Analyzing plant mechanosensitive ion channels expressed in giant E. coli spheroplasts by single-channel patch-clamp electrophysiology.

Plants possess numerous ion channels that respond to a range of stimuli, including small molecules, transmembrane voltage, and mechanical force. Many in the latter category, known as mechanosensitive (MS) ion channels, open directly in response to increases in lateral membrane tension. One of the most effective techniques for characterizing ion channel properties is patch-clamp electrophysiology, in which the current through a section of membrane containing ion channels is measured. For MS channels, this technique enables the measurement of key channel properties such as tension sensitivity, conductance, and ion selectivity. These characteristics, along with the phenotypes of genetic mutants, can help reveal the physiological roles of a particular MS channel. In this protocol, we provide detailed instructions on how to study MS ion channels using single-channel patch-clamp electrophysiology in giant E. coli spheroplasts. We first present an optimized method for preparing giant spheroplasts, then describe how to measure MS channel activity using patch-clamp electrophysiology and analyze the resulting data. We also provide recommended equipment lists, setup schematics, and useful conventions.

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 increasing 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 channel kinetics. Modest changes in gating pressure 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.

Structural mechanism for gating of a eukaryotic mechanosensitive channel of small conductance.

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.

Plant Biomechanics: No Pain, No Gain for Birch Tree Stems.

Allometric relationships between organism size and shape are often described in developmental or evolutionary terms. A new study characterizes a collapsing birch tree mutant and provides a genetic entry point into the biomechanical control of tree allometry.

Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins.

UNLABELLED: Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage. IMPORTANCE: Furin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

United in diversity: mechanosensitive ion channels in plants.

Mechanosensitive (MS) ion channels are a common mechanism for perceiving and responding to mechanical force. This class of mechanoreceptors is capable of transducing membrane tension directly into ion flux. In plant systems, MS ion channels have been proposed to play a wide array of roles, from the perception of touch and gravity to the osmotic homeostasis of intracellular organelles. Three families of plant MS ion channels have been identified: the MscS-like (MSL), Mid1-complementing activity (MCA), and two-pore potassium (TPK) families. Channels from these families vary widely in structure and function, localize to multiple cellular compartments, and conduct chloride, calcium, and/or potassium ions. However, they are still likely to represent only a fraction of the MS ion channel diversity in plant systems.

Human papillomavirus type 16 does not require cathepsin L or B for infection.

Cathepsin L (CatL) and cathepsin B (CatB) are lysosomal proteases that many viruses utilize for capsid disassembly. We tested whether CatL and CatB are required for infection by human papillomavirus type 16 (HPV16). CatL- and CatB-deficient mouse embryonic fibroblasts had higher levels of infection when compared with wild-type cells. Similar results were obtained in HaCaT keratinocytes treated with CatL- or CatB-specific small interfering RNA. Thus, CatL and CatB are not required for HPV16 infection but instead appear to restrict infection.