MYO1, a novel, unconventional myosin gene affects endocytosis and macronuclear elongation in Tetrahymena thermophila

Targeted gene disruption was used to investigate the function of MYO1, an unconventional myosin gene in Tetrahymena thermophila. Phenotypic analysis of a transformed strain that lacked a functional MYO1 gene was conducted at both 20 degrees C and 35 degrees C. At either temperature the delta MYO1 strain had a smaller cytoplasm/nucleus ratio than wild type. At 20 degrees C, delta MYO1 populations had a longer doubling time than wild type, lower saturation density, and a reduced rate of food vacuole formation. However, at 35 degrees C, these characteristics were comparable to wild type. Although micronuclear division and cytokinesis appeared normal in delta MYO1 cells, failure of the macronucleus to elongate properly resulted in unequal segregation of macronuclear DNA in cells maintained at either 20 degrees C or 35 degrees C.     

Alternate Junctions and Microheterogeneity of Tlr1, a Developmentally Regulated DNA Rearrangement in Tetrahymena thermophila

ABSTRACT. A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila , Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.     

A Genetic Screen for Vegetative Gene Expression in the Micronucleus of Tetrahymena thermophila

ABSTRACT. The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila . A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization. Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization. This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle.     

A Novel Yeast Screen for Mitotic Arrest Mutants Identifies DOC1, a New Gene Involved in Cyclin Proteolysis

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G₁-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.     

Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila

To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein. Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis.     

Regulation of the Expression of the Myosin Heavy Chain (MYH) Gene myh14 in Zebrafish Development

Marine Biotechnology - The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression...     

Exome Sequencing Identifies a Novel Gene,WNK1, for Susceptibility to Pelvic Organ Prolapse (POP)

Pelvic organ prolapse (POP) is a common gynecological disorder; however, the genetic components remain largely unidentified. Exome sequencing has been widely used to identify pathogenic gene mutations of several diseases because of its high chromosomal coverage and accuracy. In this study, we performed whole exome sequencing (WES), for the first time, on 8 peripheral blood DNA samples from representative POP cases. After filtering the sequencing data from the dbSNP database (build 138) and the 1000 Genomes Project, 2 missense variants in WNK1, c.2668G > A (p.G890R) and c.6761C> T (p.P2254L), were identified and further validated via Sanger sequencing. In validation stage, the c.2668G > A (p.G890R) variant and 8 additional variants were detected in 11 out of 161 POP patients. All these variants were absent in 231 healthy controls. Functional experiments showed that fibroblasts from the utero-sacral ligaments of POP with WNK1 mutations exhibited loose and irregular alignment compared with fibroblasts from healthy controls. In sum, our study identified a novel gene, WNK1, for POP susceptibility, expanded the causal mutation spectrums of POP, and provided evidence for the genetic diagnosis and medical management of POP in the future.     

Global soil ciliate (Protozoa, Ciliophora) diversity: a probability-based approach using large sample collections from Africa, Australia and Antarctica

Large sample collections from Africa (92 samples), Australia (157) and Antarctica (90) were investigated for soil ciliates using the non-flooded Petri dish method, which re-activates the ciliates' resting cysts from air-dried samples. Species were determined from life and by silver impregnation. The African samples were the richest, containing 507 species (240 undescribed,=47%), followed by the Australian (361 species, 154=43% undescribed) and the Antarctic (95 species, 14=15% undescribed) samples. The percentage of new species/sample was consistently low, viz. 4–8% on average, indicating that new species were considerably undersampled relative to described ones, very probably due to methodological shortcomings, i.e. usually only cysts of the more euryoecious species could be reactivated. Thus, a probability theory-based statistical approach was applied to the data sets to compensate for the underestimated number of undescribed species. This procedure indicated that, depending on the region, 70–80% of the soil ciliates are still unknown and global soil ciliate diversity amounts to at least 1330–2000 species. Several indicators, especially the constant rate at which new species have been found during a 20-year period of intensive research, suggest that this estimate is conservative. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel Features of the Light Chain of Microtubule-associated Protein MAP1B: Microtubule Stabilization,Self Interaction,Actin Filament Binding,and Regulation by the Heavy Chain

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain–induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain–light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.     

Notes on the soil ciliate biota (Protozoa, Ciliophora) from the Shimba Hills in Kenya (Africa): diversity and description of three new genera and ten new species

A very diverse ciliate community was found in nine soil samples from the Shimba Hills Nature Reserve in Kenya, equatorial Africa. The ciliates, respectively, their resting cysts, were re-activated from air-dried samples using the non-flooded Petri dish method. Species were determined from life and by silver impregnation. 34 (27%) of the 125 taxa identified had not yet been described in 1985, when the samples were collected and investigated. The richest samples, each containing 59 species, were those from a deciduous primary forest and a young secondary pine forest. The most remarkable species discovered in the Shimba Hills were Krassniggia auxiliaris, Bresslauides terricola, Gigantothrix herzogi, and Afrothrix darbyshirei. They are flagships with a very distinct morphology and easy to recognise due to their extraordinarily large body size. Krassniggia auxiliaris occurs also in Australia and probably has a restricted Gondwanan distribution, like some other ciliates. Bresslauides terricola was later found in soils from all main biogeographical regions, except for Antarctica. Gigantothrix herzogi and Afrothrix darbyshirei are still unique to the Shimba Hills. The following taxa are described in detail: Sikorops woronowiczae nov. gen., nov. spec., Arcuospathidium multinucleatum nov. spec., Dileptus similis Foissner, 1995, Plagiocampa bitricha nov. spec., Drepanomonas exigua exigua Penard, 1922, D. exigua bidentata nov. sspec., Parafurgasonia protectissima (Penard, 1922) nov. comb., P. terricola nov. spec., Brachyosoma brachypoda mucosa nov. sspec., Gigantothrix herzogi nov. gen., nov. spec., Afrothrix darbyshirei nov. gen., nov. spec., Oxytricha africana nov. spec., and O. elegans nov. spec.     

A Kinome Screen Identifies Checkpoint Kinase 1 (CHK1) as a Sensitizer for RRM1-Dependent Gemcitabine Efficacy

Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.     

A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects,Arrhythmia and Possibly Midline Defects

Background

A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5.

Methods and Results

A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface.

Conclusions

Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin.     

Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

A New Tetrahymena (Ciliophora,Oligohymenophorea) from Groundwater of Cape Town,South Africa

The identification of species within the genus Tetrahymena is known to be difficult due to their essentially identical morphology, the occurrence of cryptic and sibling species and the phenotypic plasticity associated with the polymorphic life cycle of some species. We have combined morphology and molecular biology to describe Tetrahymena aquasubterranea n. sp. from groundwater of Cape Town, Republic of South Africa. The phylogenetic analysis compares the cox1 gene sequence of T. aquasubterranea with the cox1 gene sequences of other Tetrahymena species and uses the interior‐branch test to improve the resolution of the evolutionary relationships. This showed a considerable genetic divergence of T. aquasubterranea to its next relative, T. farlyi, of 9.2% (the average cox1 divergence among bona fide species of Tetrahymena is ~ 10%). Moreover, the analysis also suggested a sister relationship between T. aquasubterranea and a big clade comprising T. farleyi, T. tropicalis, T. furgasoni and T. mobilis. The morphological data available for these species show that they share with T. aquasubterranea a pyriformis‐like life style and at least two of them, T. farleyi and T. mobilis, a similar type II silverline pattern consisting of primary and secondary meridians. Tetrahymena aquasubterranea exhibits a biphasic life cycle with trophonts and theronts, is amicronucleate, and feeds on bacteria.     

A LewisX Glycoprotein Screen Identifies the Low Density Lipoprotein Receptor-related Protein 1 (LRP1) as a Modulator of Oligodendrogenesis in Mice

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.     

Small GTPase Rho5 is a functional homologue of Rho1, which controls cell shape and septation in fission yeast

The small GTPase Rho1 plays an essential role in controlling the organization of the actin cytoskeleton and synthesis of the cell wall in the fission yeast Schizosaccharomyces pombe. Here we studied the role of Rho5 whose primary structure is very similar to that of Rho1. It was found that elevated expression of Rho5 was able to compensate for the lethality of cells lacking Rho1. Rho5 was localized to the ends of interphase cells and the mid-region of mitotic cells. Overexpression of Rho5 caused depolarization of F-actin patches and abnormal formation of the cell wall, as did Rho1. Although rho5(+) was not essential for maintaining the cell shape, rho1 rho5-double null cells showed more severe defects in cell viability than rho1-null cells. Thus, it is likely that Rho5 has an overlapping function with Rho1 in controlling cell growth and division in S. pombe.     

Myosin 1G (Myo1G) is a haematopoietic specific myosin that localises to the plasma membrane and regulates cell elasticity

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.

Structured summary

MINT-7307273: MYO1G (uniprotkb:B0I1T2) and Actin (uniprotkb:P60709) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7307283: TfR (uniprotkb:P02786) and MYO1G (uniprotkb:B0I1T2) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)