CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca2+-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.     

Calmodulin bound to stress fibers but not microtubules involves regulation of cell morphology and motility

Calmodulin (CaM) is a major cytoplasmic calcium receptor that performs multiple functions including cell motility. To investigate the mechanism of the regulation of CaM on cell morphology and motility, first we checked the distribution of CaM in the living cells using GFP-CaM as an indicator. We found that GFP-CaM showed a fiber-like distribution pattern in the cytosol of living Potorous tridactylis kidney (PtK2) cells but not in living HeLa cells. The endogenous CaM in heavily permeabilized HeLa was also found to display a fiber-like distribution pattern. Further examination showed that the distribution pattern of GFP-CaM was same as that of stress fibers, but not microtubules. Co-immunoprecipitation also showed that CaM can interact with actin directly or indirectly. The microinjection of trp peptide, a specific inhibitor of CaM, attenuated the polymerization of stress fibers and induced the alteration of cell morphology. A wound-healing assay and a single cell tracking experiment showed that CaM in PtK2 cells could increase cell motility. The data we have got from living cells suggested that CaM affect cell morphology and motility through binding to stress fibers and regulate f-actin polymerization.     

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis

Calmodulin (CaM) potentiates Ca(2+)-dependent signaling pathways in both the cytoplasm and nucleus. We have investigated the mechanism of CaM nuclear transport using tissue culture cell microinjection and a permeabilized cell import assay. The inhibition of CaM import by the translocation inhibitor wheat germ agglutinin (WGA) and by chilling, indicates that CaM import is facilitated, but because ATP depletion does not affect CaM import, the mechanism does not appear to be active. Chilling and WGA arrest persist in ATP-depleted cells, indicating that CaM is not retained in the cytoplasm by an ATP-dependent mechanism. In permeabilized cells, both Ca(2+)-CaM and Ca(2+)-free CaM are sensitive to extract-dependent WGA and chilling import inhibition. Titration experiments in microinjected and permeabilized cells indicate that a saturable cytosolic factor(s) mediates chilling and WGA arrest.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

Simple cost-effective larval injection method for dsRNA delivery to induce RNAi response in Helicoverpa armigera (Hübner)

Microinjection is considered as an effective method for dsRNA delivery in insects. It also facilitates the delivery of a precise quantity of dsRNA in the host insect, inducing an efficient RNAi response. However, the microinjection method needs prior optimization of several parameters like concentration of dsRNA, site of injection, developmental stage of insect etc. for inducing effective RNAi response. Moreover, sophisticated microinjection devices are largely expensive with high maintenance cost. The Old-World bollworm, Helicoverpa armigera (Hübner) is known to be a detrimental polyphagous pest with widespread infestations across the globe. In the present study, we demonstrate a low-cost and effective dsRNA delivery method for inducing RNAi response in H. armigera with the aid of basic insulin injection syringe and fabricated micropipette tip. In order to validate the RNAi response following dsRNA injection, we have selected three key genes from the chitin biosynthesis pathway of the insect. Besides these, argonaute 1 (ago1) was also used as an indicator gene for dsRNA-mediated RNAi induction. Delivery of dsRNA using injection with insulin syringe caused significant upregulation of the ago1 gene in the insect irrespective of any of the three target genes concerned viz. HaNAGK (3.9 fold; p < 0.001), HaAGM (6.3 fold; p < 0.001) and HaUAP (5.9 fold; p < 0.01) respectively, as compared to control injected with nuclease-free water. The dsRNA-injected insects showed aberrant developmental symptoms typical of impeded chitin synthesis, eventually leading to anomalous ecdysis with substantial mortality (up to 69.04%), as compared to controls. The described protocol reduces insect injury, enabling easy restraining of larva and quick execution of dsRNA injection with efficient RNAi response.     

Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system

Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

First Knockdown Gene Expression in Bat (Hipposideros armiger) Brain Mediated by Lentivirus

Lentivirus-mediated RNA interference (RNAi) is a potent experimental tool for investigating gene functions in vitro and in vivo. It has advantages that transgenic technology lacks. However, in vivo applications are difficult to apply in the central nervous system of non-model organisms due to the lack of a standard brain atlas and genetic information. Here, we report the development of an in vivo gene delivery system used in bat brain tissue for the first time, based on lentivirus (LV) vectors expressing short hairpin RNA (shRNA) targeting Hipposideros armiger forkhead box P2 (FoxP2). In vitro transfection into HEK 293T cell with the vector bearing the cassettes encoding FoxP2 shRNA verified the knockdown efficiency. Pseudovirus particles were administered via stereotactic intracerebral microinjection into the anterior cingulate cortex of H. armiger. FoxP2 is of major interest because of its role in sensorimotor coordination and probably in echolocation. Subsequent in situ hybridization validated the in vivo silencing of the target gene. This report demonstrates that LV-mediated expression of RNAi could achieve effective gene silencing in bats, a non-model organism, and will assist in elucidating the functions of bat genes.     

RNA interference in Caenorhabditis elegans: uptake, mechanism, and regulation

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.     

A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE protein HvSNAP34 in three types of durable, race-nonspecific resistance was observed.     

Calmodulin transmitted through gap junctions stimulates endocytic incorporation of yolk precursors in insect oocytes

In ovarian follicles of Oncopeltus fasciatus, and of Xylocopa virginica, calmodulin (CaM) of epithelial cell origin is required by oocytes for endocytic uptake of yolk precursor molecules. Furthermore, this 17-19 kDa protein is normally transported to the oocytes via gap junctions. Downregulation of gap junctions by treatment with 1 mM octanol or separation of the epithelial cells from their oocytes terminated precursor uptake, and this activity could be rescued by microinjection of 60 microM CaM, but not by injections of incubation medium, nor solutions of other molecular species tested. That endogenous CaM is required was confirmed by incubating otherwise untreated follicles in physiological salt solution (PSS) containing either calmidazolium or W-7, both known antagonists of CaM. By radioimmunoprecipitation, we show that the epithelial cells surrounding an oocyte synthesized 15 times as much calmodulin as did the oocytes they encircled. Neither octanol-treated follicles nor denuded oocytes incubated in medium containing calmodulin were able to resume endocytosis, arguing against an extracellular route. However, fluorescently labeled calmodulin microinjected into oocytes is shown to have crossed through gap junctions, making epithelial cells distinctly fluorescent.     

Small interfering RNA-mediated gene silencing in T lymphocytes

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.     

Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis

The unicellular flagellate Euglena gracilis shows a negative gravitactic behavior. This is based on physiological mechanisms which in the past have been indirectly assessed. Meanwhile, it was possible to isolate genes involved in the signal transduction chain of gravitaxis. The DNA sequences of five calmodulins were found in Euglena, one of which was only known in its protein structure (CaM.1); the other four are new. The biosynthesis of the corresponding proteins of CaM.1–CaM.5 was inhibited by means of RNA interference to determine their involvement in the gravitactic signal transduction chain. RNAi of CaM.1 inhibits free swimming of the cells and pronounced cell-form aberrations. The division of cells was also hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Blockage of CaM.3 to CaM. 5 did not impair gravitaxis. In contrast, the blockage of CaM.2 has only a transient and not pronounced influence on motility and cell form, but leads to a total loss of gravitactic orientation for more than 30 days. This indicates that CaM.2 is an element in the signal transduction chain of gravitaxis in E. gracilis. The results are discussed with regard to the current working model of gravitaxis in E. gracilis.     

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.     

Delivery of RNA Interference

Over the last few years, RNA Interference (RNAi), a naturally occurring mechanism of gene regulation conserved in plant and mammalian cells, has opened numerous novel opportunities for basic research across the field of biology. While RNAi has helped accelerate discovery and understanding of gene functions, it also has great potential as a therapeutic and potentially prophylactic modality. Challenging diseases failing conventional therapeutics could become treatable by specific silencing of key pathogenic genes. More specifically, therapeutic targets previously deemed “undruggable” by small molecules, are now coming within reach of RNAi based therapy. For RNAi to be effective and elicit gene silencing response, the double-stranded RNA molecules must be delivered to the target cell. Unfortunately, delivery of these RNA duplexes has been challenging, halting rapid development of RNAi-based therapies. In this review we present current advancements in the field of siRNA delivery methods, including the pros and cons of each method.