LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free‐living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high‐resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida‐specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free‐living marine dinoflagellates and lacks any amoeboid or other specious stages.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

REPLY TO COMMENT ON THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Free‐living, marine dinoflagellates are typified by a well‐defined, haplontic life cycle with relatively few stages. The most unusual departure from this life cycle is one reported for the heterotrophic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder. This species is alleged to have at least 24 life cycle stages including amoebae and a chrysophyte‐like cyst form ( Burkholder et al. 1992 , Burkholder and Glasgow 1997a ) not previously known in free‐living marine dinoflagellates. Litaker et al. (2002) redescribed the life cycle of P. piscicida from single‐cell isolates and found only life cycle stages typical of free‐living marine dinoflagellates. The discrepancy between these observations and the life cycle reported in the literature prompted a rigorous study to resolve the life cycle of P. piscicida. Burkholder and Glasgow (2002) took exception to this study, arguing that Litaker et al. (2002) misunderstood the life cycle of P. piscicida and ignored recent publications. We present a rebuttal of their criticisms and suggest a simple way to resolve the discrepancies in the P. piscicida life cycle.     

Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation,and organ and seed sizes in Arabidopsis

The ICK/KRP cyclin‐dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T‐DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lower‐order mutants showed no morphological phenotypes, the ick1 ick2 ick6 ick7 and ick1 ick2 ick5 ick6 ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild‐type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage‐dependent manner. Analyses also revealed increased expression of E2F‐dependent genes, and elevated RBR1 as well as an increased level of phospho‐RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.     

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.     

STIP overexpression confers oncogenic potential to human non‐small cell lung cancer cells by regulating cell cycle and apoptosis

Sip1/tuftelin‐interacting protein (STIP), a multidomain nuclear protein, is a novel factor associated with the spliceosome, yet its role and molecular function in cancer remain unknown. In this study, we show, for the first time, that STIP is overexpressed in non‐small cell lung cancer (NSCLC) tissues compared to adjacent normal lung tissues. The depletion of endogenous STIP inhibited NSCLC cell proliferation in vitro and in vivo, caused cell cycle arrest and induced apoptosis. Cell cycle arrest at the G2/M phase was associated with the expression and activity of the cyclin B1‐CDK1 (cyclin‐dependent kinase 1) complex. We also provide evidence that STIP knockdown induced apoptosis by activating both caspase‐9 and caspase‐3 and by altering the Bcl‐2/Bax expression ratio. RNA sequencing data indicated that the MAPK mitogen‐activated protein kinases, Wnt, PI3K/AKT, and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) signalling pathways might be involved in STIP‐mediated tumour regulation. Collectively, these results suggest that STIP may be a novel potential diagnostic and therapeutic target for NSCLC.     

VECTORIAL LABELING OF DINOFLAGELLATE CELL SURFACE PROTEINS1

Our objective was to identify cell surface proteins in the dinoflagellate Lingulodinium polyedrum. Proteins on the surface of living cells that had regions exposed to the external medium were labeled with Na¹²⁵I. After partial purification of membrane proteins and analysis by two‐dimensional gel electrophoresis and autoradiography, a protein of roughly 43 kDa was found to have incorporated the radiolabel. This protein was cloned using a combination of protein microsequencing and PCR amplification. The derived protein sequence in the cDNA has a signal peptide at the N‐terminal end of the protein and thus represents the first plasma membrane protein ever reported for a dinoflagellate. The function of the protein is unknown, but its cloning provides a proof of principle for the general use of vectorial labeling in identifying cell surface proteins of marine algae.     

Genome size‐dependent pcna gene copy number in dinoflagellates and molecular evidence of retroposition as a major evolutionary mechanism

Proliferating cell nuclear antigen (PCNA) plays critical roles in eukaryotic DNA replication and replication‐associated processes. It is typically encoded by one or two gene copies (pcna) in eukaryotic genomes. Recently reported higher copy numbers of pcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real‐time PCR quantification, we found a wide range of pcna copy number (2–287 copies) in 11 dinoflagellate species (n = 38), and a strong positive correlation between pcna copy number and genome size (log₁₀–log₁₀ transformed). Intraspecific pcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveying pcna copy numbers in eukaryotes, we observed a genome size threshold at 4 pg DNA, above which more than two pcna copies are found. To examine whether retrotransposition is a mechanism of pcna duplication, we measured the copy number of retroposed pcna, taking advantage of the 22‐nt dinoflagellate‐specific spliced leader (DinoSL) capping the 5′ end of dinoflagellate nuclear‐encoded mRNAs, which would exist in the upstream region of a retroposed gene copy. We found that retroposed pcna copy number increased with total pcna copy number and genome size. These results indicate co‐evolution of dinoflagellate pcna copy number with genome size, and retroposition as a major mechanism of pcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposed pcna as functional genes.     

A novel CDKN2A variant (p16L117P) in a patient with familial and multiple primary melanomas

Germline mutations in CDKN2A (p16) are commonly found in patients with family history of melanoma or personal history of multiple primary melanomas. The p16 tumor suppressor gene regulates cell cycle progression and senescence through binding of cyclin‐dependent kinases (CDK) and also regulates cellular oxidative stress independently of cell cycle control. We identified a germline missense (c.350T>C, p.Leu117Pro) CDKN2A mutation in a patient who had history of four primary melanomas, numerous nevi, and self‐reported family history of melanoma. This particular CDKN2A mutation has not been previously reported in prior large studies of melanoma kindreds or patients with multiple primary melanomas. Compared with wild‐type p16, the p16^L117P mutant largely retained binding capacity for CDK4 and CDK6 but exhibited impaired capacity for repressing cell cycle progression and inducing senescence, while retaining its ability to reduce mitochondrial reactive oxygen species. Structural modeling predicted that the Leu117Pro mutation disrupts a putative adenosine monophosphate (AMP) binding pocket involving residue 117 in the fourth ankyrin domain. Identification of this new likely pathogenic variant extends our understanding of CDKN2A in melanoma susceptibility and implicates AMP as a potential regulator of p16.     

Pyramidodinium atrofuscum gen. et sp. nov. (Dinophyceae), a new marine sand-dwelling coccoid dinoflagellate from tropical waters

A new marine sand‐dwelling coccoid dinoflagellate Pyramidodinium atrofuscum Horiguchi et Sukigara gen. et sp. nov. is described from Jellyfish Lake, Republic of Palau. The dinoflagellate alternates a non‐motile vegetative stage with a motile gymnodinioid stage within its life cycle. The non‐motile stage is dominant in the life cycle and the dinoflagellate reproduces itself by means of the production of two motile cells. The released motile cell swims only for a short period and is directly transformed into the non‐motile cell. The non‐motile cell is sessile, pyramidal in shape, with a single longitudinal ridge and a double transverse ridge. The surface of the cell wall is covered with many processes. The motile cell has a Gymnodinium‐like morphology, but no apical groove is present. An ultrastructural study revealed that the dinoflagellate possesses typical dinoflagellate organelles. Based on the unique morphology of the vegetative non‐motile stage, we propose a new genus Pyramidodinium for this dinoflagellate, with the type species Pyramidodinium atrofuscum Horiguchi et Sukigara, gen. et sp. nov.     

Pyramidodinium spinulosum sp. nov. (Dinophyceae), a sand‐dwelling non‐motile dinoflagellate from the seafloor (36 m deep) off Mageshima Island,Kagoshima, Japan

A new species of benthic marine dinoflagellate, Pyramidodinium spinulosum Horiguchi, Moriya, Pinto & Terada is described from the deep (36 m) seafloor off Mageshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The life cycle of the dinoflagellate consists of a dominant, attached, dome‐shaped, vegetative form and short‐lasting, motile cell. Asexual reproduction takes place by the formation of two motile cells within each non‐motile cell. The released motile cells swim only for a short period and transform directly into the dome‐shaped vegetative form. The duration of the cell cycle varies and can be extremely long, ranging 5–38 days under culture conditions. The non‐motile cell is enclosed by a cell wall and its surface is covered with many (80 – 130) spines of various length. The dinoflagellate is photosynthetic and contains many (more than 50) discoidal chloroplasts. Phylogenetic analysis reveals that the dinoflagellate is closely related to the type species of the genus Pyramidodinium, P. atrofuscum which also possesses a dominant, attached, non‐motile form. However, P. spinulosum can be clearly distinguished from P. atrofuscum by the cell shape (dome‐shaped vs. pyramid‐shaped) and surface ornamentation (spines vs. wart‐like processes) of the non‐motile form. Based on these morphological differences together with molecular evidence, it was concluded that this organism from a deep water sand sample should be described as a second species of the genus Pyramidodinium, P. spinulosum.     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.     

Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation

Leishmania mexicana has a large family of cyclin‐dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2‐related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co‐expression of a CRK3 transgene during rapamycin‐induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3^T178E mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.     

CDK5 protects from caspase‐induced Ataxin‐3 cleavage and neurodegeneration

Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease‐specific proteins. In case of SCA3, mutation of Ataxin‐3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin‐dependent kinase‐5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase‐dependent Ataxin‐3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease‐propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin‐3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin‐3 and other polyglutamine proteins.