Publications

Host Gene Targets For Novel Influenza Therapies Elucidated By High Throughput Rna Interference Screens

Victoria A. Meliopoulos, Lauren E. Andersen, Katherine F. Birrer, Kaylene J. Simpson, John W. Lowenthal, Andrew G. D. Bean, John Stambas, Cameron R. Stewart, S. Mark Tompkins, Victor W. van Beusechem, Iain Fraser, Musa Mhlanga, Samantha Barichievy, Queta

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.

Published in: FASEB, 2012, (1),

Involved members of the lab: Samantha Barichievy, Musa Mhlanga

Transcriptome Dysregulation By Anthrax Lethal Toxin Plays A Key Role In Induction Of Human Endothelial Cell Cytotoxicity

Monica Rolando, Caroline Stefani, Gilles Flatau, Patrick Auberger, Amel Mettouchi, Musa Mhlanga, Ulf Rapp, Antoine Galmiche and Emmanuel Lemichez

We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothe- lial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and down- regulation of XIAP both participate in LT-induced caspase-3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re-expression in LT-intoxicated cells blocks caspase-3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock-down of cortactin and rhophilin-2 under conditions of calponin-1 expression defines the minimal set of genes regulated by LT for actin cable formation. Together our data establish that the modulation of the cell transcriptome by LT plays a key role in triggering human endothelial cell toxicity.

Published in: Cellular Microbiology , 2010,

Palm And Storm: What Hides Beyond The Rayleigh Limit?

Ricardo Henriques and Musa M. Mhlanga

Super-resolution imaging allows the imaging of fluorescently labeled probes at a resolution of just tens of nanometers, surpassing classic light microscopy by at least one order of magnitude. Re- cent advances such as the development of photo-switchable fluorophores, high-sensitivity micro- scopes and single particle localization algorithms make super-resolution imaging rapidly accessi- ble to the wider life sciences research community. As we take our first steps in deciphering the roles and behaviors of individual molecules inside their living cellular environment, a new world of research opportunities beckons. Here we discuss some of the latest developments achieved with these techniques and emerging areas where super-resolution will give fundamental new “eye” sight to cell biology.

Published in: Biotechnology Journal, 2009, 4 846-857

In Vivo Colocalisation Of Oskar M Rna And Trans Acting Proteins Revealed By Quantitative Imaging Of The Drosophila Oocyte

Musa M. Mhlanga, Diana P. Bratu, Auguste Genovesio, Agata Rybarska, Nicolas Chenouard, Ulf Nehrbass, Jean-Christophe Olivo-Marin

Efficient mRNA transport in eukaryotes requires highly orchestrated relationships between nuclear and cytoplasmic proteins. For oskar mRNA, the Drosophila posterior determinant, these spatio-temporal requirements remain opaque during its multi-step transport process. By in vivo covisualization of oskar mRNA with Staufen, its putative trafficking protein, we find oskar mRNA to be present in particles distinct from Staufen for part of its transport. oskar mRNA stably associated with Staufen near the posterior pole. We observe oskar mRNA to oligomerize as hundreds of copies forming large particles which are necessary for its long range transport and localization. We show the formation of these particles occurs in the nurse cell nucleus in an Hrp48-dependent manner. We present a more refined model of oskar mRNA transport in the Drosophila oocyte.

Published in: PLOS One, 2009, 4 (7), e6241

Single Fluorescent Molecule Tracking In Live Cells

Ghislain Cabal, Jost Enninga and Musa M. Mhlanga

Biological macromolecules, such as DNA, RNA, or proteins, display a distinct motility inside living cells that is related to their functional status. Understanding the motility of individual biological macromolecules under spe- cific physiological situations gives important clues regarding the molecular role of these macromolecules. Therefore, it is important to track individual biological macromolecules in the context of living cells. Often, biological macromolecules are constituents of larger multimacromolecular assemblies that can be denomi- nated as macromolecular particles. During the last few years, various approaches based on fluorescent imaging have been developed to allow the tracking of single particles inside living cells. In this chapter, we present several such approaches to track individual (1) DNA loci, (2) ribonucleoprotein particles, or (3) membrane proteins. Particularly, we focus on the practical aspects of these approaches, allow- ing the reader to adopt the methods presented to their specific scientific problems. The methods presented are based on different principles: Tracking of chromosomal DNA loci is achieved via operator/repressor recognition using fluorescent repressor molecules. Individual ribonuleoprotein particles can be followed with small oligonucleotide sensor molecules called molecular beacons. Individual membrane proteins can be tracked via their specific labeling with antibody–quantum dot conjugates. Subsequently, we outline the principles of single particle tracking algorithms that have been developed in the field of bioinformatics, and that are crucial for a rapid, unbiased analysis of the tracked particles.

Published in: Imaging Cellular and Molecular Biological Functions, 2007, 230 -

T Rna Linked Molecular Beacons For Imaging M Rn As In The Cytoplasm Of Living Cells

Musa M. Mhlanga, Diana Y. Vargas, Cindy W. Fung, Fred Russell Kramer and Sanjay Tyagi

When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We show that when fluorescently labeled tRNAs, tRNAs with extensions at their 50 end, or chimeric molecules in which a molecular beacon possessing a 20-O-methyl- ribonucleotide backbone is linked to a tRNA, are injected into the nucleus of HeLa cells, they are exported into the cytoplasm. When these constructs are introduced into the cytoplasm, they remain cyto- plasmic. These constructs allow the distribution of both the general mRNA population and specific mRNAs to be imaged in living cells. This strategy should also be useful for enhancing the efficacy of antisense oligonucleotides by keeping them in the cytoplasm. Our observations show that the fidelity of the tRNA export system is relaxed for unnatural tRNA variants when they are introduced into the nucleus in large amounts.

Published in: Nucleic Acids Research, 2005, 33 (6), 1902-1912

Using Molecular Beacons To Detect Single Nucleotide Polymorphisms With Real Time Pcr

Musa M. Mhlanga, and Lovisa Malmberg

Detection of single-nucleotide polymorphisms (SNPs) in high- throughput studies promises to be an expanding field of molecular medicine in the near future. Highly specific, simple, and accessible methods are needed to meet the rigorous requirements of single- nucleotide detection needed in pharmacogenomic studies, linkage analysis, and the detection of pathogens. Molecular beacons pres- ent such a solution for the high-throughput screening of SNPs in homogenous assays using the polymerase chain reaction (PCR). Molecular beacons are probes that fluoresce on hybridization to their perfectly complementary targets. In recent years they have emerged as a leading genetic analysis tool in a wide range of contexts from quantification of RNA transcripts, to probes on mi- croarrays, to single-nucleotide polymorphism detection. The major- ity of these methods use PCR to obtain sufficient amounts of sample to analyze. The use of molecular beacons with other ampli- fication schemes has been reliably demonstrated, though PCR remains the method of choice. Here we discuss and present how to design and use molecular beacons to achieve reliable SNP genotyping and allele discrimination in real-time PCR. In addition, we provide a new means of analyzing data outputs from such real-time PCR assays that compensates for differences between sample condition, assay conditions, variations in fluorescent sig- nal, and amplification efficiency. The mechanisms by which molec- ular beacons are able to have extraordinary specificity are also presented.

Published in: Methods, 2001, 25 463-471