Exploration of ZmPIP2/cytoskeleton interaction and its role in the cytoskeleton organization
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- Plasma membrane intrinsic protein (PIP) is a subfamily of plant AQPs that mainly localize in the plasma membrane of the cells. They are channels that facilitate the passive diffusion of water and other small molecules across biological membranes and play crucial role in maintaining water status and other essential physiological processes in plants such as growth, development, germination to cite just a few examples. Several studies point out the fact that these channels are in close interaction with other cellular structures. Pull down assays using tagged PIPs have identified a long list of potential PIPs partners. Among them, cytoskeleton associated proteins. At this day, none of these interactions has been confirmed. It is not known if PIP directly interact with the cytoskeleton nor the functional relevance of such interactions. This thesis aimed to elucidate the direct interactions between ZmPIP2;5 and five candidate interactors: two members of the plant-specific Networked (NET) actin-binding protein superfamily (NET1A1 and NET1A2); two CLIP-associated proteins (CLASP1 and CLASP2) and a Phospholipase D (PLD). To investigate these interactions, bimolecular fluorescence complementation (BiFC) and split-ubiquitin assays (SUS) were carried on. Due to the length of the Networked genes and the impossibility to obtain the full coding sequences, the experiments were conducted with the fragments of the genes that were known to potentially interact with the candidates (i.e. NAB domains, N-terminal part and C-terminal part of the genes). No interactions were observed by BiFC nor SUS. Additionally, the interaction among VAP27-1 and NET1A NAB domains were confirmed by BiFC. The existence of a tripartite interaction NET1A-VAP27-PIP2 is still possible. Also, it is possible a competition among NET1A and PIP2 for the interaction with VAP27. These tracks must be study deeper. For the PLD a weak BiFC signal but still positive compared to the negative control was detected. This result needs to be confirmed by SUS. Of the CLASP genes, only one of the isoforms was successfully cloned, CLASP1. When tested the interaction between ZmPIP2;5 and CLASP1 proteins, no interaction was observed but interestingly, the co-expression of these genes partially modified the localization of ZmPIP2;5. It is a new avenue of investigation regarding the regulation of PIPs. In other hand, preliminary tests were performed to study the relevance of ZmPIPs in the cytoskeleton organization. To this aim, it was proposed to monitor actin and tubulin fluorescent markers in two different genetic background: the wild type (WT) and ZmPIP2;5 overexpressing line. The results were analyzed by confocal microscopy. For the tubulin, the signal was too weak. For the actin markers, a lot of variability was observed in each genetic background and the experiment must be repeated several times to build up a conclusion. This thesis provides avenues to enhance our comprehension of the interaction between ZmPIP2;5 and the cytoskeleton. The implication of ZmPIPs in cytoskeletal organization suggests novel perspective on the role of PIPs in the physiological landscape of the plant.
Citations
Mesbah, Y. (2024). Exploration of ZmPIP2/cytoskeleton interaction and its role in the cytoskeleton organization. https://dial-mem.test.bib.ucl.ac.be/handle/2078.2/39382