Biology subjects

Stanley, C. E.

Publications and source records attributed to Stanley, C. E..

11 recordsLinked to original sources

bioRxiv2026

Spores-on-a-Chip: a new sample-loading strategy for multiplexed single-cell monitoring of spore germination within a microfluidic platform

We demonstrate a new sample-loading strategy ("spot-loading") for Spores-on-a-Chip microfluidic sensors that enables multiplexed experimentation. The previous limitation of one biological sample per device is overcome through controlled sample loading at an intermediate step of the chip fabrication process. As a proof-of-concept, we use dual spore chips to compare the germination behaviours of two spore strains in two distinct microenvironments.

bioengineering↗

bioRxiv2026

A versatile method to pattern surfaces within microfluidic devices

Microfluidic devices with surface-bound biomolecular patterns enable localised detection arrays, enzymatic catalysis, and gene expression. Photolithography is a well-established method to pattern open surfaces with high spatial control. However, patterning enclosed microfluidic channels remains technically challenging. Such capability would enable in situ surface modification and precise pattern alignment to channel geometries. Here, we present a photolithographic method using commercially available reagents to pattern sealed microfluidic devices. We first coat surfaces with (3-Aminopropyl)triethoxysilane (APTES) to bond microfluidic chips and provide surface amine groups onto which photocleavable polyethylene glycol (PC PEG) compounds are bound. UV exposure using standard photolithography equipment selectively deprotects the amine groups, which can subsequently bind amine-reactive cargos. We demonstrate the versatility of this method by patterning both poly(dimethylsiloxane) (PDMS) and glass surfaces with diverse cargoes: DNA, proteins, and gold nanoparticles. We also compare covalent versus noncovalent DNA patterning. Covalently bound DNA patterns are denser and could be used for sequence-specific target DNA capture. However, noncovalently bound DNA yielded higher cell-free gene expression from surface-bound GFP templates.

systems biology↗

bioRxiv2026

Intricate asymbiotic hyphal interactions of Rhizophagus irregularis revealed with single-plane observations in a new microfluidic device

Arbuscular mycorrhizal fungi (AMF) are essential symbionts to most land plants. There is great interest in using AMF inoculation treatments to enhance restoration or agricultural efforts. However, little is known about AMF asymbiotic interactions that could shape soil ecosystems and inoculation outcomes. Studying these early traits is challenging given the obligate nature of AMF. Microfluidic devices enable high-resolution, single-plane, time-resolved observation of AMF hyphal traits in controlled, plant-less environments. The AMF-AnastomosisChip, introduced in this work, additionally separates spores and hyphae into lanes for detailed observations prior to entering a shared interaction zone. Using this device with Rhizophagus irregularis, we phenotyped key hyphal traits from germination to anastomosis. We compared the effect of a fatty acid (FA) treatment to other environmental factors in shaping AMF growth and fusion (anastomosis). Among our results, we confirm known effects of the FA treatment in increasing hyphal branching and longevity but interestingly show that multiple hyphae in a lane can negate FA effects. We also newly reveal that anastomosis occurs for 100% of interactions among hyphae from different lanes, but only for 50% of in-lane interactions, potentially linked to spore origin. Our findings validate the AMF-AnastomosisChip as a versatile platform for increasing discovery in trait-based microbial ecology, including for asymbiotic AMF interactions with environmental factors. This work sets the foundation for future studies with the AMF-AnastomosisChip on the effects of nutrient content, plant-derived molecules, or microbial community members on the AMF traits characterized in this study.

microbiology↗

bioRxiv2025

A microfluidic platform for the observation and quantification of fungal highways

Soil is a complex system characterised by intra- and inter-kingdom interactions among microbial communities. While many different types of fungal-bacterial interactions have been described, hyphal-mediated transport of bacteria via the so-called "fungal highway" (FH) has not been mechanistically described. Bacteria require a liquid film for active movement; therefore, liquid saturation is a significant limiting factor for their dispersal in soil. Hyphal networks contribute to the connectivity between discrete soil microbial populations by providing a physical network through the unsaturated soil matrix. This network serves as a scaffold for liquid transport, thus allowing bacteria to migrate further or access previously isolated spaces. Studying these interactions is challenging due to the complex and stochastic nature of the soil environment; this "black box" aspect makes it difficult to visualise interactions at the microbial scale. Microfluidic technology can provide a solution by offering precise imaging at a high resolution in a physically and chemically controlled environment. We designed a microfluidic device, the Fungal Highways Device (FHD), that allows us to culture filamentous organisms in unsaturated environments and visualise and quantify bacterial dispersal along hyphal networks at the single-cell level. We showed that Pythium ultimum is essential for Pseudomonas putida movement across an unsaturated environment, and we identified mycelial biomass and hyphal front length as key factors influencing the bacterias movement towards the outlet. We propose that the liquid transport facilitated by P. ultimum mycelium influences the FH behaviour during its interaction with P. putida.

bioengineering↗

bioRxiv2024

Visualizing Liquid Distribution Across Hyphal Networks with Cellular Resolution

Filamentous fungi and fungal-like organisms contribute to a wide range of important ecosystem functions. Evidence has shown the movement of liquid across mycelial networks in unsaturated environments, such as soil. However, tools to investigate liquid movement along hyphae at the level of the single cell are still lacking. Microfluidic devices permit the study of fungal and fungal-like organisms with cellular resolution as they can confine hyphae to a single optical plane, which is compatible with microscopy imaging over longer timescales and allows for precise control of the microchannel environment. The aim of this study was to develop a method that enables the visualization and quantification of liquid movement on hyphae of fungal and fungal-like microorganisms. For this, the Fungal-Fungal Interaction (FFI) microfluidic device was modified to allow for the maintenance of unsaturated microchannel conditions. Fluorescein-containing growth medium solidified with agar was used to track liquid transported by hyphae via fluorescence microscopy. Our key findings highlight the suitability of this novel methodology for the visualization of liquid movement by hyphae over varying time scales and the ability to quantify the movement of liquid along hyphae. Furthermore, we showed that at the cellular level, extracellular hyphal liquid movement can be bidirectional and highly dynamic, uncovering a possible link between liquid movement and hyphal growth characteristics. We envisage that this method can be applied to facilitate future research probing the parameters contributing to hyphal liquid movement and is an essential step for studying the phenomenon of fungal highways.

microbiology↗

bioRxiv2024

Microfluidic platform for microbial spore germination studies in multiple growth conditions

Spores are highly resistant dormant cells, adapted for survival and dispersal, that can withstand unfavourable environmental conditions for extended periods of time and later reactivate. Understanding the germination process of microbial spores is important in numerous areas including agriculture, food safety and health, and other sectors of biotechnology. Microfluidics combined with high-resolution microscopy allows to study spore germination at the single-cell level, revealing behaviours that would be hidden in standard population-level studies. Here, we present a microfluidic platform for germination studies where spores are confined to monolayers inside microchambers, allowing the testing of four growth conditions in parallel. This platform can be used with multiple species, including non-model organisms, and is compatible with existing image analysis software. In this study, we focused on three soil dwellers, two prokaryotes and one fungus, and revealed new insights into their germination. We studied endospores of the model bacterium Bacillus subtilis and demonstrated a correlation between spore density and germination in rich media. We then investigated the germination of the obligate-oxalotrophic environmental bacterium Ammoniphilus oxalaticus in a concentration gradient of potassium oxalate, showing that lower concentrations result in more spores germinating compared to higher concentrations. We also used this microfluidic platform to study the soil beneficial filamentous fungus Trichoderma rossicum, showing for the first time that the size of the spores and hyphae increase in response to increased nutrient availability, while germination times remain the same. Our platform allows to better understand microbial behaviour at the single-cell level, under a variety of controlled conditions. One-Sentence SummaryA microfluidic platform developed for spore germination studies in multiple growth conditions provides new insights into the germination of spores at the cellular level from three soil dwellers.

bioengineering↗

bioRxiv2023

Phospholipid isotope tracing reveals β-catenin-driven suppression of phosphatidylcholine metabolism in hepatocellular carcinoma

Background and AimsActivating mutations in the CTNNB1 gene encoding {beta}-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). HCC with CTNNB1 mutations show profound alterations in lipid metabolism including increases in fatty acid oxidation and transformation of the phospholipidome, but it is unclear how these changes arise and whether they contribute to the oncogenic program in HCC. MethodsWe employed untargeted lipidomics and targeted isotope tracing to quantify phospholipid production fluxes in an inducible human liver cell line expressing mutant {beta}-catenin, as well as in transgenic zebrafish with activated {beta}-catenin-driven HCC. ResultsIn both models, activated {beta}-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid flux analysis in human cells revealed a large reduction in phosphatidylcholine (PC) production rates as assayed by choline tracer incorporation. We developed isotope tracing lipid flux analysis for zebrafish and observed similar reductions in phosphatidylcholine synthesis flux accomplished by sex-specific mechanisms. ConclusionsThe integration of isotope tracing with lipid abundances highlights specific lipid class transformations downstream of {beta}-catenin signaling in HCC and suggests future HCC-specific lipid metabolic targets. SynopsisIn this work, we show by lipid specific isotope tracing that mutations in the oncogene CTNNB1 leads to conserved changes in lipid metabolism in hepatocellular carcinoma. These include the stimulation of fatty acid oxidation and a suppression of phosphorylcholine synthesis.

cancer biology↗

bioRxiv2023

AMF-SporeChip provides new insights into arbuscular mycorrhizal fungal pre-symbiotic hyphal growth dynamics at the cellular level.

O_LIArbuscular mycorrhizal fungi (AMF) form symbiotic associations with the majority of land plants and deliver a wide range of soil-based ecosystem services. Due to their conspicuous belowground lifestyle in a dark environment surrounded by soil particles, much is still to be learned about the influence of environmental (i.e., physical) cues on spore germination, hyphal morphogenesis and hyphopodium formation in AMF. C_LIO_LITo fill existing gaps in AMF knowledge, we developed a new microfluidic platform - termed the AMF-SporeChip - to immobilise Rhizophagus and Gigaspora spores and confront pre-symbiotic hyphae with physical obstacles. In combination with timelapse microscopy, the fungi could be examined at the cellular level and in real-time. C_LIO_LIThe AMF-SporeChip allowed us to acquire movies with unprecedented visual clarity and therefore identify various exploration strategies of AMF pre-symbiotic hyphae. We witnessed anastomosis formation involving directed hyphal growth in a "stop-and-go" manner, yielding visual evidence of pre-anastomosis signalling and decision-making. Remarkably, we also revealed a so-far undescribed reversible cytoplasmic retraction as part of a highly dynamic space navigation. C_LIO_LIOur findings demonstrated how AMF employ an intricate mechanism of space searching, involving reversible cytoplasmic retraction, branching and directional changes. In turn, the AMF-SporeChip is expected to open many future frontiers for AMF research. C_LI

microbiology↗

bioRxiv2020

Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae

The association of the agriculturally significant phytopathogenic fungus Rhizopus microsporus with the bacterial endosymbiont Burkholderia rhizoxinica is a remarkable example of bacteria controlling host physiology and reproduction. Here, we show that a group of transcription activator-like effectors (TALEs) called Burkholderia TALE-like proteins (BATs) from B. rhizoxinica are essential for the establishment of the symbiosis. Mutants lacking BAT proteins are unable to induce host sporulation. Utilising novel microfluidic devices in combination with fluorescence microscopy we observed the accumulation of BAT-deficient mutants in specific fungal side-hyphae with accompanying increased fungal re-infection. High-resolution live imaging revealed septa biogenesis at the base of infected hyphae leading to compartmental trapping of BATdeficient endobacteria. Trapped endosymbionts showed reduced intracellular survival, suggesting a protective response from the fungal host against bacteria lacking specific effectors. These findings underscore the involvement of BAT proteins in maintaining a balance between mutualism and antagonism in bacterial-fungal interactions and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.

microbiology↗

bioRxiv2019

Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch from cell elongation to tip growth through polarization of the growth machinery to a pre-defined root hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain resembles the establishment of cell polarity in other eukaryotic systems [1-3]. Rho-type GTPases of plants (ROPs) are among the first molecular determinants of the RHID [4, 5] and later play a central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the RHID in the cell [7-9] or regulate ROP activity [10-18]. The molecular players that target ROPs to the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the growth machinery assembly in polarizing hair cells and found that positioning of molecular players and outgrowth are temporally separate processes that are each controlled by specific ROP guanine nucleotide exchange factor (GEFs). A functional analysis of trichoblast-specific GEFs revealed GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, while GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role of GEF3 to serve as a membrane landmark during cell polarization. Our findings suggest that morphogenetic programs in plants employ distinct regulatory modules for the alignment and activation of the cellular growth machinery.

cell biology↗

bioRxiv2017

An organ-on-a-chip approach for investigating root-environment interactions in heterogeneous conditions

Plants adapt their root morphology in response to changing environmental conditions, yet it remains largely unknown to what extent developmental adaptations are based on systemic or cell-autonomous responses. We present the dual-flow-RootChip (dfRootChip), a microfluidic organ-on-a-chip platform for asymmetric perfusion of Arabidopsis roots to investigate root-environment interactions under simulated environmental heterogeneity. Applications range from root guidance, monitoring of physiology and development under asymmetric conditions, tracing molecular uptake and selective drug treatments to local inoculation with microbes. We measured calcium responses in roots treated with biotic and abiotic elicitors and observed elicitor-specific signal propagation across the root from treated to untreated cells. We provide evidence for non-autonomous positive regulation of hair growth across the root upon exposure to unfavourable conditions on the opposite side. Our approach sheds light on lateral coordination of morphological adaptation and facilitates studies on root physiology, signalling and development in heterogeneous environments at the organ level.

plant biology↗