Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER–plasma membrane contact sites in Arabidopsis thaliana
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Evaluation Summary:
The study is of interest to researchers in the field of cell biology, especially mechanosensing. The work identifies a new context to evaluate the activity of MSL proteins in mechanosensing by identifying two novel suppressors of MSL10 as components of the ER-PM contact sites (EPCS). The work has significance for both the plant and the animal science community providing the basics for various avenues of further research in the area of mechanobiology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum–plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele ( msl10-3G, MSL10 S640L ), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.
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Evaluation Summary:
The study is of interest to researchers in the field of cell biology, especially mechanosensing. The work identifies a new context to evaluate the activity of MSL proteins in mechanosensing by identifying two novel suppressors of MSL10 as components of the ER-PM contact sites (EPCS). The work has significance for both the plant and the animal science community providing the basics for various avenues of further research in the area of mechanobiology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Codjoe and colleagues report a combined proteomic and genetic analysis of MSL protein function in the context of mechanosensing in Arabidopsis leaf epidermis. The study identifies MSL10 as being associated with proteins residing in ER-PM contact sites (EPCSs). This is a novel and interesting observation and offers a new context in which to evaluate MSL activity in mechanosensing. It is striking that genetic suppressor analysis of a gain of function msl10 allele also identifies two components of EPCSs as suppressors.
This firmly associates MSL10 with EPCS. However, beyond this association, the study does not identify a clear mechanism of action or even relevance of EPCS localization or relevance of the MSL10/VAP27/SYT1 interaction. There is some indication based on synthetic lethality between msl10 loss of …
Reviewer #1 (Public Review):
Codjoe and colleagues report a combined proteomic and genetic analysis of MSL protein function in the context of mechanosensing in Arabidopsis leaf epidermis. The study identifies MSL10 as being associated with proteins residing in ER-PM contact sites (EPCSs). This is a novel and interesting observation and offers a new context in which to evaluate MSL activity in mechanosensing. It is striking that genetic suppressor analysis of a gain of function msl10 allele also identifies two components of EPCSs as suppressors.
This firmly associates MSL10 with EPCS. However, beyond this association, the study does not identify a clear mechanism of action or even relevance of EPCS localization or relevance of the MSL10/VAP27/SYT1 interaction. There is some indication based on synthetic lethality between msl10 loss of function and VAP27 or SYT1 overexpression that the interaction is relevant, but most direct assays for localization are negative. As a consequence, there is much interesting speculation in the discussion, but I find this somewhat unsatisfying.
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Reviewer #2 (Public Review):
In this work, the authors aim to elucidate a function of the protein MSL10 within plant cells. This protein so far has only been studied on an isolated protein level or in whole plants and has been implicated in relaying mechanical information. For this, the authors wanted to find proteins interacting with MLS10 and involved in the signalling process of MSL10. The authors successfully identify novel protein interaction partners for MSL10. Some of them show a physical interaction with MSL10 whereas other proteins do not physically but show a function together with MSL10.
Interestingly the proteins found are involved in contact sites between the plasma membrane (PM) -where MLS10 is localised- and the endoplasmic reticulum (ER). MLS10 also has an impact on the abundance of these contact sites. As these contact …
Reviewer #2 (Public Review):
In this work, the authors aim to elucidate a function of the protein MSL10 within plant cells. This protein so far has only been studied on an isolated protein level or in whole plants and has been implicated in relaying mechanical information. For this, the authors wanted to find proteins interacting with MLS10 and involved in the signalling process of MSL10. The authors successfully identify novel protein interaction partners for MSL10. Some of them show a physical interaction with MSL10 whereas other proteins do not physically but show a function together with MSL10.
Interestingly the proteins found are involved in contact sites between the plasma membrane (PM) -where MLS10 is localised- and the endoplasmic reticulum (ER). MLS10 also has an impact on the abundance of these contact sites. As these contact sites undergo some rearrangement upon mechanical pressure, the authors hypothesised that MSL10 could be involved in this response but this did not turn out to be the case.
Overall the work leads to a discussion and a proposed model for MLS10 signalling. Despite the model not being fully proven and elucidated, the work provides the basics for various avenues of further research.
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