Biology subjects

Haswell, E. S.

Publications and source records attributed to Haswell, E. S..

5 recordsLinked to original sources

bioRxiv2023

Mechanosensitive ion channel MSL8 is required for pulsatile growth and cell wall dynamics in Arabidopsis pollen tubes

HIGHLIGHTPollen tube growth requires tight control of apical wall expansion. We present evidence for a mechanosensitive ion channel, MSL8, as a braking signal in growth dynamics through cell wall regulation. The male gametophyte in flowering plants, pollen, both performs the critical role of fertilization and represents a unique and accessible system for interrogating plant cell mechanics. Pollen endures multiple mechanical hurdles during its lifecycle: desiccation in the anther, rapid rehydration on the stigma, and germination to produce a rapidly growing pollen tube that will eventually reach the ovule. A key component in this robust mechanical system is MscS-Like 8 (MSL8), a mechanosensitive ion channel. We previously proposed that that MSL8 serves as an "osmotic safety valve", regulating pressure in the germinating pollen tube by releasing anions in response to plasma membrane tension, thereby preventing pollen tube rupture. However, we subsequently identified defects in the cell walls of msl8 mutant pollen, suggesting that it plays a role independent of osmoregulation, a conclusion also supported by mathematical modeling. Here, we show that pollen tubes lacking MSL8 channel function by genetic knockout or channel-blocking point mutation lose major growth pauses, have altered pectin esterification patterns, and are sensitive to pectin crosslinking. Together, these data suggest a mechanism whereby tension-gated ion release through mechanosensitive channels regulates apoplastic function and cell wall dynamics.

plant biology↗

bioRxiv2022

The soluble N-termini of mechanosensitive ion channels MSL8, MSL9, and MSL10 are environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations. In the past several decades, IDRs have emerged as a core component of many proteomes, comprising [~]30% of all eukaryotic protein sequences. IDRs are ubiquitous throughout different biological pathways, with a notable enrichment in responses to environmental stimuli such as abiotic stress. However, the diversity of IDR-based systems that biology has evolved to respond to different stimuli is expansive, warranting the exploration of IDRs present in unique molecular contexts. Here, we identify and characterize intrinsic disorder in the soluble, cytoplasmic N-terminal domains of three members of the MscS-Like (MSL) family of mechanosensitive ion channels, MSL8, MSL9 and MSL10. In plants, MSL channels are proposed to mediate the reactions to cell swelling, pathogenic invasion, and touch. A series of bioinformatic tools unanimously predicted that the cytosolic N-termini of MSLs are intrinsically disordered. We confirmed this prediction for the N-terminus of MSL10 (MSL10N) via circular dichroism spectroscopy. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE) and underwent structural changes and alterations to homotypic interaction favorability in the presence of molecular crowding agents. Lastly, in vitro imaging of condensates indicated that MSL8N, MSL9N and MSL10N have sharply differing propensities for condensate formation both inherently and in response to salt, temperature, and molecular crowding. Altogether, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond disparately to changes in their physiochemical environment.

biochemistry↗

bioRxiv2022

OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels

Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive sudden osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing green fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, OzTrac sensors reported osmolality changes as a proportional change in emission ratio of the two fluorescent protein domains. Live-cell imaging revealed accumulation of fluorescent sensors in internal aggregates presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

cell biology↗

bioRxiv2021

Interactions between a mechanosensitive channel and cell wall integrity signaling influence pollen germination in Arabidopsis thaliana

Cells employ multiple systems to maintain cellular integrity, including mechanosensitive (MS) ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like (MSL)8 is an MS channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination and tube growth. New CRISPR/Cas9 and artificial microRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS, and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP over-expressors. These data show that MS ion channels modulate callose deposition in pollen and provides evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.

plant biology↗

bioRxiv2016

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in a msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild type Ler background, MSL8I711S suppressed pollen germination, similar to wild type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required to for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

plant biology↗