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. Recent advances such as the development of photo-switchable fluorophores, high-sensitivity microscopes and single particle localization algorithms make super-resolution imaging rapidly accessible to the wider life sciences research community. All are a subject of on going research in our laboratory. We have two instruments to perform this work, one of which is dedicated just for super-resolution microscopy work.
Gene expression is an essential process for living organisms, and is regulated at many levels. In recent years the importance of the role of nuclear architecture in such regulation has become increasingly evident, although many facets of nuclear spatial regulation of transcription remain unclear. The expression status of genes has been shown to be related to their sub-nuclear position within the eukaryotic nucleus in many organisms. This relocation of gene loci as they are activated or inactivated has been postulated to be the locus moving to and from hypothesized “transcription factories”.
Subcellular localization of messenger RNA (mRNA) provides a mechanism for the precise control over where and when protein products are synthesized and operate, providing spatial and temporal regulation of gene activity. Asymmetric distribution of mRNAs in the cytoplasm was first visualized using in situ hybridization techniques in the 1980s. Research in this field has demonstrated that mRNA localization is more common than previosuly assumed and as such many key players in mRNA localization have been identified. However, future challenges involve determining a detailed molecular understanding of the interactions that govern the localization of mRNAs and the transport of mRNAs to various subcellular compartments.
Currently nearly 40 million people worldwide are infected with HIV and despite 25 years since its discovery, science is only beginning to understand the myriad complex interactions between this virus and its human host. There is considerable heterogeneity in the HIV genome and numerous subtypes have been described since the epidemic began. In addition, recombination events between different subtypes have led to the emergence of ‘circulating recombinant forms’ (CRFs) of HIV. Subtype C accounts for 50% of all new HIV infections in sub-Saharan Africa but different subtypes or CRFs predominate in geographically distinct regions. North America, Canada and much of western Europe displays subtype B HIV infections, South America predominantly has B/F recombinant subtypes and South-East Asia (excluding China) has AE/B CRF infections. Recent advances in high-throughput microscopy coupled with genome-wide screening have uncovered hundreds of human host factors that are required by HIV during various stages of infection. Importantly, while these studies have provided valuable insight into HIV-host interactions, the assays were all completed using laboratory-adapted HIV strains from a subtype B background only. The use of these HIV molecular clones may have biased the selection of host factors to those ‘generic’ to HIV infection. Alternatively, only subtype B-specific host factors may have been identified. To dissect out potential differences in host factors required for infection with discrete HIV subtypes, we will be conducting genome-wide RNAi screens using fluorescence microscopy to identify positive hits.
In biology several important processes occur at a spatial dimension currently beyond the reaches of conventional light microscopy. The majority of the molecular players implicated in gene expression and its regulation are outside the resolution grasp of most optical microscopy techniques. These include the study of RNA transcription, nuclear architecture and chromosomal dynamics associated with the induction or repression of gene expression within the eukaryotic cell nucleus. In this project, the aim is to confront biological questions such as what is the connection between the spatio-temporal location of RNA transcription, the sub-nuclear gene territories and how it is co-regulated by the activity of gene networks as well as “noise” or stochasticity in gene expression. With this goal we will take advantage of the most recent techniques in super-resolution far-field light microscopy and develop novel methods to achieve live-cell sub-nuclear quantitative map of some of the key factors modulating RNA transcription and gene expression.