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Recent characterizations of MscS and its homologs provide insight into the basis of ion selectivity in mechanosensitive channels

The bacterial mechanosensitive channel MscS provides an excellent model system for the study of mechanosensitivity and for investigations into the cellular response to hypoosmotic shock. Numerous studies have elucidated the structure, function and gating mechanism of Escherichia coli MscS, providing a wealth of information for the comparative analysis of MscS family members in bacteria, archaea, fungi and plants. We recently reported the electrophysiological characterization of MscS-Like (MSL)10, a MscS homolog from the model flowering plant Arabidopsis thaliana. Here we summarize our results and briefly compare MSL10 to previously described members of the MscS family. Finally, we comment on how this and other recently published studies illuminate the possible mechanisms by which ion selectivity is accomplished in this fascinating family of channels

Functional Analysis of Conserved Motifs in the Mechanosensitive Channel Homolog MscS-Like2 from Arabidopsis thaliana

The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)9N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology

Expressing and Characterizing Mechanosensitive Channels in Xenopus Oocytes

The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes

MscS-Like10 is a stretch-activated ion channel from Arabidopsis thaliana with a preference for anions

Like many other organisms, plants are capable of sensing and responding to mechanical stimuli such as touch, osmotic pressure, and gravity. One mechanism for the perception of force is the activation of mechanosensitive (or stretch-activated) ion channels, and a number of mechanosensitive channel activities have been described in plant membranes. Based on their homology to the bacterial mechanosensitive channel MscS, the 10 MscS-Like (MSL) proteins of Arabidopsis thaliana have been hypothesized to form mechanosensitive channels in plant cell and organelle membranes. However, definitive proof that MSLs form mechanosensitive channels has been lacking. Here we used single-channel patch clamp electrophysiology to show that MSL10 is capable of providing a MS channel activity when heterologously expressed in Xenopus laevis oocytes. This channel had a conductance of ∼100 pS, consistent with the hypothesis that it underlies an activity previously observed in the plasma membrane of plant root cells. We found that MSL10 formed a channel with a moderate preference for anions, which was modulated by strongly positive and negative membrane potentials, and was reversibly inhibited by gadolinium, a known inhibitor of mechanosensitive channels. MSL10 demonstrated asymmetric activation/inactivation kinetics, with the channel closing at substantially lower tensions than channel opening. The electrophysiological characterization of MSL10 reported here provides insight into the evolution of structure and function of this important family of proteins

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 pathogen invasion-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 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 several previously demonstrated responses to hypo-osmotic shock, including a cytoplasmic calcium transient within the first few seconds, accumulation of ROS within the first 30 min, and increased transcript levels of mechano-inducible genes within 60 min. We also show that cell swelling induces programmed cell death within 3 h 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

MscS-like Proteins Control Plastid Size and Shape in Arabidopsis thaliana

Background Mechanosensitive (MS) ion channels provide a mechanism for the perception of mechanical stimuli such as sound, touch, and osmotic pressure. The bacterial MS ion channel MscS opens in response to increased membrane tension and serves to protect against cellular lysis during osmotic downshock. MscS-like proteins are found widely in bacterial and archaeal species and have also been identified in fission yeast and plants. None of the eukaryotic members of the family have yet been characterized. Results Here, we characterize two MscS-like (MSL) proteins from Arabidopsis thaliana, MSL2 and MSL3. MSL3 can rescue the osmotic-shock sensitivity of a bacterial mutant lacking MS-ion-channel activity, suggesting that it functions as a mechanosensitive ion channel. Arabidopsis plants harboring insertional mutations in both MSL3 and MSL2 show abnormalities in the size and shape of plastids, which are plant-specific endosymbiotic organelles responsible for photosynthesis, gravity perception, and numerous metabolic reactions. MSL2-GFP and MSL3-GFP are localized to discrete foci on the plastid envelope and colocalize with the plastid division protein AtMinE. Conclusions Our data support a model wherein MSL2 and MSL3 control plastid size, shape, and perhaps division during normal plant development by altering ion flux in response to changes in membrane tension. We propose that MscS family members have evolved new roles in plants since the endosymbiotic event that gave rise to plastids

Plastids and pathogens: Mechanosensitive channels and survival in a hypoosmotic world

In bacteria, MscS-type mechanosensitive channels serve to protect cells from lysis as they swell during extreme osmotic stress. We recently showed that two MscS homologs from Arabidopsis thaliana serve a similar purpose in the epidermal plastids of the leaf, indicating that the plant cell cytoplasm can present a dynamic osmotic challenge to the plastid. MscS homologs are predicted to be targeted to both plastids and mitochondrial envelopes and have been found in the genomes of intracellular pathogens. Here we discuss the implications of these observations, and propose that MS channels provide an essential mechanism for osmotic adaptation to both intracellular and the extracellular environments

A role for mechanosensitive channels in chloroplast and bacterial fission.

The division site in both chloroplasts and bacteria is established by the medial placement of the FtsZ ring, a process that is in part regulated by the evolutionarily conserved components of the Min system. We recently showed that mechanosensitive ion channels influence FtsZ ring assembly in both Arabidopsis thaliana chloroplasts and in Escherichia coli; in chloroplasts they do so through the same genetic pathway as the Min system. Here we describe the effect of heterologous expression of the Arabidopsis MS channel homolog MSL2 on FtsZ ring placement in E. coli. We also discuss possible molecular mechanisms by which MS channels might influence chloroplast or bacterial division

A force of nature: molecular mechanisms of mechanoperception in plants

The ability to sense and respond to a wide variety of mechanical stimuli-gravity, touch, osmotic pressure, or the resistance of the cell wall-is a critical feature of every plant cell, whether or not it is specialized for mechanotransduction. Mechanoperceptive events are an essential part of plant life, required for normal growth and development at the cell, tissue, and whole-plant level and for the proper response to an array of biotic and abiotic stresses. One current challenge for plant mechanobiologists is to link these physiological responses to specific mechanoreceptors and signal transduction pathways. Here, we describe recent progress in the identification and characterization of two classes of putative mechanoreceptors, ion channels and receptor-like kinases. We also discuss how the secondary messenger Ca2+ operates at the centre of many of these mechanical signal transduction pathways