The FERONIA (FER)-LLG1 co -receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal -induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non -cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress -induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER plant growth and survival.

Shipworms, historically known for their destruction of wooden ships and coastal structures, play a key role in carbon and nutrient cycling in aquatic ecosystems. Despite extensive research, the exact mechanisms underlying their ability to digest wood have remained elusive, particularly regarding the degradation of lignin, a major component of wood that resists breakdown. Here we employ a combination of microbial culture, metagenomics, and FISH-probe microscopy analyses to explore previously overlooked aspects of their digestive system. Specifically, we identify the presence of bacterial symbionts within the typhlosole, a specialized structure within the main wood digestive organ (cecum), challenging the long-held belief that shipworm foreguts are nearly sterile environments. The discovery of Alteromonas species in bacterial clusters within the typhlosole suggests a symbiotic relationship that may play a crucial role in the digestion of woody substrates, offering a potential explanation for the shipworm's ability to degrade lignocellulose without ligninases from the host or gill symbionts. These findings provide insight into the digestive physiology and host defense of shipworms, but also suggests that the typhlosole in other related organisms might also harbor symbionts. This study also opens new insights into research into the ecological and biotechnological applications of shipworms and their gut symbionts, enhancing our understanding of lignocellulose degradation in marine environments.

Myosin transduces chemical energy from ATP into mechanical work to generate force and/or motion. Key to this process is the coupling of the powerstroke and the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved remain unclear. We determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10 pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30 mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7 nm for the WT myosin. Repeating these experiments using a construct with a mutation in the Switch I region of the active site (S217A) increased myosin's sensitivity to load five-fold (d = 3.5 nm). Thus, these findings provide a quantitative measure of the force-dependence of Pi-rebinding to myosin and suggest that this effect involves the switch I element of the nucleotide-binding pocket.

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (P i ) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated P i on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of P i -induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM P i , but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of P i from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the P i -induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of P i -rebinding.

Abstract Myosin is a molecular motor responsible for generating the force and/or motion that drive many intracellular processes, from muscle contraction to vesicular transport. It is powered by its ability to convert the chemical energy, released from the hydrolysis of ATP, into mechanical work. The key event in the transduction process is the coupling of the force-generating powerstroke with the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved in this coupling remain unclear. Therefore, we determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7nm for the WT myosin. Repeating these experiments using a construct with a mutation (S217A) in a key region (Switch I) of the nucleotide-binding site increased myosin’s sensitivity to load five-fold (d = 3.5nm). Thus, these findings provide a quantitative measure of the force-dependent nature of Pi-rebinding to myosin’s active site and suggest that this effect involves the switch I element of the nucleotide-binding pocket. These findings, therefore, provide important new insights into the mechanisms through which this prototypical motor enzyme couples the release of chemical energy to the generation of force and/or motion.

Mechanical interactions between cells have been shown to play critical roles in regulating cell signaling and communications. However, the precise measurement of intercellular forces is still quite challenging, especially considering the complex environment at cell-cell junctions. In this study, we report a fluorescence lifetime-based approach to image and quantify intercellular molecular tensions. Using this method, tensile forces among multiple ligand-receptor pairs can be measured simultaneously. We first validated our approach and developed lifetime measurement-based DNA tension probes to image E-cadherin-mediated tension on epithelial cells. These probes were then further applied to quantify the correlations between E-cadherin and N-cadherin tensions during an epithelial-mesenchymal transition process. The modular design of these probes can potentially be used to study the mechanical features of various physiological and pathological processes.

The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among various lipids and proteins. Currently, it is still challenging to image these short-lived membrane complexes, especially in living cells. In this work, we present a DNA-based probe, termed "DNA Zipper", which allows the membrane order and pattern of transient interactions to be imaged in living cells using standard fluorescence microscopes. By fine-tuning the length and binding affinity of DNA duplex, these probes can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. The correlation between membrane order and the activation of T-cell receptor signaling has also been studied. These programmable DNA probes function after a brief cell incubation, which can be easily adapted to study lipid interactions and membrane order during different membrane signaling events.