NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration.

During a study of the genetics of nuclear migration in the filamentous fungus Aspergillus nidulans, we cloned a gene, nudF, which 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.     

Neuronal migration: unraveling the molecular pathway with humans, mice, and a fungus

This review highlights the utility of comparative genetics in understanding the molecular mechanisms that underlie neuronal migration. It is apparent from studies in humans, mice, and a fungus that nuclear migration is a key component of neuronal migration and that both are dependent on a dynamic microtubule network. In vertebrates regulation of this network involves a complex pathway that is dependent on extracellular guidance cues, membrane-bound receptors, intracellular signaling molecules, proteins associated with microtubules, and the components of microtubules themselves.     

Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. Unlike other Cdks that promote cell cycle, Cdk5 is activated in postmitotic neurons and critically regulates neuronal migration by phosphorylating its substrates during brain development. Recently, we found that Cdk5 phosphorylates focal adhesion kinase (FAK) at Serine 732 in vitro and is responsible for this phosphorylation in the developing brain. Our experiments using a phospho-specific antibody and an S732-unphosphorylatable mutant FAK suggest that S732 phosphorylation may regulate a centrosome-associated microtubule structure to promote nuclear translocation, a critical step in neuronal migration. S732 phosphorylation does not directly impact on the kinase activity of FAK, but appears to prevent the accumulation of FAK at the centrosome. Our study reveals a similarity between Cdk5 and Cdk1 in the regulation of neuronal migration and cell division, respectively. In addition, our study implicates FAK in a signaling pathway that directly regulates microtubules.     

Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans

The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.     

NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development

NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.     

NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein

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, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, 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. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.     

Ultrastructural basis of biotrophic relationships between brown rust fungus and common wheat

The ultrastructural research of relationships between common wheat and brown rust fungus Puccinia triticina demonstrated that the biotrophy was based on the induction of plant cytoplasmic aggregate organization, nuclear migration to haustorium and stimulation of mesophyll cell metabolism. Resistant reactions in the form of hypersensitivity and depositions on plant cell walls were suppressed up to fungal sporogenesis. Cytoplasmic reorganization and nuclear taxis to haustorium were not registered in the epidermal and vascular bundle sheath cells.     

Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.     

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.     

Colocalization of doublecortin with the microtubules: an ex vivo colocalization study of mutant doublecortin

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. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.     

Nuclear migration in a nud mutant of Aspergillus nidulans is inhibited in the presence of a quantitatively normal population of cytoplasmic microtubules

Nuclear migration was studied in germinating conidia of a temperature-sensitive mutant of the fungus Aspergillus nidulans. At the restrictive temperature motility was demonstrably impaired because significantly fewer nuclei migrated into the germ tube relative to a population of similarly sized germlings grown at the permissive temperature. Further comparison of these populations showed that the mutant was leaky in that an increasing number of nuclei migrated as the total nuclear content increased in each germling. The restrictive temperature also induced elevated mitotic asynchrony and increased numbers of nuclei per germling. Serial section-based reconstruction of the microtubules in a freeze-substituted germling showed that they were not attached to the nucleus-associated organelles, were approximately parallel to the long axis of the germ tube, and seemed to be randomly distributed between the central and peripheral cytoplasm. Five germlings from each temperature were selected for quantitative analysis of cytoplasmic microtubules. All 10 germlings had typical nuclear migration phenotypes. No significant temperature-related difference in microtubule density was found. We conclude that inhibition of nuclear migration in the mutant is the effect of some defect other than the failure of cytoplasmic microtubules to assemble to their normal population density. We also suggest that nuclear motility is not dependent on mitosis-related microtubules.     

The role of fractions from Paracoccidioides brasiliensis in the genesis of inflammatory response

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.     

神经元的迁移机制

在脊椎动物的脑发育过程中,未成熟的神经元在时间和空间上的精确迁移到达最后行使功能的目的地,是中枢神经系统发育的一个重要阶段.最近的研究结果表明这一过程涉及到一系列分子事件,包括细胞表面分子的相互作用、离子通道的激活和细胞骨架的作用等.对这些事件的了解不但有助于了解神经元迁移的机制,而且对阐明由于神经元异常迁移而引起的脑紊乱失调等病理现象的机理都是必要的.     

Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima "Pink Banana."     

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.     

Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.     

APC nuclear membrane association and microtubule polarity

Background information. Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. Results. We previously showed, using primary fibroblasts and astrocytes in in vitro scratch‐induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus‐ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. Conclusions. We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Mouse disabled 1 regulates the nuclear position of neurons in a Drosophila eye model

Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Here we show that Disabled 1 (Dab1), which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, we observed that a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhanced the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduced the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling which regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons.