The Structure of the Coiled-Coil Domain of Ndel1 and the Basis of Its Interaction with Lis1, the Causal Protein of Miller-Dieker Lissencephaly

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Structure (Impact Factor: 5.62). 12/2007; 15(11):1467-81. DOI: 10.1016/j.str.2007.09.015
Source: PubMed


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

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    • "After an unstructured loop, the C-terminal region folds into a beta propeller domain, composed by seven WD40 repeats. Experimental evidence indicates that the LisH domain is essential for in vivo homodimerization (Kim et al., 2004; Mateja et al., 2006), and contains a binding site for known LIS1 protein interactors, such as Nde1 (Derewenda et al., 2007). Both the LisH domain and the coiled-coil motif are known structural mediators of protein–protein interactions and mass spectrometry evidence indicates that the LisH domain and its flanking regions in LIS1 are rich in post-translational modifications (PTMs), suggesting functional regulation (http://www.phosphosite "
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    ABSTRACT: LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling towards the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1. Copyright © 2015. Published by Elsevier Inc.
    Fungal Genetics and Biology 07/2015; 82. DOI:10.1016/j.fgb.2015.07.009 · 2.59 Impact Factor
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    • "The elution volume of Ndel11–201 in isolation (Fig. 3C, blue trace) is compatible with either a monomer with elongated shape or an oligomer. To distinguish between the two possibilities, we performed Static Light Scattering analysis of the same construct, which revealed that Ndel11–201 is dimeric in solution (data not included; Derewenda et al., 2007). The elution profile of p6004480–5183 presents two peaks (red trace). "
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    ABSTRACT: Apical neural progenitors (aNPs) drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600) in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis.
    Biology Open 05/2014; 3(6). DOI:10.1242/bio.20147807 · 2.42 Impact Factor
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    • "The deletion of NDE1 could act at another, for example submicroscopic, level; it is also possible that the deletion unmasks a recessive mutation on the remaining allele, but this is unlikely given the normal appearance and cognitive abilities of our two patients and previous examination of this possibility [10]. Alternatively, loss of NDE1 in patients with heterozygous NDE1 deletion may possibly be compensated by other proteins with similar functions such as LIS1 or NDE1-related protein 1 (NDEL1), a homolog of NDE1 [14], [15], [49]. "
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    ABSTRACT: 16p13.11 genomic copy number variants are implicated in several neuropsychiatric disorders, such as schizophrenia, autism, mental retardation, ADHD and epilepsy. The mechanisms leading to the diverse clinical manifestations of deletions and duplications at this locus are unknown. Most studies favour NDE1 as the leading disease-causing candidate gene at 16p13.11. In epilepsy at least, the deletion does not appear to unmask recessive-acting mutations in NDE1, with haploinsufficiency and genetic modifiers being prime candidate disease mechanisms. NDE1 encodes a protein critical to cell positioning during cortical development. As a first step, it is important to determine whether 16p13.11 copy number change translates to detectable brain structural alteration. We undertook detailed neuropathology on surgically resected brain tissue of two patients with intractable mesial temporal lobe epilepsy (MTLE), who had the same heterozygous NDE1-containing 800 kb 16p13.11 deletion, using routine histological stains and immunohistochemical markers against a range of layer-specific, white matter, neural precursor and migratory cell proteins, and NDE1 itself. Surgical temporal lobectomy samples from a MTLE case known not to have a deletion in NDE1 and three non-epilepsy cases were included as disease controls. We found that apart from a 3 mm hamartia in the temporal cortex of one MTLE case with NDE1 deletion and known hippocampal sclerosis in the other case, cortical lamination and cytoarchitecture were normal, with no differences between cases with deletion and disease controls. How 16p13.11 copy changes lead to a variety of brain diseases remains unclear, but at least in epilepsy, it would not seem to be through structural abnormality or dyslamination as judged by microscopy or immunohistochemistry. The need to integrate additional data with genetic findings to determine their significance will become more pressing as genetic technologies generate increasingly rich datasets. Detailed examination of brain tissue, where available, will be an important part of this process in neurogenetic disease specifically.
    PLoS ONE 04/2012; 7(4):e34813. DOI:10.1371/journal.pone.0034813 · 3.23 Impact Factor
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