Biology subjects

Haswell, E.

Publications and source records attributed to Haswell, E..

9 recordsLinked to original sources

bioRxiv2022

Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana

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 both a proteomic screen and a forward genetic screen; both unexpectedly implicated ER-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, MSL10S640L), 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 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 EPCS provides new insight into the function and regulation of this type of subcellular compartment.

plant biology↗

bioRxiv2021

In vitro experiments and kinetic models of pollen hydration show that MSL8 is not a simple tension-gated osmoregulator

Pollen, a neighbor-less cell that contains the male gametes, undergoes multiple mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously showed that the pollen-specific mechanosensitive ion channel MscS-Like (MSL)8 is essential for pollen survival during hydration and proposed that it functions as a tension-gated osmoregulator. Here we test this hypothesis with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell and then stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model wherein MSL8 acts as a simple tension-gated osmoregulator does not replicate this behavior. A better fit was obtained from variations of the model wherein MSL8 inactivation is independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the Cell Wall Strengthening Model best simulated experimental responses. Finally, expression of a non-conducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that, contrary to our hypothesis and to known MS ion channel function in single-cell systems, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular-scale model system and illustrate how mathematical models can correct intuitive hypotheses.

plant biology↗

bioRxiv2021

The Mechanosensitive Ion Channel MSL10 Modulates Susceptibility to Pseudomonas syringae in Arabidopsis thaliana

Plants sense and respond to molecular signals associated with the presence of pathogens and their virulence factors. Mechanical signals generated during pathogenic invasion may also be important, but their contributions have rarely been studied. Here we investigate the potential role of a mechanosensitive ion channel, MscS-Like (MSL)10, in defense against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. We previously showed that overexpression of MSL10-GFP, phospho-mimetic versions of MSL10, and the gain-of-function allele msl10-3G all produce dwarfing, spontaneous cell death, and the hyperaccumulation of reactive oxygen species. These phenotypes are shared by many autoimmune mutants and are frequently suppressed by growth at high temperature in those lines. Here, we found that the same was true for all three MSL10 hypermorphs. In addition, we show that the SGT1/RAR1/HSP90 co-chaperone complex was required for dwarfing and ectopic cell death, PAD4 and SID2 were partially required, and the immune regulators EDS1 and NDR1 were dispensable. All MSL10 hypermorphs exhibited reduced susceptibility to infection by P. syringae strain Pto DC3000, Pto DC3000 expressing the avirulence genes avrRpt2 or avrRpm1, but not Pto DC3000 hrpL, and showed an accelerated induction of PR1 expression compared to wild-type plants. Null msl10-1 mutants were delayed in PR1 induction and displayed modest susceptibility to infection by COR-deficient Pst. Finally, stomatal closure was reduced in msl10-1 loss-of-function mutants in response to Pst COR-. These data show that MSL10 modulates pathogen responses and begin to address the possibility that mechanical signals are exploited by the plant for pathogen perception.

plant biology↗

bioRxiv2020

Regulation of Vacuole Morphology by PIEZO Channels in Spreading Earth Moss

The perception of mechanical force is a fundamental property of most, if not all cells. PIEZO channels are plasma membrane-embedded mechanosensitive calcium channels that play diverse and essential roles in mechanobiological processes in animals1,2. PIEZO channel homologs are found in plants3,4, but their role(s) in the green lineage are almost completely unknown. Plants and animals diverged approximately 1.5 billion years ago, independently evolved multicellularity, and have vastly different cellular mechanics5. Here, we investigate PIEZO channel function in the moss Physcomitrium patens, a representative of one of the first land plant lineages. PpPIEZO1 and PpPIEZO2 were redundantly required for normal growth, size, and shape of tip-growing caulonema cells. Both were localized to vacuolar membranes and facilitated the release of calcium into the cytosol in response to hypoosmotic shock. Loss-of-function ({Delta}Pppiezo1/2) and gain-of-function (PpPIEZO2-R2508K and -R2508H) mutants revealed a role for moss PIEZO homologs in regulating vacuole morphology. Our work here shows that plant and animal PIEZO homologs have diverged in both subcellular localization and in function, likely co-opted to serve different needs in each lineage. The plant homologs of PIEZO channels thus provide a compelling lens through which to study plant mechanobiology and the evolution of mechanoperceptive strategies in multicellular eukaryotes.

plant biology↗

bioRxiv2020

Charged Pore-lining Residues are Required for Normal Channel Kinetics in the Eukaryotic Mechanosensitive Ion Channel MSL1

Mechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single channel patch-clamp electrophysiology in E. coli, we show that altering the size and charge of R326 and D327 leads to dramatic changes in open state dwell time. Modest changes in gating pressure and open state stability were also observed while no effects on channel rectification or conductance were detected. MSL1 channel variants had differing physiological function in E. coli hypoosmotic shock assays, without clear correlation between function and particular channel characteristics. Taken together, these results demonstrate that altering pore-lining residue charge and size disrupts normal channel state stability and gating transitions, and led us to propose the "sweet spot" model. In this model, the transition to the closed state is facilitated by attraction between R326 and D327 and repulsion between R326 residues of neighboring monomers. In the open state, expansion of the channel reduces inter-monomeric repulsion, rendering open state stability influenced mainly by attractive forces. This work provides insight into how unique charge-charge interactions can be combined with an otherwise conserved structural feature to help modulate MS channel function.

physiology↗

bioRxiv2020

The Mechanosensitive Ion Channel MSL10 Potentiates Responses to Cell Swelling in Arabidopsis Seedlings

The ability to respond to unanticipated increases in volume is a fundamental property of cells, essential for cellular integrity in the face of osmotic challenges. Plants must manage cell swelling during flooding, rehydration, and pathogenesis-but little is known about the mechanisms by which this occurs. It has been proposed that plant cells could sense and respond to cell swelling through the action of mechanosensitive ion channels. Here we develop and characterize a new assay to study the effects of cell swelling on Arabidopsis thaliana seedlings and to test the contributions of the mechanosensitive ion channel MscS-Like10 (MSL10). The assay incorporates both cell wall softening and hypo-osmotic treatment to induce cell swelling. We show that MSL10 is required for previously demonstrated responses to hypo-osmotic shock, including a cytoplasmic calcium transient within the first few seconds, accumulation of ROS within the first 30 minutes, and increased transcript levels of mechano-inducible genes within 60 minutes. We also show that cell swelling induces programmed cell death within 3 hours, also in a MSL10-dependent manner. Finally, we show that MSL10 is unable to potentiate cell swelling-induced death when phosphomimetic residues are introduced into its soluble N-terminus. Thus, MSL10 functions as a phospho-regulated membrane-based sensor that connects the perception of cell swelling to a downstream signaling cascade and programmed cell death.

plant biology↗

bioRxiv2019

Interactions between the N- and C- termini of mechanosensitive ion channel AtMSL10 support a three-step mechanism for activating its signaling function

Although a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to GFP triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag and could be also induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus are transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants. HIGHLIGHTCell death is triggered by mutations in either the cytoplasmic N- or C-terminus of AiMSLlu. Our proposed model explains how membrane tension may activate signaling through the interaction of these two domains.

plant biology↗

bioRxiv2018

Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL1, MSL2, and MSL3 reveal a role for inter-organellar communication in plant development

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between three Mechanosensitive channels of Small conductance-Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bacterial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to the inner mitochondrial membrane, while MSL2 and MSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion in MSL1, both in wild type and in msl2 msl3 mutant backgrounds. msl1 single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling in msl2 msl3 double mutants was exacerbated in the msl1 msl2 msl3 triple mutant. However, the introduction of the msl1 lesion into the msl2 msl3 mutant background suppressed other msl2 msl3 mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version of MSL1. In yeast-based interaction studies, MSL1 interacted with itself, but not with MSL2 or MSL3. These results establish that the abnormalities observed in msl2 msl3 double mutants is partially dependent on the presence of functional MSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

plant biology↗

bioRxiv2018

Nonpolar Residues in the Presumptive Pore-Lining Helix of Mechanosensitive Channel MSL10 Influence Channel Behavior and Confirm a Non-Conducting Function

Mechanosensitive (MS) ion channels provide a universal mechanism for sensing and responding to increased membrane tension. MscS-Like(MSL)10 is a relatively well-studied MS ion channel from Arabidopsis thaliana that is implicated in cell death signaling. The relationship between the amino acid sequence of MSL10 and its conductance, gating tension, and opening and closing kinetics remain unstudied. Here we identify several nonpolar residues in the presumptive pore-lining transmembrane helix of MSL10 (TM6) that contribute to these basic channel properties. F553 and I554 are essential for wild type channel conductance and the stability of the open state. G556, a glycine residue located at a predicted kink in TM6, is essential for channel conductance. The increased tension sensitivity of MSL10 compared to close homolog MSL8 may be attributed to F563, but other channel characteristics appear to be dictated by more global differences in structure. Finally, MSL10 F553V and MSL10 G556V provided the necessary tools to establish that MSL10s ability to trigger cell death is independent of its ion channel function.

plant biology↗