A dual-state emission luminogen for lipid droplet imaging and photodynamic therapy.

Organic luminogens with dual-state emission (DSE) have garnered widespread attention due to their versatility in the forms of both dilute solutions and solids. Despite the growing interest, most research on DSE focuses primarily on molecule design and photophysical investigation, with limited exploration of their practical applications. In this study, we introduce a novel fluorescent molecule, PCT, featuring a distinct D-π(A)-D' electronic structure. PCT exhibited efficient DSE properties, with high quantum yields in both dilute solutions (ΦTHF = 52.3 %) and solid-state (Φsolid = 74.6 %). Taking advantage of PCT's lipophilicity, we demonstrated its potential for targeted lipid droplet (LD) imaging in living cells and its utility in monitoring LD depletion during cellular starvation. To further enhance its applicability in photodynamic therapy (PDT), PCT was encapsulated within the amphiphilic triblock copolymer Pluronic F127, forming PCT@F127 nanoparticles with improved colloidal stability. These nanoparticles efficiently generated singlet oxygen (1O2) under white light irradiation, achieving a 1O2 quantum yield of 57.2 %. In vitro studies on MCF-7 cells revealed significant 1O2 generation and potent phototoxicity, leading to marked cell apoptosis and necrosis. These results underscore PCT's multifunctionality as a DSEgen, with promising applications in both bioimaging and PDT.

A Fluorescence Lifetime-Based Novel Method for Accurate Lipid Quantification of BODIPY Vital-Stained C. elegans.

Lipid droplets (LDs) are organelles associated with lipid storage and energy metabolism, thus their morphology and quantity are of significant research interest. While commercially available BODIPY dye effectively labels LDs in various cell types, it also labels lysosome-related organelles (LROs) in C. elegans, leading to non-specific LD quantification. Here, we report that the fluorescent signals of BODIPY exhibit distinct fluorescence lifetime patterns for LROs and LDs, which can be captured, visualized, and filtered by fluorescence lifetime imaging microscopy. Furthermore, we proposed and validated a method based on fluorescence lifetime that can improve the accuracy of fat storage quantification in BODIPY vital-staining worms, which holds broad applications, including rapid and accurate LD quantification in forward genetic screening. Additionally, our method enables observing dynamic LD-LRO interactions in living worms, a unique capability of BODIPY vital-staining. Our findings highlight distinct BODIPY fluorescence lifetime characteristics of LDs and LROs, providing a valuable tool for future research on LDs, LROs, or their interactions.

Development of a monoclonal antibody to study MARCH6, an E3 ligase that regulates proteins that control lipid homeostasis.

MARCH6, also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum (ER) where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of MARCH6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

Experimental study of liquid drop impact on granular medium: Drop spreading/splashing and particle ejection.

The impact of a liquid drop on a granular medium is a common phenomenon in nature and engineering. The possible splashing droplets and ejected particles could pose a risk of pathogen transmission if the water source or granular medium is contaminated. This work studies the liquid drop impact on the granular medium using high-speed photography and considers the effects of liquid properties, drop impact characteristics, and granular medium properties. Four flow regimes, including direct penetration, prompt splashing, spreading, and corona splashing, are observed and a regime map is created to identify their thresholds. The spreading regime can eject a large number of particles, and the corona splashing regime can produce splashing droplets in addition to the ejected particles. For the splashing droplets, their median diameters and velocities are in the ranges 0.11 to 0.21 and 0.15 to 0.37 of the diameter and velocity of the impact drop, and their median splashing angles range from 14° to 27°. Two particle ejection mechanisms are observed, falling squeeze and forward collision, driven by the collapsing and forward spreading of the liquid lamella, respectively. The particles ejected by the latter mechanism have larger ejection velocities, angles and distances from the impact center, which can facilitate their long-range transmission. In addition, the process of spreading and retracting of the lamella formed by the drop impact is also studied, and it is found that the maximum spreading diameter of the lamella is proportional to the crater diameter. These results improve the understanding of the phenomenon after the drop impact on the granular medium and the characteristics of the splashing droplets and ejected particles, contributing to the prediction and risk assessment of contaminated particle transmission.

Biomimetic Transparent Slippery Surface for the Locomotion of Photocontrol Droplets and Bubbles.

Directed transportation and collection of liquids and bubbles play a vital role in the survival of ecosystems. Among them, the optical response control is widely used in the fields of microfluidic chips and chemical synthesis because of its high remote operation and fast response speed. However, due to poor light transmission, the development direction of traditional near-infrared (NIR) absorbing materials in the field of visualization is limited, and there are few reports of manufacturing an operating platform that can realize the directional movement of droplets/bubbles on a single platform. Here, a transparent photo-responsive PBFS platform is prepared for droplet and bubble manipulation by coating the etched glass substrate with Prussian blue (PB) nanocubes. When near-infrared (NIR) irradiation on the PBFS platform, PB nanocubes trigger heat production by photothermal means, due to the action of Marangoni force, the surface tension on the left and right sides of the droplets and bubbles is not uniform, forming a surface tension gradient, thereby driving the movement of the droplets and bubbles. The control platform has good application potential in the field of microchemical reaction and biomedical engineering and brings new solutions to the field of transparent photothermal materials.

Fabrication of Conical Microstructure Array for Stable Droplet Generation Over Wide Flow Rate Range.

Droplet generators with the ability to resist flow fluctuations are of importance for microfluidic chip analysis systems. However, obtaining stably desired-size droplets is still a bugbear since even slight fluctuations can cause polydisperse droplets. In this study, a high-performance droplet generator is achieved with a functional conical array housed in the junction of the channels. The conical microstructures are fabricated through the selective etching of the scratched silicon nitride/silicon (Si3N4/Si) substrate in potassium hydroxide (KOH) etchant, where the combination of lateral and normal material removal contributes to the structure formation. It is found that the key role of the conical microstructures is to regulate the flow rate of the continuous phase, which allows droplet generation to turn to the necking phase and enables droplets to shed more easily. It is also noted that the droplet generator with such a conical array can produce monodisperse droplets in wide-range flow, providing new insights for high-quality device design.

Obesity facilitated colon cancer progression is mediated by increased diacylglycerol o-acyltransferases 1 and 2 (DGAT1/2) levels.

BACKGROUND & AIMS: The obesity epidemic is associated with increased colon cancer progression. As lipid droplets (LDs) fuel tumor growth, we aim to determine the significance of diacyltransferases, DGAT1/2, responsible for LDs biogenesis, in obesity-mediated colonic tumorigenesis. METHODS: Human colon cancer samples, colon cancer cells, colonospheres, and ApcMin/+ colon cancer mouse model on a high-fat diet were employed. For DGAT1/2 inhibition, enzymatic inhibitors and siRNA were used. Expression, pathways, cell cycle, and growth were assessed. Bioinformatic analyses of CUT&RUN and RNAseq data were performed. RESULTS: DGAT1/2 levels in human colon cancer tissue are significantly elevated with disease severity and obesity (vs normal). Their levels are increased in human colon cancer cells (vs non-transformed) and further enhanced by fatty acids prevalent in obesity; augmented DGAT2 expression is MYC-dependent. Inhibition of DGAT1/2 improves FOXO3 activity by attenuating PI3K, resulting in reduced MYC-dependent DGAT2 expression and LDs accumulation, suggesting feedback. This inhibition attenuated growth in colon cancer cells and colonospheres via FOXO3/p27kip1 cell cycle arrest and reduced colonic tumors in ApcMin/+ mice on a high-fat diet. Transcriptomic analysis revealed that DGAT1/2 inhibition targeted metabolic and tumorigenic pathways in human colon cancer and colon cancer crypts, stratifying human colon cancer samples from normal. Further analysis revealed that this inhibition is predictive of advanced disease-free state and survival in colon cancer patients. CONCLUSION: This is a novel mechanism of DGAT1/2-dependent metabolic and tumorigenic remodeling in obesity-facilitated colon cancer, providing a platform for the future development of effective treatments for colon cancer patients.

A novel bacterial effector protein mediates ER-LD membrane contacts to regulate host lipid droplets.

Effective intracellular communication between cellular organelles occurs at dedicated membrane contact sites (MCSs). Tether proteins are responsible for the establishment of MCSs, enabling direct communication between organelles to ensure organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). Here, we identify a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

Planarian Mucus: A Novel Source of Pleiotropic Cytotoxic and Cytostatic Agents against Cancer Cells.

Biological evolution has generated a vast array of natural compounds produced by organisms across all domains. Among these, secondary metabolites, selected to enhance an organism's competitiveness in its natural environment, make them a reservoir for discovering new compounds with cytotoxic activity, potentially useful as novel anticancer agents. Slime secretions, the first barrier between epithelial surfaces and the surrounding environment, frequently contain cytotoxic molecules to limit the growth of parasitic organisms. Planarians, freshwater Triclads, continuously secrete a viscous mucus with multiple physiological functions. The chemical composition of planarian mucus has been only partially elucidated, and there are no studies reporting its cytotoxic or cytostatic effects. In this study, we developed a protocol for collecting mucus from Dugesia japonica specimens and we demonstrated that it inhibits the growth of cancer cells by activating cytostatic and ROS-dependent cytotoxic mechanisms inducing lipid droplet accumulation and mitochondrial membrane reorganization. Although further research is needed to identify the specific chemicals responsible for the anticancer activity of planarian mucus, this work opens up numerous research avenues aimed at better understanding the mechanisms of action of this product for potential therapeutic applications.

Mimicking a Cellular Crowding Environment for Enzyme-Free Paper-Based Nucleic Acid Tests at the Point of Care.

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases in the same location where the patient receives treatment. Communicable diseases, such as human immunodeficiency virus (HIV), disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource-intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes, producing large DNA nanostructures, capable of single-molecule detection in cellulo. These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here, we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub-pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an in cellulo reaction in vitro by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.

Single Cell Droplet-Based Efficacy and Transcriptomic Analysis of a Novel Anti-KLRG1 Antibody for Elimination of Autoreactive T Cells.

Progress in developing improvements in the treatment of autoimmune disease has been gradual, due to challenges presented by the nature of these conditions. Namely, the need to suppress a patient's immune response while maintaining the essential activity of the immune system in controlling disease. Targeted treatments to eliminate the autoreactive immune cells driving disease symptoms present a promising new option for major improvements in treatment efficacy and side effect management. Monoclonal antibody therapies can be applied to target autoreactive immune cells if the cells possess unique surface marker expression patterns. Killer cell lectin like receptor G1 (KLRG1) expression on autoreactive T cells presents an optimal target for this type of cell depleting antibody therapy. In this study, we apply a variety of in vitro screening methods to determine the efficacy of a novel anti-KLRG1 antibody at mediating specific natural killer (NK) cell mediated antibody-dependent cellular cytotoxicity (ADCC). The methods include single-cell droplet microfluidic techniques, allowing timelapse imaging and sorting based on cellular interactions. Included in this study is the development of a novel method of sorting cells using a droplet-sorting platform and a fluorescent calcium dye to separate cells based on CD16 recognition of cell-bound antibody. We applied this novel sorting method to visualize transcriptomic variation between NK cells that are or are not activated by binding the anti-KLRG1 antibody using RNA sequencing. The data in this study reveals a reliable and target-specific cytotoxicity of the cell depleting anti-KLRG1 antibody, and supports our droplet-sorting calcium assay as a novel method of sorting cells based on receptor activation.

Calcineurin activation improves cell survival during amino acid starvation in lipid droplet-deficient yeasts.

Lipid droplets (LD) are storage sites for neutral lipids that can be used as a source of energy during nutrient starvation, but also function as hubs for fatty acid (FA) trafficking between organelles. In the yeast Saccharomyces cerevisiae, the absence of LD causes a severe disorganization of the endomembrane network during starvation. Here we show that cells devoid of LD respond to amino acid (AA) starvation by activating the serine/threonine phosphatase calcineurin and the nuclear translocation of its target protein Crz1. This activation was inhibited by treatments that restore a normal endomembrane organization, i.e. inhibition of FA synthesis with cerulenin or deletion of the inhibitory transcription factor Opi1. Activation of calcineurin increased the lifespan of LD-deficient cells during AA starvation. Indeed, deletion of its regulatory or catalytic subunits accelerated cell death. Surprisingly, calcineurin activation appeared to be calcium-independent. An increase in intracellular calcium was observed in LD-deficient cells during AA starvation, but its inhibition by genetic deletion of MID1 or YVC1 did not affect calcineurin activity. In contrast, calcineurin activation required the direct regulator of calcineurin Rcn1 and its activating (GSK-3)-related protein kinase Mck1.

An intelligent NIR fluorescent dye with dual response to pH and viscosity for investigating the interactions between LDs and mitochondria.

The interaction between lipid droplets and mitochondria plays a pivotal role in biological processes including cellular stress, metabolic homeostasis, cellular autophagy and apoptosis. Deciphering the complex interplay between lipid droplets and mitochondria is essential for gaining insights into the fundamental workings of the cell and can have broad implications for the development of therapeutic strategies for various diseases, including metabolic disorders, neurodegenerative diseases, and cancer. In this study, we develop a pH and viscosity-responsive near-infrared (NIR) fluorescent probe PTOH to investigate the interaction between lipid droplets and mitochondria. This probe demonstrates a significant enhancement in fluorescence intensity at 470 nm when the pH increases, while under acidic conditions, its fluorescence intensity at 730 nm intensifies by a factor of 35 with rising system viscosity. Cell imaging experiments revealed that PTOH can effectively discriminate between normal and cancerous cells, as well as detect intracellular pH and viscosity alterations induced by drugs. Additionally, PTOH is utilized to visualize the interaction between lipid droplets and mitochondria and to differentiate between cellular autophagy and apoptosis phenomena, providing a valuable tool for elucidating the mechanisms underlying lipid droplet-mitochondria interactions and their associated diseases.

Micro-droplet printed ion-selective membrane sensors for in situ monitoring of marine heavy metal ions.

Fast, accurate, and reliable techniques for marine toxic heavy metal ions (HMI) detection are critical for the ecological environment and human health. One of the fatal drawbacks of traditional ion selective electrochemical sensors is that the modification of electrode cannot be accurately quantified, resulting in poor repeatability of the detection electrode and large error between the multi-electrode detection results. In order to tackle this challenge, this study presents ultra-fine micro-droplet printed electrodes for the in-situ detection of Cd2+, a carcinogenic and toxic HMI commonly found in the ocean. The ion selective membrane casting liquid was dispersed into tiny droplets with a diameter of micron through microfluidic technology, and the microdroplets were precisely arranged on the electrode surface. As a result, the modification error of electrode was reduced to pL level (accurate to 10 pL), which greatly improved the repeatability between electrodes prepared in different batches. The results of experiments with pure electrolyte, interference ions and artificial seawater indicated that the micro-droplet printed sensors possessed excellent properties of accuracy, precision, repeatability, and anti-interference. This novel micro-droplet printed sensor has the potential to capture an accurate picture of nearshore HMI in heterogeneous environments under shock conditions.

Genome-Wide Association Study of Cuticle and Lipid Droplet Properties of Cucumber (Cucumis sativus L.) Fruit.

The fruit surface is a critical first line of defense against environmental stress. Overlaying the fruit epidermis is the cuticle, comprising a matrix of cutin monomers and waxes that provides protection and mechanical support throughout development. The epidermal layer of the cucumber (Cucumis sativus L.) fruit also contains prominent lipid droplets, which have recently been recognized as dynamic organelles involved in lipid storage and metabolism, stress response, and the accumulation of specialized metabolites. Our objective was to genetically characterize natural variations for traits associated with the cuticle and lipid droplets in cucumber fruit. Phenotypic characterization and genome-wide association studies (GWAS) were performed using a resequenced cucumber core collection accounting for >96% of the allelic diversity present in the U.S. National Plant Germplasm System collection. The collection was grown in the field, and fruit were harvested at 16-20 days post-anthesis, an age when the cuticle thickness and the number and size of lipid droplets have stabilized. Fresh fruit tissue sections were prepared to measure cuticle thickness and lipid droplet size and number. The collection showed extensive variation for the measured traits. GWAS identified several QTLs corresponding with genes previously implicated in cuticle or lipid biosynthesis, including the transcription factor SHINE1/WIN1, as well as suggesting new candidate genes, including a potential lipid-transfer domain containing protein found in association with isolated lipid droplets.

Preparation and Molecular Dynamic Simulation of Superfine CL-20/TNT Cocrystal Based on the Opposite Spray Method.

In view of the current problems of slow crystallization rate, varying grain sizes, complex process conditions, and low safety in the preparation of CL-20/TNT cocrystal explosives in the laboratory, an opposite spray crystallization method is provided to quickly prepare ultrafine explosive cocrystal particles. CL-20/TNT cocrystal explosive was prepared using this method, and the obtained cocrystal samples were characterized by electron microscopy morphology, differential thermal analysis, infrared spectroscopy, and X-ray diffraction analysis. The effects of spray temperature, feed ratio, and preparation method on the formation of explosive cocrystal were studied, and the process conditions of the pneumatic atomization spray crystallization method were optimized. The crystal plane binding energy and molecular interaction forces between CL-20 and TNT were obtained through molecular dynamic simulation, and the optimal binding crystal plane and cocrystal mechanism were analyzed. The theoretical calculation temperature of the binding energy was preliminarily explored in relation to the preparation process temperature of cocrystal explosives. The mechanical sensitivity of ultrafine CL-20/TNT cocrystal samples was tested. The results showed that choosing acetone as the cosolvent, a spraying temperature of 30 °C, and a feeding ratio of 1:1 was beneficial for the formation and growth of cocrystal. The prepared CL-20/TNT cocrystal has a particle size of approximately 10 µm. The grain size is small, and the crystallization rate is fast. The impact and friction sensitivity of ultrafine CL-20/TNT cocrystal samples were significantly reduced. The experimental process conditions are simple and easy to control, and the safety of the preparation process is high, providing certain technical support for the preparation of high-quality cocrystal explosives.

Lipid droplets and small extracellular vesicles: More than two independent entities.

Despite increasing knowledge about small extracellular vesicle (sEV) composition and functions in cell-cell communication, the mechanism behind their biogenesis remains unclear. Here, we reveal for the first time that sEV biogenesis and release into the microenvironment are tightly connected with another important organelle, Lipid Droplets (LDs). The correlation was observed in several human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results demonstrated that external stimuli such as radiation, pH, hypoxia or lipid-interfering drugs, known to affect the number of LDs/cell, similarly influenced sEV secretion. Importantly, through multiple omics data, at both mRNA and protein levels, we revealed RAB5C as a potential important molecular player behind this organelle connection. Altogether, the potential to fine-tune sEV biogenesis by targeting LDs could significantly impact the amount, cargos and properties of these sEVs, opening new clinical perspectives.

YOLO-PBESW: A Lightweight Deep Learning Model for the Efficient Identification of Indomethacin Crystal Morphologies in Microfluidic Droplets.

Crystallization is important to the pharmaceutical, the chemical, and the materials fields, where the morphology of crystals is one of the key factors affecting the quality of crystallization. High-throughput screening based on microfluidic droplets is a potent technique to accelerate the discovery and development of new crystal morphologies with active pharmaceutical ingredients. However, massive crystal morphologies' datum needs to be identified completely and accurately, which is time-consuming and labor-intensive. Therefore, effective morphologies' detection and small-target tracking are essential for high-efficiency experiments. In this paper, a new improved algorithm YOLOv8 (YOLO-PBESW) for detecting indomethacin crystals with different morphologies is proposed. We enhanced its capability in detecting small targets through the integration of a high-resolution feature layer P2, and the adoption of a BiFPN structure. Additionally, in this paper, adding the EMA mechanism before the P2 detection head was implemented to improve network attention towards global features. Furthermore, we utilized SimSPPF to replace SPPF to mitigate computational costs and reduce inference time. Lastly, the CIoU loss function was substituted with WIoUv3 to improve detection performance. The experimental findings indicate that the enhanced YOLOv8 model attained advancements, achieving AP metrics of 93.3%, 77.6%, 80.2%, and 99.5% for crystal wire, crystal rod, crystal sheet, and jelly-like phases, respectively. The model also achieved a precision of 85.2%, a recall of 83.8%, and an F1 score of 84.5%, with a mAP of 87.6%. In terms of computational efficiency, the model's dimensions and operational efficiency are reported as 5.46 MB, and it took 12.89 ms to process each image with a speed of 77.52 FPS. Compared with state-of-the-art lightweight small object detection models such as the FFCA-YOLO series, our proposed YOLO-PBESW model achieved improvements in detecting indomethacin crystal morphologies, particularly for crystal sheets and crystal rods. The model demonstrated AP values that exceeded L-FFCA-YOLO by 7.4% for crystal sheets and 3.9% for crystal rods, while also delivering a superior F1-score. Furthermore, YOLO-PBESW maintained a lower computational complexity, with parameters of only 11.8 GFLOPs and 2.65 M, and achieved a higher FPS. These outcomes collectively demonstrate that our method achieved a balance between precision and computational speed.

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets.

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

Zygospore formation in Zygnematophyceae predates several land plant traits.

Recent research on a special type of sexual reproduction and zygospore formation in Zygnematophyceae, the sister group of land plants, is summarized. Within this group, gamete fusion occurs by conjugation. Zygospore development in Mougeotia, Spirogyra and Zygnema is highlighted, which has recently been studied using Raman spectroscopy, allowing chemical imaging and detection of changes in starch and lipid accumulation. Three-dimensional reconstructions after serial block-face scanning electron microscopy (SBF-SEM) or focused ion beam SEM (FIB-SEM) made it possible to visualize and quantify cell wall and organelle changes during zygospore development. The zygospore walls undergo strong modifications starting from uniform thin cell walls to a multilayered structure. The mature cell wall is composed of a cellulosic endospore and exospore and a central mesospore built up by aromatic compounds. In Spirogyra, the exospore and endospore consist of thick layers of helicoidally arranged cellulose fibrils, which are otherwise only known from stone cells of land plants. While starch is degraded during maturation, providing building blocks for cell wall formation, lipid droplets accumulate and fill large parts of the ripe zygospores, similar to spores and seeds of land plants. Overall, data show similarities between streptophyte algae and embryophytes, suggesting that the genetic toolkit for many land plant traits already existed in their shared algal ancestor. This article is part of the theme issue 'The evolution of plant metabolism'.