The Conjusome-A Transient Organelle Linking Genome Rearrangements in the Parental and Developing Macronuclei.

The conjusome plays an important role in the conjugation events that occur in Tetrahymena thermophila. The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the PDD1 allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, Stylonichia. In general, conjusomes have features that resemble germ line P-granules.

Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium.

BACKGROUND: Cells show directed migration response to electric signals, namely electrotaxis or galvanotaxis. PI3K and PTEN jointly play counterbalancing roles in this event via a bilateral regulation of PIP3 signaling. PI3K has been proved essential in anterior signaling of electrotaxing cells, whilst the role of PTEN remains elusive. METHODS: Dictyostelium cells with different genetic backgrounds were treated with direct current electric signals to investigate the genetic regulation of electrotaxis. RESULTS: We demonstrated that electric signals promoted PTEN phosphatase activity and asymmetrical translocation to the posterior plasma membrane of the electrotaxing cells. Electric stimulation produced a similar but delayed rear redistribution of myosin II, immediately before electrotaxis started. Actin polymerization is required for the asymmetric membrane translocation of PTEN and myosin. PTEN signaling is also responsible for the asymmetric anterior redistribution of PIP3/F-actin, and a biased redistribution of pseudopod protrusion in the forwarding direction of electrotaxing cells. CONCLUSIONS: PTEN controls electrotaxis by coordinately regulating asymmetric redistribution of myosin to the posterior, and PIP3/F-actin to the anterior region of the directed migration cells.

AKR1C3 mediates pan-AR antagonist resistance in castration-resistant prostate cancer.

BACKGROUND: Antiandrogens are effective therapies that block androgen receptor (AR) transactivation and signaling in over 50% of castration-resistant prostate cancer (CRPC) patients. However, an estimated 30% of responders will develop resistance to these therapies within 2 years. JNJ-pan-AR is a broad-spectrum AR antagonist that inhibits wild-type AR as well as several mutated versions of AR that have emerged in patients on chronic antiandrogen treatment. In this work, we aimed to identify the potential underlying mechanisms of resistance that may result from chronic JNJ-pan-AR treatment. METHODS: The LNCaP JNJR prostate cancer subline was developed by chronically exposing LNCaP parental cells to JNJ-pan-AR. Transcriptomic and proteomic profiling was performed to identify potential drivers and/or biomarkers of the resistant phenotype. RESULTS: Several enzymes critical to intratumoral androgen biosynthesis, Aldo-keto reductase family 1 member C3 (AKR1C3), UGT2B15, and UGT2B17 were identified as potential upstream regulators of the JNJ-pan-AR resistant cells. While we confirmed the overexpression of all three enzymes in the resistant cells only AKR1C3 expression played a functional role in driving JNJ-pan-AR resistance. We also discovered that AKR1C3 regulates UGT2B15 and UGT2B17 expression in JNJ-pan-AR resistant cells. CONCLUSIONS: This study supports the rationale to further investigate the benefits of AKR1C3 inhibition in combination with antiandrogens to prevent CRPC disease progression.

Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 µM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M-1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.

Live-cell imaging of small nucleolar RNA tagged with the broccoli aptamer in yeast.

The development of the RNA 'vegetable' aptamers, Spinach and Broccoli, has simplified RNA imaging, especially in live cells. These RNA aptamers interact with a fluorophore (DFHBI or DFHBI-1T) to produce a green fluorescence signal. Although used in mammalian and Escherichia coli cells, the use of these aptamers in yeast has been limited. Here we describe how the Saccharomyces cerevisiae snoRNA, snR30, was tagged with the Spinach or the Broccoli aptamers and observed in live cells. The ability to observe aptamer fluorescence in polyacrylamide gels stained with a fluorophore or with a microplate reader can ease preliminary screening of the aptamers in different RNA scaffolds. In snR30 a tandem repeat of the Broccoli aptamer produced the best signal in vitro. Multiple factors in cell preparation were vital for obtaining a good fluorescence signal. These factors included the clearance of the native unmodified snR30, the amount and length of dye incubation and the rinsing of cells. In cells, the aptamers did not interfere with the structure or essential function of snR30, as the tagged RNA localized to the nucleolus and directed processing of ribosomal RNA in yeast. High-resolution images of the tagged snoRNA were obtained with live cells immobilized by a microcompressor.

Real-time visualization of chromatin modification in isolated nuclei.

Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.

Recent advances in imaging subcellular processes.

Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology.

A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells.

Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.

On-chip open microfluidic devices for chemotaxis studies.

Microfluidic devices can provide unique control over both the chemoattractant gradient and the migration environment of the cells. Our work incorporates laser-machined micro and nanofluidic channels into bulk fused silica and cover slip-sized silica wafers. We have designed "open" chemotaxis devices that produce passive chemoattractant gradients without an external micropipette system. Since the migration area is unobstructed, cells can be easily loaded and strategically placed into the devices with a standard micropipette. The reusable monolithic glass devices have integral ports that can generate multiple gradients in a single experiment. We also used cover slip microfluidics for chemotaxis assays. Passive gradients elicited from these cover slips could be readily adapted for high throughput chemotaxis assays.We have also demonstrated for the first time that cells can be recruited into cover slip ports eliciting passive chemoattractant gradients. This proves, in principle, that intravital cover slip configurations could deliver controlled amounts of drugs, chemicals, or pathogens as well as recruit cells for proteomic or histological analysis in living animals while under microscopic observation. Intravital cover slip fluidics will create a new paradigm for in vivo observation of biological processes.

Open access microfluidic device for the study of cell migration during chemotaxis.

Cells sense and interpret chemical gradients, and respond by localized responses that lead to directed migration. An open microfluidic device (OMD) was developed to provide quantitative information on both the gradient and morphological changes that occurred as cells crawled through various microfabricated channels. This device overcame problems that many current devices have been plagued with, such as complicated cell loading, media evaporation and channel blockage by air bubbles. We used a micropipette to set up stable gradients formed by passive diffusion and thus avoided confounding cellular responses produced by shear forces. Two versions of the OMD are reported here: one device that has channels with widths of 6, 8, 10 and 12 µm, while the other has two large 100 µm channels to minimize cellular interaction with lateral walls. These experiments compared the migration rates and qualitative behavior of Dictyostelium discoideum cells responding to measurable cAMP and folic acid gradients in small and large channels. We report on the influence that polarity has on a cell's ability to migrate when confined in a channel. Polarized cells that migrated to cAMP were significantly faster than the unpolarized cells that crawled toward folic acid. Unpolarized cells in wide channels often strayed off course, yet migrated faster than unpolarized cells in confined channels. Cells in channels farthest from the micropipette migrated through the channels at rates similar to cells in channels with higher concentrations, suggesting that cell speed was independent of mean concentration. Lastly, it was found that the polarized cells could easily change migration direction even when only the leading edge of the cell was exposed to a lateral gradient.

Directional sensing during chemotaxis.

Cells have the innate ability to sense and move towards a variety of chemoattractants. We investigate the pathways by which cells sense and respond to chemoattractant gradients. We focus on the model system Dictyostelium and compare our understanding of chemotaxis in this system with recent advances made using neutrophils and other mammalian cell types, which share many molecular components and signaling pathways with Dictyostelium. This review also examines models that have been proposed to explain how cells are able to respond to small differences in ligand concentrations between the anterior leading edge and posterior of the cell. In addition, we highlight the overlapping functions of many signaling components in diverse processes beyond chemotaxis, including random cell motility and cell division.