Vytaute Starkuviene

The genome sequence of the extreme thermophile Thermus thermophilus

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.

A microscope-based screening platform for large-scale functional protein analysis in intact cells.

A modular microscope‐based screening platform, with applications in large‐scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome‐wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large‐scale, cell‐based functional proteomic projects.

The potential of high-content high-throughput microscopy in drug discovery

Fluorescence microscopy is a powerful method to study protein function in its natural habitat, the living cell. With the availability of the green fluorescent protein and its spectral variants, almost any gene of interest can be fluorescently labelled in living cells opening the possibility to study protein localization, dynamics and interactions. The emergence of automated cellular systems allows rapid visualization of large groups of cells and phenotypic analysis in a quantitative manner. Here, we discuss recent advances in high‐content high‐throughput microscopy and its potential application to several steps of the drug discovery process.

Hypothalamic miR-103 Protects from Hyperphagic Obesity in Mice

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Golgi apparatus studied in vitreous sections

Cryo‐electron microscopy of vitrified specimen is the method of choice to explore cellular ultrastructure at high resolution as close as possible to the native state and environment. In this study, we investigated the Golgi apparatus – the main organelle of the secretory pathway. Cultured mammalian cells were fixed by high‐pressure freezing, sectioned in vitreous ice and subjected to cryo‐electron microscopy and cryo‐electron tomography. Although the overall morphology of Golgi stacks was comparable to well prepared and plastic‐embedded samples, in detail we reached much higher resolution in terms of distinction between biological structures based on their native density. On cisternal buds and peri‐Golgi vesicles – some associated with microtubules – we detected two different subtypes of COPI coats: (1) a homogenous coat and (2) an inhomogeneous spiky coat, providing an 8–9 nm regularity, clearly distinct from clathrin coat. Next, we monitored the secretion of cargo, namely, procollagen I, through the Golgi complex. Temporally correlated with fluorescence microscopy, we performed three‐dimensional cryo‐electron tomography analysis and detected Golgi cisternae enlarged to saccules, containing cargo and showing inter‐cisternal connections. Our work provides a first step towards the high‐resolution description of the secretory pathway in native vitrified samples and describes the challenges associated with this attempt.

miR-17-5p regulates endocytic trafficking through targeting TBC1D2/Armus.

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.

Correlative light microscopy for high-content screening.

High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for scalable experiments. The workflow consists of a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently re-localizing those cells with micrometer precision for confocal and super-resolution imaging. As a case study, we visualized and quantified cis- and trans-Golgi markers at increasing resolution.

Differential Requirements for ts-O45-G and Procollagen Biosynthetic Transport

Emerging experimental evidence favours the existence of cargo sorting occurring upon the endoplasmic reticulum (ER) exit. Recent studies revealed that, in contrast to the conventional secretory marker ts‐O45‐G, procollagen (PC I) exits the ER at sites not coated with coat protein II and is transported to the Golgi complex in carriers devoid of coat protein I. Here, we investigated whether PC I trafficking requires a different molecular machinery in comparison with the ts‐O45‐G. By combining colocalization of the cargoes with endogenous markers, downregulation of transport machinery by RNA interference and knock‐ins by complementary DNA over‐expression, we provide strong evidence that PC I and ts‐O45‐G have common but also different molecular requirements during pre‐ and post‐Golgi trafficking events.

Transfected cell microarrays: an efficient tool for high-throughput functional analysis

Transfected cell microarrays are considered to be a breakthrough methodology for high-throughput and high-content functional genomics. Here, recent advances in the cell microarray field are reviewed, along with its potential to increase the speed of determining gene function. These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level.