related proteins have also been suggested to link dynein to MT plus ends. Surprisingly, loss of Nip100p did not diminish MT-association of dynein, but in fact caused enhanced Bik1p- and Pac1p-dependent localization of dynein to aMT plus ends in anaphase. Therefore, at least in budding yeast, Nip100p does not appear to play the major role in recruiting dynein to aMT plus ends (Sheeman, 2003).

There are differences in the apparent role of LIS1-related proteins in different systems. (1) LIS1-related proteins have also been implicated in recruitment of dynein to the cell cortex in some cell types. One way to reconcile the current findings with previous work is to postulate that the plus ends of aMTs deliver LIS1 to the cell cortex in these cells. If this explanation is correct, then (at least in the Drosophila oocyte), once delivered to the cortex, LIS1 must interact stably with a cortical component, because LIS1 cortical localization is not lost after disruption of MTs by colchicine treatment. (2) In Aspergillus, dynein appears to localize normally to MT plus ends in the absence of the LIS1 ortholog NUDF. At this point, the basis for these species-specific differences is not known; however, in Aspergillus dynactin might assume a more important role in dynein localization than in budding yeast (Sheeman, 2003).

The targeting of dynein to MT plus ends raises interesting questions about the regulation of dynein in vivo. Because of the rapid speed of minus end-directed movement of cytoplasmic dynein, it seems likely that dynein could only accumulate at MT plus ends if the motor activity were inhibited and/or if dynein processivity were extremely low. The results support this idea. (1) Anaphase cells lacking components of the dynactin complex display a marked accumulation of the dynein heavy chain on the plus ends of aMTs. (2) Concomitant with this plus-end enhancement, there was a decrease in the amount of dynein associated with the SPBs in these cells. (3) An inactivating mutation in a dynein P loop domain also enhances dynein localization to aMT plus ends. These findings suggest that dynactin promotes the ability of dynein to translocate from the plus end of an aMT to the minus end (SPB). Additional levels of control on the rate of recruitment of dynein to the MT plus end, on dynein motor activity, or on the activity of dynactin may also exist. Cell cycle regulation at any of these levels might account for the finding that in dynactin mutants, dynein accumulation at aMT plus ends is primarily seen during anaphase (Sheeman, 2003).

Cloning and expression of mammalian LIS1

The Miller-Dieker syndrome, a disorder of neuronal migration, is caused by deletions of chromosome 17p13.3. A gene on 17p13.3, named LIS-1, has been identified as the causative gene for this cerebral anomaly. The gene product, LIS-1 protein, has been localized in control normal subjects and patients with Miller-Dieker syndrome, using specific antibodies raised against synthetic peptide fragments of LIS-1 protein. Western blot analyses have identified LIS-1 protein as a 45-kd, heparin-binding protein abundant in the cytosolic fraction. The protein is restricted to the central nervous system and detectable in brains of controls at all ages, from the early fetal to adult period. Immunostaining demonstrates the widespread distribution of LIS-1 protein in the brain and spinal cord of controls and a loss of immunoreactivity in individuals with Miller-Dieker syndrome. These results are consistent with the notion that a deficiency of LIS-1 protein is the direct cause of the brain malformation and that the protein plays a critical role in neuronal migration (Mizuguchi, 1995).

The LIS1 protein associates with PAF, the enzyme that hydrolyzes platelet-activating factor

Platelet-activating factor (PAF) is involved in a variety of biological and pathological processes and PAF acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is widely distributed in plasma and tissue cytosols. One isoform of PAF acetylhydrolase present in bovine brain cortex is a heterotrimer comprising subunits with relative molecular masses of 45K, 30K and 29K. The complementary DNA for the 45K subunit has been isolated. Sequence analysis reveals a striking identity (99%) of the subunit with a protein encoded by the causative gene (LIS-1) for Miller-Dieker lissencephaly, a human brain malformation manifested by a smooth cerebral surface and abnormal neuronal migration. This indicates that the LIS-1 gene product is a human homolog of the 45K subunit of intracellular PAF acetylhydrolase. These results raise the possibility that PAF and PAF acetylhydrolase are important in the formation of the brain cortex during differentiation and development (Hattori, 1994).

A hemizygous deletion of LIS1, the gene encoding alphaLis1 protein, causes Miller-Dieker syndrome (MDS). MDS is a developmental disorder characterized by neuronal migration defects resulting in a disorganization of the cerebral and cerebellar cortices. alphaLis1 binds to two other proteins (beta and gamma) to form a heterotrimeric cytosolic enzyme that hydrolyzes platelet-activating factor (PAF). The existence of heterotrimers is implicated from copurification and crosslinking studies carried out in vitro. To determine whether such a heterotrimeric complex could be present in tissues, an investigation was carried out to see whether the alphaLis1, beta, and gamma genes are coexpressed in the developing and adult brain. Murine cDNAs were isolated and in situ hybridization shows that in developing brain tissues alphaLis1, beta, and gamma genes are coexpressed. This suggests that alphaLis1, beta, and gamma gene products form heterotrimers in developing neuronal tissues. In the adult brain, alphaLis1 and beta mRNAs continue to be coexpressed at high levels while gamma gene expression is greatly diminished. This reduction in gamma transcript levels is likely to result in a decline of the cellular concentration of alphaLis1, beta, and gamma heterotrimers. The developmental expression pattern of alphaLis1, beta, and gamma genes is consistent with the neuronal migration defects seen in MDS; regions containing migrating neurons such as the developing cerebral and cerebellar cortices express these genes at a particularly high level. Furthermore, a correlation was uncovered between gamma gene expression, granule cell migration, and PAF hydrolytic activity in the cerebellum. In this tissue gamma gene expression and PAF hydrolysis peaks at postnatal days 5 and 15, a period during which neuronal migration in the cerebellum is most extensive. Mechanisms by which PAF could affect neuronal migration are discussed (Albrecht, 1996).

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signaling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extra-cellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homolog of the beta-subunit of trimeric G proteins. The crystal structure is reported of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer (Ho, 1997).

Lissencephaly patients are born with severe brain malformations and suffer from recurrent seizures. LIS1, the gene mutated in isolated lissencephaly patients, is a subunit of the heterotrimeric cytosolic enzyme platelet-activating factor acetylhydrolase (PAF-AH), interacts with tubulin, and affects microtubule dynamics. In order to gain molecular insights into the possible involvement of LIS1 in seizures in lissencephaly patients, seizures were induced in rats by injection of kainate. PAF-AH activity is markedly reduced as early as 30 min following initiation of seizures, making this parameter a sensitive indicator of seizure events. PAF-AH activity returns to and surpasses control values 1 week following initiation of seizures. Expression of LIS1 in the dentate gyrus changes significantly in a manner similar to that of PAF-AH enzymatic activity. This is the first correlation found between LIS1 expression and PAF-AH activity. Furthermore, the expression of the alpha2 catalytic subunit, which is the major PAF-AH catalytic subunit in rat adult brain, changes in a dramatic fashion. An additional higher-mobility LIS1 cross-reactive band was detected in samples isolated a week following seizure occurrence. This LIS1 isoform is enriched in the microtubule-associated fraction. It is proposed that LIS1 expression is an important factor in regulation of PAF-AH activity. Reductions in LIS1 protein levels found in lissencephaly patients may render them more susceptible to seizures (Shmueli, 1999).

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, the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins were examined. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrate that all human LIS1 mutations examined result 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 (Sweeney, 2000).

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. The structure of LIS1 in complex with the alpha2/alpha2 PAF-AH homodimer is reported. One LIS1 homodimer binds symmetrically to one alpha2/alpha2 homodimer via the highly conserved top faces of the LIS1 ß 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. These data suggest that the LIS1 molecule undergoes major conformational rearrangement when switching from a complex with the acetylhydrolase to the one with Ndel1 (Tarricone, 2004).

The crystal structure of LIS1 in complex with PAF-AH provides a new powerful tool to elucidate the function of LIS1 and its interactions. The LIS1 binding signature in the alpha1 and alpha2 subunits of PAF-AH can be exploited to design PAF-AH alleles impaired in LIS1 binding. The ß propeller scaffold revealed by structural analysis, in contrast, will allow the design of mutational strategies to address the interaction of LIS1 with dynein, Ndel1, CLIP-170, and PAF-AH. An important conclusion from the present study is that LIS1 and its interacting partners form dimers and that the complexes of LIS1 with Ndel1 and PAF-AH are tetrameric. The quaternary organization of LIS1 and its binding partners has far-reaching implications for how mutation might affect LIS1 function. In the simplest model, dimerization of the binding partners doubles the binding energy relative to that in equivalent monovalent interactions. In the specific case of the LIS1/alpha2/alpha2 interaction, this might explain why monovalent LIS1 species (lacking the N-LIS1 domain) are unable to interact strongly with PAF-AH despite being structurally stable. Although the N-LIS1 dimerization domain seems to be directly involved in the LIS1/Ndel1 interaction, the alpha4 helix and the ß propeller might provide additional contacts whose contribution to the stability of the complex, once again, will depend on the dimerization of LIS1. In this respect, it is useful to bear in mind that the incorporation of stable loss-of-function LIS1 mutants in dimeric complexes with wild-type LIS1 might have more dramatic damaging effects than those caused by structurally destabilizing mutations, which are likely to result in the removal of the misfolded protein product. In the latter situation, less abundant but functional dimers might still form, while in the former situation, ~75% of the LIS1 dimers will contain at least one mutated allele and be dysfunctional. This might explain why most disease-causing mutations consist of deletions or nonsense point mutations, resulting in a truncated and, therefore unstable, LIS1 protein or why missense mutations destabilize the propeller's fold, while only very few mutations identified in lissencephaly patients affect what are likely to be key functional residues on the highly conserved propeller's top surface. It is suspected that the incorporation of such mutant alleles in a dimer with the wild-type protein may have a dominant-negative effect, resulting in more severe phenotypes relative to structurally unstable mutants, whose rapid degradation would simply result in LIS1 haploinsufficiency. Studies to clarify this point are in progress (Tarricone, 2004 and references therein).

LIS1 interacts with mNudE

Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene: both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons. LIS1 is highly expressed in the ventricular zone and the cortical plate, and its product, Lis1, has seven WD repeats. Several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2, and with tubulin; it also reduces microtubule catastrophe events in vitro. A yeast two-hybrid screen has been used to isolate new Lis1-interacting proteins and a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans, has been found. The specificity of the mammalian NudC-Lis1 interaction has been demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture (S. M. Morris, 1998).

In both Aspergillus nidulans and the mouse, studies of the nuclear distribution gene C (NudC) have strongly suggested that the NudC protein interacts with NudF, which is the product of NudF, a homolog of human LIS1 (also know as PAFAH1B1), one of the causative genes for classical lissencephaly. The human NUDC gene and its two processed pseudogenes have been isolated. The human NUDC gene is highly conserved and its predicted amino acid sequence shows 94% identity to mouse NudC and 95% identity to rat NudC. The genomic structure of NUDC, its chromosomal localization, and expression pattern in human tissues were characterized. NUDC consists of at least 9 exons ranging from 66 bp to 266 bp in size and 8 introns from 92 bp to 2.0 kb in length, and the total genomic region spans about 8 kb. NUDC was mapped to 1p34-p35 by fluorescence in situ hybridization. Northern analysis shows a major 1.6 kb transcript in all fetal and adult tissues examined. Primers that amplify individual exons of NUDC have been developed for mutation analysis (Matsumoto, 1999).

LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. A LIS1 interacting protein has been identified, encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC (Feng, 2000).

LIS1 is a product of the causative gene for type I lissencephaly characterized by a smooth brain surface due to a defect in neuronal migration during brain development and a regulatory subunit of platelet-activating factor acetylhydrolase (PAF-AH). It is also a mammalian homolog of the fungal nuclear distribution (nud) gene, nudF, which controls the migration of fungal nuclei. Using the two-hybrid system, a novel LIS1-interacting protein, rat NUDE (rNUDE) has been identified: it is a mammalian homolog of another fungal nud gene product, NUDE, and Xenopus mitotic phosphoprotein 43, which is phosphorylated in a cell cycle-dependent manner. rNUDE and the catalytic subunits of PAF-AH interact with the N- and C-termini of LIS1, respectively. However, these proteins, instead of simultaneously binding to LIS1, appear to bind to LIS1 in a competitive manner. These results suggest that LIS1 functions in nuclear migration by interacting with multiple intracellular proteins in mammals (Kitagawa, 2000).

Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, it has been demonstrated that mammalian LIS1 regulates dynein functions. NUDEL is a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. These findings point to cross-talk between two prominent pathways that regulate neuronal migration (Niethammer, 2000).

Mutations in mammalian Lis1 result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. 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 (Sasaki, 2000).

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), it was 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 (Shu, 2004).

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains unanswered. Overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end-directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end-directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain (Liang, 2004).

Ablation of the LIS1-interacting protein Nde1 (formerly mNudE) in mouse produces a small brain (microcephaly), with the most dramatic reduction affecting the cerebral cortex. While cortical lamination is mostly preserved, the mutant cortex has fewer neurons and very thin superficial cortical layers (II to IV). BrdU birthdating reveals retarded and modestly disorganized neuronal migration; however, more dramatic defects on mitotic progression, mitotic orientation, and mitotic chromosome localization in cortical progenitors were observed in Nde1 mutant embryos. The small cerebral cortex seems to reflect both reduced progenitor cell division and altered neuronal cell fates. In vitro analysis demonstrated that Nde1 is essential for centrosome duplication and mitotic spindle assembly. The data show that mitotic spindle function and orientation are essential for normal development of mammalian cerebral cortex (Feng, 2004).

The phenotype of Nde1-/- mice closely resembles defects produced by Lis1 mutations in mouse in affecting both neurogenesis and neuronal migration. Although homozygous Lis1 knockout mice are periimplantation lethal, compound heterozygous mice expressing 35% of the wildtype level of Lis1 protein show severe defects in both neuronal migration and neurogenesis. Among various defects, a mitotic delay with increased numbers of M phase cells and with mispositioned mitotic nuclei was also found in the Lis1 mutant embryos, which is similar to observations in Nde1 homozygous mutant mice. While LIS1 has been known to be required for cortical neuronal migration, the molecular function of LIS1 has been of intense interest and controversy. In addition to cell migration, LIS1 has also been implicated in cell division, through the observation that Lis1 null cells in Drosophila show proliferation defects and decreased LIS1 activity induces mitotic arrest in mammalian cell culture. Therefore, it is highly likely that LIS1 and Nde1 are in the same molecular and genetic pathway in regulating progenitor cell mitosis. In contrast, the restriction of Nde1 loss-of-function phenotype to the six-layered neocortex and specifically to neurons originating from the cortical ventricular precursors correlates very well with the observation that haploinsufficiency of LIS1 in humans mostly presents as a cerebral cortex-specific defect with little impact on other brain structures such as the cerebellum and basal ganglia. This suggests that cortical progenitor cell proliferation defects may also underlie the pathogenesis of lissencephaly in humans. Since neural progenitor cell division occurs prior to and is tightly coupled to neuronal migration, the defects in the mode and duration of progenitor proliferation may have a strong impact on subsequent neuronal migration processes. Moreover, a significant reduction of nestin-positive cells was observed in mice by E15, which suggests that the mutant Nde1 mice may also have defective radial glia processes due to mitotic arrest of both neural and radial glial progenitors. The potential radial glia defect could also contribute to the migration difficulties of neurons in these mice. Since both Nde1 homozygous and Lis1 heterozygous mice show modest neuronal migration abnormalities, further study of the correlation between cortical progenitor mitosis and cortical neuronal migration with genetic models of Nde1 and Lis1 double mutations would allow a better understanding of the mechanism of both molecules in the pathological basis of lissencephaly (Feng, 2004).

Cenp-F is a nuclear matrix component that localizes to kinetochores during mitosis and is then rapidly degraded after mitosis. Unusually, both the localization and degradation of Cenp-F require it to be farnesylated. Cenp-F is required for kinetochore-microtubule interactions and spindle checkpoint function; however, the underlying molecular mechanisms have yet to be defined. Cenp-F interacts with Ndel1 and Nde1, two human NudE-related proteins implicated in regulating Lis1/Dynein motor complexes. 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, these data demonstrate that Ndel1 and Nde1 play distinct roles to ensure chromosome alignment and segregation (Vergnolle, 2007).

Other LIS1 protein interactions

Interactions have been demonstrated between certain pleckstrin homology (PH) domains and regions containing the so-called WD40 (or beta-transducin) repeats of the beta subunit of trimeric G-proteins (G beta), a finding that is here extended to the PH domains of the src-related tyrosine kinase TecIIa and the GTPase dynamin. To examine the possibility that WD40 repeats in molecules other than G beta might also bind PH domains PAFAH-45, the protein product of the Lis-1 gene, which contains 7 WD40 repeats, was examined. Purified PAFAH-45 binds PH domain constructs in vitro. Protein constructs expressing all 7 WD40 repeats of PAFAH-45 but lacking the N-terminal non WD40 region also bind PH domains of beta-adrenergic receptor kinase, beta-spectrin, TecIIa and dynamin but with a differing hierarchy of affinities than that seen with G beta. PAFAH-45 WD40 repeats will reduce the binding of PH domains to brain G beta and brain G beta gamma will reduce the binding of PH domains to PAFAH-45. These data support the hypothesis that PH domain/WD40 interactions are involved in a wide variety of important protein/protein interactions (Wang, 1995).

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. LIS1 interaction with microtubules was detected by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicate that physiological concentrations of LIS1 indeed reduce microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. These new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. It is postulated that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients (Sapir, 1997).

LIS1 is found in a complex with two protein kinases: 1) a T-cell Tat-associated kinase, which contains casein-dependent kinase (CDK) activating kinase (CAK), as well as CAK-inducing activity and with 2) a spleen protein-tyrosine kinase similar to the catalytic domain of p72syk. Since phosphorylation is one of the ways to control cellular localization and protein-protein interactions, an investigation was carried out to see whether LIS1 undergoes this post-translational modification. LIS1 has been shown to be a developmentally regulated phosphoprotein. Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis reveals that LIS1 is phosphorylated on serine residues. Alkaline phosphatase treatment reduces the number of visible LIS1 isoforms. In-gel assays demonstrate a 50-kDa LIS1 kinase that is enriched in microtubule-associated fractions. In vitro, LIS1 is phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested. It is suggested that LIS1 activity may be regulated by phosphorylation (Sapir, 1999a).

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), that 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. Colocalization and direct interaction between CLIP-170 and LIS1 is demonstrated in this study. 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 (Coquelle, 2002).

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. Mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. LIS1 has been localized to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. 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. 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, these data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities (Tai, 2002).

Characterization of LIS1 mutations

Classical lissencephaly (smooth brain) or generalized agyria-pachygyria is a severe brain malformation that results from an arrest of neuronal migration at 9-13 weeks gestation. It has been observed in several malformation syndromes including Miller-Dieker syndrome (MDS) and isolated lissencephaly sequence (ILS). A gene containing beta-transducin like repeats, now known as LIS1, was previously mapped to the ILS/MDS chromosome region on 17p13.3. The classical lissencephaly critical region has been localized to the LIS1 gene locus by molecular analysis of key ILS and MDS patients. The structure of LIS1, consists of 11 exons, and the presence of subtle mutations have been sought in 19 ILS patients who show no gross rearrangements of LIS1. Single strand conformational polymorphism (SSCP) analysis reveals band-shifts for three patients, each involving a different coding exon: these band-shifts were not observed in their respective parental DNAs. Sequence analysis has identified these de novo mutations as dA to dG transition in exon VI at nucleotide 446, a dC to dT transition in exon VIII at nucleotide 817, and a 22 bp deletion at the exon IX-intron 9 junction from nucleotide 988 to 1,002+7, which causes skipping of exon IX in the mature LIS1 transcript. These changes are predicted to result in an H149R amino acid substitution, an R273X premature translation termination, and abolition of amino acids 301-334, in the respective LIS1 proteins. These data thus confirm LIS1 as the gene responsible for classical lissencephaly in ILS and MDS (Lo Negro, 1997).

Heterozygous mutation or deletion of the beta subunit of platelet-activating factor acetylhydrolase (PAFAH1B1, also known as LIS1) in humans is associated with type I lissencephaly, a severe developmental brain disorder thought to result from abnormal neuronal migration. To further understand the function of PAFAH1B1, three different mutant alleles in mouse Pafah1b1 have been produced. Homozygous null mice die early in embryogenesis soon after implantation. Mice with one inactive allele display cortical, hippocampal and olfactory bulb disorganization resulting from delayed neuronal migration by a cell-autonomous neuronal pathway. Mice with further reduction of Pafah1b1 activity display more severe brain disorganization as well as cerebellar defects. These results demonstrate an essential, dosage-sensitive neuronal-specific role for Pafah1b1 in neuronal migration throughout the brain, and an essential role in early embryonic development. The phenotypes observed are distinct from those of other mouse mutants with neuronal migration defects, suggesting that Pafah1b1 participates in a novel pathway for neuronal migration (Hirotsune, 1998).

Heterozygous mutation or deletion of Pafab1b1 (LIS1) in humans is associated with syndromes with type 1 lissencephaly, a severe brain developmental disorder resulting from abnormal neuronal migration. Lis1 heterozygous mutant mice have been created by gene targeting. Heterozygous mutant mice are viable and fertile, but display global organizational brain defects as a result of impaired neuronal migration. To assess the functional impact of the mutation, Lis1 heterozygous mice and their wild-type littermates were evaluated on a wide variety of behavioral tests. Lis1 mutant mice display abnormal hindpaw clutching responses and are impaired on a rotarod test. Lis1 heterozygous mice are also impaired in the spatial learning version of the Morris water task. Impaired motor behavior and spatial learning and memory in Lis1 mutant mice indicates that impaired neuronal migration can have functional effects on complex behavioral responses. The behavioral findings also support the use of the Lis1 mutant mice as a model from human type 1 lissencephaly (Paylor, 1999).

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. The consequences of the point mutation, deletion mutation and one of the reported truncations have been studied. In order to study LIS1 structure function, an additional point mutation and other truncations have been introduced in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of these results, it is suggested that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein (Sapir, 1999b).

Lissencephaly (agyria-pachygyria) is a human brain malformation manifested by a smooth cerebral surface and abnormal neuronal migration. Identification of the gene(s) involved in this disorder would facilitate molecular dissection of normal events in brain development. Type 1 lissencephaly occurs either as an isolated abnormality or in association with dysmorphic facial appearance in patients with Miller-Dieker syndrome. About 15% of patients with isolated lissencephaly and more than 90% of patients with Miller-Dieker syndrome have microdeletions in a critical 350-kilobase region in chromosome 17p13.3. These deletions are hemizygous, so haplo-insufficiency for a gene in this interval is implicated. A gene (LIS-1, lissencephaly-1) in 17p13.3 that is deleted in Miller-Dieker patients has been cloned. Non-overlapping deletions involving either the 5' or 3' end of the gene were found in two patients, identifying LIS-1 as the disease gene. The deduced amino-acid sequence shows significant homology to beta-subunits of heterotrimeric G proteins, suggesting that it could possibly be involved in a signal transduction pathway crucial for cerebral development (Reiner, 2000).

Human cortical heterotopia and neuronal migration disorders result in epilepsy; however, the precise mechanisms remain elusive. Severe neuronal dysplasia and heterotopia occurs throughout the granule cell and pyramidal cell layers of mice containing a heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birth-dating analysis using bromodeoxyuridine reveals that neurons in Lis1+/- murine hippocampus are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice are stunted and possess fewer dendritic branches, whereas dentate granule cells are hypertrophic and form spiny basilar dendrites from which the principal axon emerge. Both somatostatin- and parvalbumin-containing inhibitory neurons are heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission are severely disrupted, revealing hyperexcitability at Schaffer collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition, the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission is less than that of wild type. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K(+)](o) model of epilepsy. In Lis1+/- hippocampus, intense interictal bursting is observed on elevation of extracellular potassium to 6.5 mM, a condition that results in only minimal bursting in wild type. These anatomical and physiological hippocampal defects may provide a neuronal basis for seizures associated with lissencephaly (Fleck, 2000).

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active lipid mediator. Expression of PAF receptor has been demonstrated in neurons and microglia. PAF is produced in the brain from its precursor, and degraded by the enzyme PAF acetylhydrolase. LIS1 is a regulatory subunit of PAF acetylhydrolase, and is identical to a gene whose deletion causes the human neuronal migration disorder, type I lissencephaly. Indeed, Lis1 mutant mice display defects in neuronal migration and layering in vivo, and also in cerebellar granule cell migration in vitro. However, the roles of PAF and the PAF receptor in the neuronal migration remain to be determined. PAF receptor-deficient mice exhibited histological abnormalities in the embryonic cerebellum. PAF receptor-deficient cerebellar granule neurons migrated more slowly in vitro than wild-type neurons, consistent with the observation that a PAF receptor antagonist reduced the migration of wild-type neurons in vitro. Synergistic reduction of neuronal migration was observed in a double mutant of PAF receptor and LIS1. Unexpectedly, PAF affects the migration of PAF receptor-deficient neurons, suggesting a receptor-independent pathway for PAF action. The PAF receptor-independent response to PAF is abolished in granule neurons derived from the double mutant mice. Thus, these results suggest that the migration of cerebellar granule cells is regulated by PAF through receptor-dependent and receptor-independent pathways, and that LIS1 is a pivotal molecule that links PAF action and neuronal cell migration both in vivo and in vitro (Tokuoka, 2003).

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. A mouse mutant (LIS1GT/GT) was generated by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa (Nayernia, 2003).

Lis1 regulates asymmetric division in hematopoietic stem cells and in leukemia

Cell fate can be controlled through asymmetric division and segregation of protein determinants, but the regulation of this process in the hematopoietic system is poorly understood. This study shows that the dynein-binding protein Lis1 is critically required for hematopoietic stem cell function and leukemogenesis. Conditional deletion of Lis1 (also known as Pafah1b1) in the hematopoietic system led to a severe bloodless phenotype, depletion of the stem cell pool and embryonic lethality. Further, real-time imaging revealed that loss of Lis1 caused defects in spindle positioning and inheritance of cell fate determinants, triggering accelerated differentiation. Finally, deletion of Lis1 blocked the propagation of myeloid leukemia and led to a marked improvement in survival, suggesting that Lis1 is also required for oncogenic growth. These data identify a key role for Lis1 in hematopoietic stem cells and mark its directed control of asymmetric division as a critical regulator of normal and malignant hematopoietic development (Zimdahl, 2014).

Lis-1 is required for dynein-dependent cell division processes in C. elegans embryos

The role was investigated of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. Apparent null alleles of lis-1 were identified, that result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. Antibodies were raised against LIS-1, and transgenic animals were generated expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, it was found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. It was established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, it was found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another (Cockell, 2004).

Mutants of Doublecortin, a Lissencephaly-interacting protein, give rise to a neuronal migration disorder

X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. A novel CNS gene has been characterized encoding a 40 kDa predicted protein that has been named Doublecortin. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion (des Portes, 1998).

X-linked lissencephaly and 'double cortex' are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. A novel 10 kb brain-specific cDNA, interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein, has been named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration (Gleeson, 1998).

The development of functional layers in the brain involves spatially and temporally regulated gene expression. Through cDNA library screening, genes have been identified that are expressed in a neural-specific manner during brain development. Sequencing and expression data indicate that one of the clones, 18C15, is the chick homolog of doublecortin, a human X-linked gene found to be mutated in subcortical laminar heterotopia (double cortex syndrome) and lissencephaly. The 18C15 mRNA contains multiple motifs that are known to regulate mRNA stability in response to inductive signals, and these motifs are conserved between the chick and human sequences. Doublecortin is found to be expressed at peak levels during early development of the cerebellum and forebrain, and is expressed in other regions including the tectum, spinal cord, and dorsal root ganglia. This study demonstrates both spatial and temporal regulation of doublecortin expression in the chick, which is associated with early events in brain development, including neuronal migration (Hannan, 1999).

X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, the protein has been analyzed upon transfection of COS cells. Doublecortin has been found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an iso-electric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and previous work on another lissencephaly gene (LIS1) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis (Horesh, 1999).

The doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. Sedimentation and microscopy studies demonstrate that Doublecortin is associated with microtubules (MTs) and it is postulated that Doublecortin is a novel MAP. Data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development (Francis, 1999).

Mutations in either LIS1 or DCX are the most common cause for type I lissencephaly. LIS1 and DCX interact physically both in vitro and in vivo. Epitope-tagged DCX transiently expressed in COS cells can be co-immunoprecipitated with endogenous LIS1. Furthermore, endogenous DCX can be co-immunoprecipitated with endogenous LIS1 in embryonic brain extracts, demonstrating an in vivo association. The two protein products also co-localize in transfected cells and in primary neuronal cells. In addition, homodimerization of DCX occurs in vitro. Using fragments of both LIS1 and DCX, the domains of interaction were mapped. LIS1 and DCX interact with tubulin and microtubules. These results suggest that addition of DCX and LIS1 to tubulin enhances polymerization in an additive fashion. In in vitro competition assays, when LIS1 is added first, DCX competes with LIS1 in its binding to microtubules, but when DCX is added prior to the addition of LIS1 it enhances the binding of LIS1 to microtubules. It is concluded that LIS1 and DCX cross-talk is important to microtubule function in the developing cerebral cortex (Caspi, 2000).

Mutations in the X-linked gene doublecortin (DCX) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified: the sequence differences include nonsense, splice site and missense mutations throughout the gene. DCX interacts and stabilizes microtubules. A detailed sequence analysis of DCX and DCX-like proteins from various organisms has been undertaken and an evolutionarily conserved Doublecortin (DC) domain has been defined. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. It is hypothesized that these repeats may be important for microtubule binding. DCX or DCLK (KIAA0369) repeats have been expressed in vitro and in vivo. The results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, seven of the reported mutations were overexpressed in COS7 cells and their effects on the microtubule cytoskeleton have been examined. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development (Sapir, 2000).

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. Mouse DCX colocalizes with the microtubules and a mutant doublecortin expression study has provided evidence that doublecortin's conformational structure is important for its subcellular localization. The results of this study suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development (Yoshiura, 2000).

Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. The two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro and in vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a beta-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif (Taylor, 2000).

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. A yeast two-hybrid screen was carried out to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments show that these interactions are mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions is suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction is further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking (Friocourt, 2001).

Doublecortin (DCX) is a microtubule-associated protein that is required for normal neocortical and hippocampal development in humans. Mutations in the X-linked human DCX gene cause gross neocortical disorganization (lissencephaly or 'smooth brain') in hemizygous males, whereas heterozygous females show a mosaic phenotype with a normal cortex as well as a second band of misplaced (heterotopic) neurons beneath the cortex ('double cortex syndrome'). A mouse carrying a targeted mutation in the Dcx gene was generated. Hemizygous male Dcx mice show severe postnatal lethality; the few that survive to adulthood are variably fertile. Dcx mutant mice show neocortical lamination that is largely indistinguishable from wild type and show normal patterns of neocortical neurogenesis and neuronal migration. In contrast, the hippocampus of both heterozygous females and hemizygous males shows disrupted lamination that is most severe in the CA3 region. Behavioral tests show defects in context and cued conditioned fear tests, suggesting that deficits in hippocampal learning accompany the abnormal cytoarchitecture (Corbo, 2002).

Mutations in the doublecortin gene (DCX) in humans cause malformation of the cerebral neocortex. Paradoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. Electroporation of plasmids encoding short hairpin RNA was used to create interference (RNAi) of DCX protein in utero; DCX is shown to be required for radial migration in developing rat neocortex. RNAi of DCX causes both cell-autonomous and non-cell autonomous disruptions in radial migration, and creates two disruptions in neocortical development: (1) many neurons prematurely stop migrating to form subcortical band heterotopias within the intermediate zone and then white matter; (2) many neurons migrate into inappropriate neocortical lamina within normotopic cortex. In utero RNAi can therefore be effectively used to study the specific cellular roles of DCX in neocortical development and to produce an animal model of double cortex syndrome (Bai, 2003).

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. Evidence is presented that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, a serine has been identified within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity (Schaar, 2004).

Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules (Tanaka, 2004a).

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, in vitro neuronal migration assays were combined with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increases migration rates. Dcx overexpression rescues the migration defect in Lis1+/- neurons. Lis1 localizes predominantly to the centrosome, and after disruption of microtubules, redistributes to the perinuclear region. Dcx outlined microtubules extending from the perinuclear 'cage' to the centrosome. Lis1+/- neurons display increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition results in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects are rescued by Dcx overexpression, and Dcx is 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 (Tanaka, 2004b).

Lissencephaly-1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.