Date of Award

Winter 12-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type



Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. A plasma membrane-localized MS channel from Arabidopsis thaliana, MscS-Like (MSL)10, senses cell swelling and initiates a signaling cascade that triggers programmed cell death. Whereas the channel properties of MSL10 have been well studied, how MSL10 signals remains largely unknown. I worked collaboratively to show that important lesions for cell death signaling in the cytosolic N- and C-terminal domains of MSL10 interact genetically. I also helped show that ionic flux through MSL10 is dispensable for signaling, which suggested that MSL10 activates other proteins to trigger cell death. I searched for such signaling partners of MSL10 using a candidate approach—testing the signaling contributions of immune modulators EDS1 and NDR1—as well as unbiased proteomic and forward genetic screens. Both screens unexpectedly implicated ER-plasma membrane contact sites (EPCSs) in MSL10 function. I subsequently found that MSL10 enhances the size of EPCSs in adult leaves. The proteomic screen revealed that MSL10 co-immunoprecipitates with multiple proteins associated with EPCSs, including VAMP-associated proteins (VAP)27-1 and VAP27-3 and synaptotagmin 1 (SYT)1. Of these, only VAP27-1 and VAP27-3 interact directly with MSL10, and this interaction is likely constitutive. Interactions between plasma membrane-localized MSL10 and ER-localized VAP27-1/3 would, by definition, create EPCSs. Co-localization analyses indicate that only a subpopulation of MSL10 interacts with VAP27-1 and VAP27-3 at EPCSs, with the majority of MSL10 being evenly distributed throughout the plasma membrane. In the forward genetic screen, I isolated suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L). Two of these suppressors had missense mutations in SYT5 and SYT7 genes that I confirmed cause suppression of msl10-3G-dependent dwarfing and ectopic cell death phenotypes. SYT5 and SYT7 were previously reported to interact with SYT1 at EPCSs. I found that the suppressor mutations do not appreciably change SYT5 or SYT7 stability or localization, nor does MSL10 directly interact with SYT5 and SYT7. Although the mechanism by which SYT5 and SYT7 attenuate MSL10 signaling remains unknown, the fact that they do so indicates that the association of MSL10 with EPCS proteins has functional relevance for its signaling function. Using confocal microscopy, I also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Specifically, SYT1 EPCSs were expanded, but not those containing SYT5, SYT7, VAP27-1, or VAP27-3. Since EPCSs labelled by a generic marker are even larger in msl10-3G suppressor mutants, I conclude that EPCS expansion is not a cause of msl10-3G-triggered cell death. I found synthetically lethal interactions between MSL10 mutant alleles and VAP27-3 and SYT1 overexpression, further suggesting that the association of MSL10 with EPCS proteins has biological relevance. Taken together, these results indicate that MSL10 can be found at EPCSs and functions there, 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. I anticipate that future studies will test if mechanical information from the cell wall and plasma membrane is conveyed directly to the ER through MSL10.


English (en)

Chair and Committee

Elizabeth S. Haswell

Committee Members

Susan K. Dutcher, Richard D. Vierstra, Barbara N. Kunkel, Dmitri A. Nusinow,