The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein

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. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue 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 homologue 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.     

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.     

Roles of NUDE and NUDF proteins of Aspergillus nidulans: insights from intracellular localization and overexpression effects

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes ("comets") that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudE gene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditional nudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF and nudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between the nudF and apsA genes suggests a role for NUDF at the cell cortex.     

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.     

BICD2, dynactin,and LIS1 cooperate in regulating dynein recruitment to cellular structures

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.     

Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.     

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

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

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.     

The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.     

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.     

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

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

RanBPM, a novel interaction partner of the brain-specific protein p42IP4/centaurin alpha-1

The protein p42^IP4 (aka Centaurin α-1) is highly enriched in the brain and has specific binding sites for the membrane lipid phosphatidylinositol 3,4,5-trisphosphate and the soluble messenger inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄; IP4). p42^IP4 shuttles between plasma membrane, cytosol and cell nucleus. However, the molecular function of p42^IP4 is still largely unclear. Here, we report a novel interaction partner for p42^IP4, Ran binding protein in microtubule-organizing center (RanBPM). RanBPM is ubiquitously expressed and seems to act as scaffolding and modulator protein. In our studies, we established this interaction in vitro and in vivo . The in vivo interaction was demonstrated with endogenous RanBPM from rat brain. Both proteins co-localize in transfected HEK 293 cells. We could show that the interaction does not require additional proteins. D-Ins(1,3,4,5)P₄, a specific ligand for p42^IP4, is a concentration-dependent and stereoselective inhibitor of this interaction; the l -isoform is much less effective. We found that mainly the SPRY domain of RanBPM mediates the p42^IP4-RanBPM association. The ARFGAP domain of p42^IP4 is important for the interaction, without being the only interaction site. Recently, p42^IP4 and RanBPM were shown to be involved in dendritic differentiation. Thus, we hypothesize that RanBPM could act as a modulator together with p42^IP4 in synaptic plasticity.     

MYND domain specific interaction of the melanin-concentrating hormone receptor 1 interacting zinc-finger protein with alpha- and beta-tubulin

MIZIP was originally identified as a highly conserved zinc-finger protein from human brain interacting with the C-terminus of the melanin-concentrating hormone receptor 1. However, the cellular functions of MIZIP are still not known. Here, we focussed on the identification of associated proteins using affinity purification from human cells. This resulted in the identification of alpha- and beta-tubulin. The interaction was confirmed in vitro and in vivo using GST pull-down and immunoprecipitation assays, and was mapped to the MYND zinc-finger of MIZIP and to the N-terminus of tubulin. Immunoprecipitation and immunocytochemistry analyses demonstrate that MIZIP binds to tubulin but not to cellular microtubules in vivo and that ectopic expression of MIZIP does not interfere with the overall structure of the microtubular cytoskeleton. Our results suggest that MIZIP might play an important role in mammalian cells by associating with tubulin and thus might provide a link between MCHR1 and tubulin functions.     

Structure of Tctex-1 and its interaction with cytoplasmic dynein intermediate chain

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.     

The interaction of the histone H1-related protein phi 0 with chromatin

Protein phi 0 is a unique protein which is present in the sperm of the sea cucumber, Holothuria tubulosa. It associates with histones, but its physiological role is unknown. From its amino acid composition and sequence, protein phi 0 can be considered as an H1-related protein. In this paper, we have studied its interaction with chicken erythrocyte chromatin particles of different complexity, from core particles to polynucleosomes. Addition of protein phi 0 results in marked chromatin insolubilization. The higher the molecular weight of the chromatin fragment, the lower is the phi 0/nucleosome molar ratio at which precipitation occurs, so that complete insolubilization of polynucleosomes is achieved at a phi 0/nucleosome molar ratio which is identical to that found in mature H. tubulosa spermatozoa. We have also found that the interaction of protein phi 0 with chromatin is cooperative. These findings contribute to clarification of the peculiar physico-chemical properties shown by H. tubulosa sperm chromatin and the role played by the phi 0 protein.