- The overall focus of this thesis is on the distribution of specific lipids and membrane proteins of the external and internal membranes of plant cells, in the context of the roles that those lipids and proteins may play in microbe-plant interactions. The work includes the development of several new tools, the refinement of some existing tools, and the highlighting of several poorly appreciated artifacts that are common to such studies.
This thesis is comprised of five chapters. In Chapter 1, I would like to step back to have a general overview, including initial questions we asked, the way we interpreted unexpected results. In Chapter 2 “Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering through BiFC interactions in plants”, I demonstrated that the heterogeneous network of patches produced in FLS2-StRem1.3 BiFC complexes corresponded to ER-PM tethering, which resulted from the non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3. This work confirmed that membrane targeting of integral membrane proteins (IMPs) such as FLS2 requires either co-translational or post-translational integration into the ER membrane before trafficking to their membrane destination. These observations suggest a re-visit of several previous studies which have reported heterogeneous patch-like distributions when using IMPs and PMPs in BiFC experiments.
In Chapter 3 “Manipulating Tethering of Multivesicular Bodies and the Tonoplast to the Plasma Membrane Through BiFC Interactions in Plants”, I strengthened the evidence that the patch-like distributions observed when combining PtdIns(3)P biosensors with StREM1.3, resulted from tethering of MVBs and the tonoplast to the PM. I also observed that the membrane binding domains of the E3 ubiquitin-ligases SAUL1 (AtPUB44) and AtPUB43, could tether MVBs and the tonoplast to the PM, suggesting a possible functional role for these proteins in MVB-PM tethering, such as the secretion of exosomes.
Although my observations reported in Chapter 2 and Chapter 3 led to new insights into membrane organism in plant cells, they also highlighted the risk of using BiFC assays to study membrane protein interactions in plants, which without proper controls could lead to misinterpretation, or cause unrecognized alterations in cellular structure and membrane organization. In chapter 4 “Fluorescent Protein mEos3.2 Shows Low Self-Association in Bimolecular Fluorescence Complementation Assays in Plants”, I show that the mEOS3.2 BiFC probe, split at residue 164E, also produced minimal non-specific detectable BiFC signals when transiently expressed in Nicotiana benthamiana leaf cortical cells, but produced excellent signals with interacting protein partners. I also demonstrated that the re-assembled mEos3.2 could still photo-convert from green to red, which aided in distinguishing specific BiFC signals from background, and could allow the visualization of BiFC complexes at nanometer spatial resolution using photo-activated localization microscopy (PALM) imaging.
In chapter 5 “In vivo Super-Resolution Imaging of the Dynamics of PtdIns(4)P in the Plasma Membrane Of Plant Cells”, I successfully extended the application of mEos3.2 to study the spatiotemporal dynamics of a lipid species, PtdIns(4)P, in the plasma membrane of plant cells at single-molecule resolution using Single Particle Tracking PALM imaging (sptPALM). This work demonstrated the advantages of sptPALM compared to traditional imaging methods, such as Fluorescence Recovery After Photobleaching (FRAP), for studying the molecular dynamics of the plasma membrane. In addition, my work refined the specificity of the PtdIns(4)P biosensor FAPP1.