SFB796 - Joint project 02

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Begomoviral transport proteins in human cells

 

Project summary

The LBR:GFP marker was used in transient expression experiments in tobacco plants to visualize the plant nuclear envelope (PNE). The LBR:GFP marker consists of the first 238 amino acids of the human lamin B receptor (LBR), fused to the green fluorescent protein (GFP). GFP fluorescence accumulated only at the PNE of leaf epidermal cells, although plants do not possess any lamin proteins. Abutilon mosaic virus (AbMV; genus begomovirus) induce alteration of plant nuclear architecture which can be monitored by the LBR:GFP marker. Co-expression experiments have shown that the two transport proteins of AbMV (MP and NSP) are responsible for those alterations, e.g. intense necking, vesicle formation and budding. Successful LBR:GFP targeting anchoring in PNE suggests, that underlying mechanism may be common in plants and humans. We predicted that NSP and MP also re-localize LBR in the human cell and therefore we addressed the question and investigate if the begomoviral transport proteins also have a similar impact on the human nuclear envelope.

We have visualized the nuclear shuttling of NSP by microscopy, using a transient expression assay in plant epidermis cells. Other studies established that this mechanism of nuclear shuttling is not only conserved in plants, but as well as in human cells and yeast. Preliminary results have shown that NSP transiently expressed in HeLa cells localized to the same compartment as in plant cells, into the nucleus. To our surprise, MP appeared to be associated with the microtubules network and intermediate filaments in HeLa cells whereas in plants it is targeted to the plasma membrane. Interestingly, the microtubules network showed a re-organized and abnormal phenotype in the presence of MP, indicating an interference with cytokinesis. To determine which domains of AbMV-MP are necessary and sufficient for its capacity to associate at microtubules, we constructed a set of deletion mutants. Only the deletion mutant MP aa 1-180 exhibited the same phenotype as the wild type MP, leading to the consumption that only the oligomerization domain of MP is dispensable for microtubule targeting. We also ruled out that the fluorescent tag is responsible for this phenomenon or the transfected cell type and could show that MPs from other geminiviruses induce the same phenotype, whereas MPs from non-geminiviruses do not induced abnormal bundled microtubules.  




Figure: Rhodamine phalloidin (red) and Dapi (blue) stained HeLa cell transiently expressing AbMV MP fused to eYFP (yellow).

In the future, we will test MP microtubules stabilizing ability and proliferation inhibitor and investigate the underlying mechanism. In addition, we want to investigate whether NSP and MP have an impact on the human lamin layer. We have identified the binding partner(s) of the LBR:GFP marker in plant nuclei and now we want to gain further insight into the plant nucleus inner membrane architecture.  

The project will not only broaden our understanding in the plant nucleus inner membrane architecture and interactome, but also reveal conserved structural features of the plant and human nucleus. Despite the fact that plant NE has a unique composition, we address the question if NE protein targeting, retention and anchoring mechanism may be common in animal and plant cells. The proposed research will also clarify the observed remodeling effect of MP on the microtubule cytoskeleton in human cells. To examine the influence of MP and NSP on the human cell will also broaden our current understanding in the mechanism within the plant cell.