Periodic Reporting for period 1 - PIPContactSite (Roles of maize PIP2 aquaporins in the structural organization of the cell)
Periodo di rendicontazione: 2022-06-01 al 2024-10-31
Diffusion of water through cell membranes is facilitated by aquaporins (AQPs). Regulation of AQP activity provides plants with the means to modify rapidly and reversibly water membrane permeability1. Plasma membrane (PM) intrinsic proteins (PIPs) are a subfamily of AQPs located mainly in the PM of plant cells. In the PM some channels are restricted in microdomains whereas others move freely (Li et al, Plant Cell 2011 23: 3780–3797). Additionally, PIPs seem to be in close interaction with other cellular structures such as the actin filaments (Hosy et al, Molecular Plant 2015; 8: 339– 342) and we showed that PM-located PIPs interact with the endoplasmic reticulum (ER) resident VAP27s at ER–PM contact sites (EPCS) (Fox et al, New Phytologist 2020; 228: 973-988). Altogether, the evidence points to the existence of PM-organized PIP channels in interaction with proteins from other cellular structures. However, in-depth investigation integrating all the components (PIP microdomains-EPCS-cytoskeleton) is missing.
In this context, we proposed the PIPContactSite project, whose the main objective was to determine whether ZmPIP2s mediate PM interaction with other cellular structures (ER, cytoskeleton) and elucidate the physiological impact of those interactions under stress. Our central hypothesis is that ZmPIP2 interaction with EPCS and the cytoskeleton contributes to an efficient response to stimuli by its participation in the structural organization of the cell. If this is confirmed, it would mean that PIPs are not only playing a role as channels, but also as a scaffold for PM reorganization to initiate cellular response upon stress. The specific objectives of the project included:
1. Comprehensive characterization of the role of ZmPIP2s in the organization of ZmVAP-loaded EPCS.
2. Exploration of ZmPIP2 interactions and roles in the cytoskeleton organization.
3. Highlighting ZmPIP2;5 interactions in EPCS and cytoskeleton.
In this context, we proposed the PIPContactSite project, whose the main objective was to determine whether ZmPIP2s mediate PM interaction with other cellular structures (ER, cytoskeleton) and elucidate the physiological impact of those interactions under stress. Our central hypothesis is that ZmPIP2 interaction with EPCS and the cytoskeleton contributes to an efficient response to stimuli by its participation in the structural organization of the cell. If this is confirmed, it would mean that PIPs are not only playing a role as channels, but also as a scaffold for PM reorganization to initiate cellular response upon stress. The specific objectives of the project included:
1. Comprehensive characterization of the role of ZmPIP2s in the organization of ZmVAP-loaded EPCS.
2. Exploration of ZmPIP2 interactions and roles in the cytoskeleton organization.
3. Highlighting ZmPIP2;5 interactions in EPCS and cytoskeleton.
Our work addressed the scientific objectives comprehensively:
1. Role in EPCS Organization: The interaction between ZmPIP2s and ZmVAP proteins was studied by ratiometric Bimolecular Fluorescence Complementation (rBIFC) and Split-ubiquitin assays (SUS). ZmPIP2;5 interacted with all tested ZmVAP isoforms. And all the ZmPIP2 isoforms tested interacted with ZmVAP27-1. Even though, favorite pairs may exist as the rBiFC quantification suggested. Notably, ZmPIP2;5 also interacted with another ER tethering, ZmSYT1, but the rBIFC signal was weaker than the rBIFC of the interactions with ZmVAPs. We did not observe any effect on PIP-VAP interactions upon salt stress.
2. EPCS Phenotyping in ZmPIP2 Knockouts: We generated maize cell lines knocked out for ZmPIP2 genes using CRISPR/Cas9 technology. Phenotypic analysis revealed impaired growth and osmolarity response. We will analyze soon the intracellular organization of this mutant.
3. Cytoskeleton Interactions: While no direct interaction between ZmPIP2;5 and cytoskeletal candidates (NET1A1, CLASP1, PLD) was observed, our results indicated potential indirect interactions mediated by VAP27 proteins.
Despite delays caused by technical challenges, including issues with resistance marker selection in genetic editing and difficulties cloning certain candidate genes, most deliverables and milestones were achieved or addressed through contingency measures. This work lays a robust foundation for future investigations into the structural and functional dynamics of aquaporins.
1. Role in EPCS Organization: The interaction between ZmPIP2s and ZmVAP proteins was studied by ratiometric Bimolecular Fluorescence Complementation (rBIFC) and Split-ubiquitin assays (SUS). ZmPIP2;5 interacted with all tested ZmVAP isoforms. And all the ZmPIP2 isoforms tested interacted with ZmVAP27-1. Even though, favorite pairs may exist as the rBiFC quantification suggested. Notably, ZmPIP2;5 also interacted with another ER tethering, ZmSYT1, but the rBIFC signal was weaker than the rBIFC of the interactions with ZmVAPs. We did not observe any effect on PIP-VAP interactions upon salt stress.
2. EPCS Phenotyping in ZmPIP2 Knockouts: We generated maize cell lines knocked out for ZmPIP2 genes using CRISPR/Cas9 technology. Phenotypic analysis revealed impaired growth and osmolarity response. We will analyze soon the intracellular organization of this mutant.
3. Cytoskeleton Interactions: While no direct interaction between ZmPIP2;5 and cytoskeletal candidates (NET1A1, CLASP1, PLD) was observed, our results indicated potential indirect interactions mediated by VAP27 proteins.
Despite delays caused by technical challenges, including issues with resistance marker selection in genetic editing and difficulties cloning certain candidate genes, most deliverables and milestones were achieved or addressed through contingency measures. This work lays a robust foundation for future investigations into the structural and functional dynamics of aquaporins.
The outcomes of this research represent a substantial advance beyond the existing knowledge of ZmPIP2 aquaporins, with promising implications for further exploration.
We have elucidated the molecular interactions of ZmPIP2s with key tethering proteins (VAPs and SYT1) and their potential indirect links to cytoskeletal components. The observation of extensive interaction among PIPs and VAPs reinforced the idea of an important structural interaction between these protein families. This has positioned ZmPIP2s as critical elements in plant physiology, not only because of their roles in transport, but also because of their contribution to the cellular organization.
Genome-edited maize cell lines lacking ZmPIP2s have been established. Further characterization of these lines by electron microscopy will undoubtedly contribute to our understanding of the structural roles of PIPs. Additionally, their study, not only in the context of this project, will potentially lead to a better understanding of PIP crucial roles, which may provide a tool for agricultural improvements.
We have elucidated the molecular interactions of ZmPIP2s with key tethering proteins (VAPs and SYT1) and their potential indirect links to cytoskeletal components. The observation of extensive interaction among PIPs and VAPs reinforced the idea of an important structural interaction between these protein families. This has positioned ZmPIP2s as critical elements in plant physiology, not only because of their roles in transport, but also because of their contribution to the cellular organization.
Genome-edited maize cell lines lacking ZmPIP2s have been established. Further characterization of these lines by electron microscopy will undoubtedly contribute to our understanding of the structural roles of PIPs. Additionally, their study, not only in the context of this project, will potentially lead to a better understanding of PIP crucial roles, which may provide a tool for agricultural improvements.