Biology subjects

Leng, W.

Publications and source records attributed to Leng, W..

9 recordsLinked to original sources

bioRxiv2025

Visualizing sub-organellar lipid distribution using correlative light and electron microscopy

Lipids and proteins compartmentalize biological membranes into nanoscale domains which are crucial for signaling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here, we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labeling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules, and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.

cell biology↗

bioRxiv2023

Cell free expression in proteinosomes prepared from native protein-PNIPAAm conjugates

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalisation with an open, semi-permeable membrane. Critically, to date, there have been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, we present a novel approach to the fabrication of proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. We show that these native proteinosomes offer an excellent alternative as artificial cell chassis. Significantly, the native proteinosomes are stable under high salt conditions and can consequently support cell free transcription and translation. The native proteinosomes offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, we demonstrate the integration of proteinosomes into higher order cellular architectures with membrane free compartments and liposomes. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

synthetic biology↗

bioRxiv2021

A local insulin reservoir ensures developmental progression in condition of nutrient shortage in Drosophila

Insulin/IGF signalling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides (Ilp) is produced by brain neurosecretory cells and exerts systemic functions. Here, we describe the local uptake and storage of Ilps in the Corpora Cardiaca (CC), a group of alpha cell homolog that produces the glucagon-like hormone AKH. Dilp uptake relies on the expression of Impl2, an IGF-BP that accumulates in the CCs. During nutrient shortage, this specific reserve of Ilps is released and activates IIS in a paracrine manner in the prothoracic gland, securing accelerated entry into pupal development through the production of the steroid hormone ecdysone. We therefore uncover a sparing mechanism whereby local Ilp storage and release activates the production of steroids and ensures early developmental progression in adverse food conditions. Highlights- Dilps are uptaken by CC cells through the IGF-BP Imp-L2 - the CC-Dilp store is released upon nutrient shortage and activates IIS through CC projections on the PG - upon nutrient shortage, IIS activation in the PG ensures an accelerated transition from larval feeding stage to metamorphosis.

developmental biology↗

bioRxiv2020

A WDR35/IFT121-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins a and {beta}', and demonstrate an accumulation of coat-less vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.

cell biology↗

bioRxiv2019

Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting {beta}Heavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

cell biology↗

bioRxiv2018

Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. The mechanism by which starved cells regulate the process of protein synthesis is largely unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants of eIF2B suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through protein assembly is a widespread mechanism that allows cells to adapt to fluctuating environments.

cell biology↗

bioRxiv2018

Adaptive reorganization of the cytoplasm upon stress in budding yeast

Yeast cells, when exposed to stress, can enter a protective state in which cell division, growth and metabolism are downregulated. They remain viable in this state until nutrients become available again. How cells enter this protective survival state and what happens at a cellular and subcellular level is largely unknown. In this study, we used electron tomography to investigate the stress-induced ultrastructural changes in the cytoplasm of yeast cells. After ATP depletion, we observed a significant cytosolic compaction and an extensive cytoplasmic reorganization, as well as the emergence of distinct membrane-bound and membrane-less organelles. By using correlative light and electron microscopy (CLEM), we further demonstrate that one of these membrane-less organelles is generated by the reversible polymerization into large bundles of filaments of the eukaryotic translation initiation factor 2B (eIF2B), an essential enzyme in the initiation of protein synthesis. The changes we observe are part of a stress-induced survival strategy, allowing yeast cells to save energy, protect proteins from degradation, and inhibit protein functionality by forming assemblies of said proteins.

cell biology↗

bioRxiv2018

The splicing regulator Prp31 prevents retinal degeneration in Drosophila by regulating Rhodopsin levels

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery, but the molecular consequences that link defects in splicing factor genes to the aetiology of the disease remain to be elucidated. Mutations in PRPF31, one of the splicing factors, are linked to RP11. To get insight into the mechanisms by which mutations in this gene lead to retinal degeneration, we induced mutations in the Drosophila orthologue Prp31. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Prp31 mutant flies show a high degree of phenotypic variability, similar as reported for human RP11 patients. Degeneration is associated with increased accumulation of rhodopsin 1, both in the rhabdomere and in the cell body. In fact, reducing rhodopsin levels by raising animals in a carotenoid-free medium not only suppressed rhodopsin accumulation, but also retinal degeneration. In addition, our results underscore the relevance of eye color mutations on phenotypic traits, in particular whilst studying a complex process such as retinal degeneration.\n\nArticle SummaryRetinitis pigmentosa (RP) is a human disease affecting 1.6 million people worldwide. So far >50 genes have been identified that are causally related to RP. Mutations in the splicing factor PRPF31 are linked to RP11. We induced mutations in the Drosophila orthologue Prp31 and show that flies heterozygous for Prp31 undergo light-dependent retinal degeneration. Degeneration is associated with increased accumulation of the light-sensitive molecule, rhodopsin 1. In fact, reducing rhodopsin levels by dietary intervention suppressed retinal degeneration. We believe that this model will help to better understand the aetiology of the human disease.

genetics↗

bioRxiv2017

Mutations in the Drosophila splicing regulator Prp31 as a model for Retinitis pigmentosa 11

Retinitis pigmentosa is a clinically heterogeneous disease affecting 1.6 million people worldwide. A growing number of identified disease-causing genes are associated with the spliceosome, but the molecular consequences that link defects in splicing factor genes to the aetiology of the disease remain to be elucidated. In this paper, we present a Drosophila model for Retinitis pigmentosa 11, a human disease caused by mutations in the splicing factor PRPF31. Here, we induced mutations in the Drosophila orthologue Prp31. Mutant flies are viable and show a normal eye phenotype when kept under regular light conditions. However, when exposed to constant light, photoreceptors of mutant flies degenerate, thus resembling the human disease phenotype. Degeneration could be shown to be associated with increased oxidative stress. This increase was in agreement with severe dysregulation of genes involved in oxidation/reduction processes, as revealed by high throughput transcriptome sequencing. In fact, light induced photoreceptor cell degeneration could be attenuated by experimentally reducing oxidative stress. A comparable decrease in retinal degeneration was achieved by raising mutant larvae on a vitamin A-depleted medium, thereby reducing rhodopsin levels. Finally, transcriptome data further uncovered an overall retention of introns in mRNAs. Among those, mRNAs of genes involved in synapse assembly, growth and stability were most prominent. These results point to a multifactorial genesis of light induced degeneration in retinae of Prp31 mutant flies, including transcriptional and splicing dysregulation, oxidative stress and defects in vitamin A metabolism.

genetics↗