Past Research

Plasmodium falciparum serves as a suitable model organism for the study of these relocalisation events as sub-nuclear localisation of var genes has been shown to be related to the transcriptional status of the genes. In addition, several other virulence factor genes have been demonstrated to be co-regulated through out the parasitic life cycle.

Use of single molecule FISH and other super resolution methods to probe for transcripts of these loci is expected to demonstrate whether or not such co-regulated genes are co-localized to a sub-nuclear transcription factory during active expression. The presence or exclusion of transcriptionally silent loci at such transcriptional foci may also reveal the involvement of other regulatory mechanisms in gene expression. Such results are hoped to elucidate the pertinence of the transcription factory theory in P. falciparum pathogenicity.

The Drosophila melanogaster oocyte offers an ideal model system to study mRNP interactions with proteins involved in transport. Though genetic data has identified hnRNP and other proteins to be key during the transport process, for many their specific roles remain unknown. In the oocyte, the composition of the mRNP particle during transcription, transport, and eventual translation, ultimately determines the successful formation of the anterior-posterior and dorso-ventral body axes. In the case of oskar mRNA, which specifies the inchoate pole plasm as well as abdominal and germline determinants, its transport, spatial positioning and eventual translation require the occurrence of precise nuclear and cytoplasmic events .

Several proteins which repress translation of osk during its transport have also been identified, acting to aggregate osk transcripts during the transport process, a process that is likely conserved for many other mRNAs across species. More trans-acting factors are known for oskar than for any other localized transcript. How trans-acting proteins contribute to oskar transport is poorly understood. Understanding the spatio-temporal relationship between oskar and these trans-acting proteins necessitates approaches permitting the direct observation of such dynamic events in vivo. Advances that facilitate the tracking and covisualization of trans-acting factors with individual mRNP particles in real-time, permit the precise description of transport events and how they are influenced by these factors. They assist in deciphering the highly orchestrated behavior between mRNA and proteins, several questions, remain: (i) the biophysical properties of the transport from the point of transcription to eventual localization (ii) the temporal requirement of specific nuclear or cytoplasmic proteins, and (iii) the principal nature and composition of the oskar particle during its transport.

Though fluorescent proteins have been used to label proteins implicated in the transport of oskar, a central challenge has remained; the inability to image the key player in the process, the mRNA. We will tackle these problems systematically introducing new methods in imaging, probe development and analytical tools that will assist in answering each of these questions. This will permit an unprecedented spatio-temporal resolution to be achieved and elucidate basic biological mechanisms that are involved in translational control and long-range transport. First we will utilize our expertise in super resolution microscopy, specifically in PALM/STORM to achieve 3D resolutions of oskar mRNA in the drosophila oocyte that will be in the tens of nanometers and of a similar scale in the Z axis. Secondly, we build on our developments of a new class of molecular beacons that are genetically encodable. These probes will be delivered using a transgene and expressed via constitutive or inducible promoters and be able to report the presence of a specific RNA sequence purely from their binding with the target sequence. Third, we will engineer proteins that interact with oskar mRNA with fluorescent labels that make them compatible with PALM/STORM. These approaches will be used where possible in live cells. Fixed cells will be used to obtain high resolution maps comparable to electron microscopy but using PALM/STORM.

Together these technical advancements will breach the largest barrier to a deeper biological insight of the mechanisms implicated in long range mRNA transport process. Coupled with new algorithmic and analytical tools, we will be able to develop new models of how oskar mRNA undergoes its highly choreographed “dance” with numerous differing proteins over the length of the oocyte. We will investigate how well conserved these “actors” are in other long range mRNA transport milieu, such as in migratory cells types. By using siRNA approaches we will target specific proteins in other model systems that have been shown to be involved in RNA transport in fly, and establish convergent and divergent conservation of transport mechanisms. Thus our ambition is to visualize oskar mRNA from its birth in the nucleus and the proteins it interacts with from then on until its localization at the posterior pole of the oocyte and the proteins that facilitate its translation.

The development of new methodologies for in vivo super-resolution spatio-temporal localization and tracking of RNA transcripts constitutes an appealing target for the study of dynamics of gene expression modulation and sub-nuclear transcription complex assembly mapping. The application of these methods will not only unfold new information on some of the mechanisms involved in gene expression but will also allow us to relate how the relocalization of non-constitutively expressed genes such as the GAL family modulate RNA transcription depending on their nuclear positioning.

The overall goal of this project it to understand the spatio-temporal behavior and relation between the gene loci position, gene cascade activation and associated RNA transcripts inside live-cells using amongst others the GAL gene family as a model developing novel technologies that help us achieve this objectives. Current microscopy approaches, in live cells, have focused on using statistical mapping to understand genome organization and fluorescent microscopy techniques to visualize chromosome repositioning within the nucleus. These approaches have fallen short when it has come to the study of gene expression and transcription resorting to fluorescent in situ hybridization (FISH) to take snap shots of these events in fixed cells, albeit at times with single molecule resolution.

Gene expression is a fundamentally stochastic process, and in some instances appears purposefully so. This makes the construction and modeling of gene networks especially difficult. Transcription is thought to occur in bursts with little known as to their source. One may be the existence of pre-initiation complexes formed on the promoter that may permit multiple rounds of RNA Polymerase II transcription. Such complexes could form in so-called “transcription factories” where active genes would be recruited. Some evidence for transcription factories does exist though at best 3 differing transcripts have been imaged in putative factories. Super resolution microscopy could be most informative in this field if one could achieve the ability to image the majority of the species of transcripts at single molecule level within these factories allowing us to understand the contents of the transcriptional machinery in real time. Techniques such as Photo-activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) are taking the first steps for evolving into the live-cell imaging domain setting up the path for multi-color 4D (3D plus time) quantitative imaging of single molecules at a sub-diffraction resolution (under 100 nm) in living cells.

At the current state of technology a few limitations exist in these techniques that undermine their use in the study of the sub-nuclear architecture and dynamics: the lack of DNA and RNA probes compatible with the stochastic photo-switching dynamics required by the techniques, the high degree of cell damage caused by laser cycling during imaging and fast-dynamic imaging suitable with the time resolution of the gene modulating processes occurring within the cell nucleus. By taking as a model published work on the relation of gene networks and gene territories we intend to tackle each one of these restrains creating new answers on the dynamic behavior and regulation of gene expression through the use of light microscopy.