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Cell biology

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bioRxiv2018

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

cell biology↗

bioRxiv2018

Plate-based assay of AP-3 traffic in budding yeast

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening. SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathways importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

cell biology↗

bioRxiv2018

LRH-1 Mitigates Intestinal Inflammatory Disease by Maintaining Epithelial Homeostasis and Cell Survival

Epithelial dysfunction and loss of intestinal crypts are defining features of inflammatory bowel disease (IBD). However, current therapies primarily target the immune system and not the epithelium. The nuclear receptor LRH-1 encoded by Nr5a2 is expressed in intestinal epithelium and is thought to contribute to epithelial renewal. Here we investigate how loss and gain of LRH-1 impacts the intestinal epithelium in healthy and inflammatory conditions. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death and weakens the epithelial barrier. Loss of LRH-1 also distorts the cellular composition of the epithelium, resulting in an expansion of Paneth and goblet cells, and a decrease in enteroendocrine cells. Human LRH-1 (hLRH-1) not only rescues epithelial integrity, but when overexpressed, mitigates inflammatory damage in mouse and human intestinal organoids, including those from IBD patients. Finally, hLRH-1 greatly reduces disease severity in a mouse model of T cell-mediated colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNF-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.

cell biology↗

bioRxiv2018

Comparison of antioxidant activity between cyanidin-3-O-glucoside (C3G) liposome and cyanidin-3-O-glucoside (C3G) in 2D and 3D cell cultures

The 2D cell culture is the predominant in vitro model for numerous studies. However, 2D cell cultures may not accurately reflect the functions of three-dimensional (3D) tissues, which have extensive cell-cell and cell-matrix interactions; thus, using 2D cell cultures may lead to inaccurate experimental results. Therefore, to obtain adequate and detailed information about the antioxidant activity of cyanidin-3-O-glucoside (C3G) and C3G liposomes in the 2D and 3D cell culture models, we used in this study H2O2 to construct the cell damage model and assess the antioxidant activity of C3G and C3G liposomes on Caco-2 cells cultured in the 3D model. We also measured the cell viability, cell morphology, and activity of glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content of Caco-2 cells treated with H2O2, C3G, and C3G liposomes. Results showed that cells cultured in the 3D culture model formed a 3D structure and tight spheroids and showed increased cell activity and IC50. The C3G and C3G liposomes can enhance the activity of GSH, SOD, and T-AOC but decrease the MDA content. At the same time, the effect was more obvious in the 3D cell culture model than in the cells cultured in the 2D model. This study revealed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. A realistic mechanism study of antioxidant activity of C3G and C3G liposomes in the 3D cell model, which acts as an intermediate stage bridging the in vitro 2D and in vivo models, was observed.

cell biology↗

bioRxiv2018

Enterovirus 71 structural viral protein 1 promotes mouse Schwann cell autophagy via endoplasmic reticulum stress-mediated peripheral myelin protein 22 upregulation

Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease-related deaths due to fatal neurological complications. The clinical observations and animal models found the early invasion of nervous system, and the demyelinating phenomenon was observed. As one of the receptors of EV71 structural viral protein 1 (VP1), SCARB2 mainly exists on the myelin sheath. EV71 VP1 can promote viral replication through inducing autophagy in neuron cells. This study aims to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells (MSCs). An EV71 VP1-expressing vector (pEGFP-C3-VP1) was generated and transfected into MSCs. Transmission electron microscopy (TEM) and Western blot analysis of the autophagy marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) were used to assess autophagy in the cells. Real-time PCR and immunofluorescent staining were performed to determine the expression of PMP22. Small interfering RNA against PMP22 was employed to investigate the role of PMP22 in MSCs autophagy. Selective endoplasmic reticulum (ER) stress inhibitor salubrinal (SAL) was employed to determine whether PMP22 is mediated by ER stress. Our results demonstrated that VP1 played a promotive role in MSC autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3B and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by SAL. VP1 promotes MSC autophagy through upregulating ER stress-mediated PMP22 expression. VP1/ER stress/ PMP22 axis in autophagy may be a potential therapeutic target for EV71 infection-induced fatal neuronal damage.

cell biology↗

bioRxiv2018

Direct reprogramming of human epithelial cells into organoids by miR-106a-3p

The 3D cultures provide more insight into cell-to-cell and cell-to-matrix interactions, better mimicking the environment where stem cells reside compared to traditional 2D cultures. Although the precise molecular pathways involved in the regulation of stem and progenitor cell fate remain unknown, it is widely accepted that transcription factors play a crucial role as intrinsic regulators in these fate decisions. In this study, we carried out a microRNA screen to track the behaviour of adult stem/progenitor cells derived from human mammary epithelial cells grown in 3D cultures. We identified miR-106a-3p, which enriches the adult stem cell-like lineage and promotes the expansion of 3D cultures. Transcriptomic analysis showed that this miRNA regulates transcription factors such as REST, CBFB, NF-YA, and GATA3, thereby enhancing the maintenance of adult stem/progenitor cells in human epithelial cells. These data reveal a clear transcriptional program that governs the maintenance of adult stem/progenitor cells and controls their fate.

cell biology↗

bioRxiv2018

Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in Drosophila melanogaster

Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. Here, we report a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. We show that reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. We also report that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. Our results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation.\n\nBrief summary statementThe authors show that the membrane phospholipid PIP2, and the kinase that produces PIP2 called Skittles, are needed for normal ciliary transition zone morphology and function in the Drosophila male germline.

cell biology↗

bioRxiv2018

Inward tubulation of the plasma membrane expedites membrane exchange and receptor presentation

The plasma membrane is a crucial barrier between the cell and its external environment, and it also enables efficient passage of materials and information. Membrane vesicle trafficking allows precise delivery of materials but is rather inefficient. The mechanism for efficient membrane exchange remains elusive. Here we describe inward tubulation of the plasma membrane (PM tubes) that extends deep into the cytoplasm. These widespread PM tubes elongate along microtubules and are stabilized by actin filaments and cholesterol. PM tubes are preferred sites for connection between the endoplasmic reticulum and the plasma membrane. PM tubes facilitate receptor presentation at the surface of cells, possibly also shortening the distance for transported cargo to reach the external environment.\n\nIn BriefA new type of tubular membrane structures was discovered in cells, revealing a shortcut that cells employ to expedite material exchange with their external environment.\n\nHighlightsO_LIInward tubulation of the plasma membrane (PM tubes), transiently interacts with the Golgi apparatus\nC_LIO_LIMicrotubule side-binding proteins pull PM tubes, while actin filaments and cholesterol stabilize PM tubes\nC_LIO_LIPM tubes are preferred sites where ER-PM contacts form in response to increased cytoplasmic calcium concentration\nC_LIO_LIPM tubes are preferred sites for the surface presentation of GLUT1 upon glucose deprivation\nC_LI

cell biology↗

bioRxiv2018

MEF2C is a new regulator of the human articular chondrocyte phenotype

MEF2C plays a role in diverse tissues, most notably heart, brain, eyes and developing bones. Here we report for the first time that MEF2C is present and active in the permanent articular cartilage in humans which lines and protects our joints throughout life. We show that MEF2C directly targets cartilage master regulator gene SOX9, and SOX9, in turn, regulates MEF2C in a novel positive feedback loop maintaining high expression levels of both transcription factors, and consequently stabilising the articular chondrocyte phenotype and helping prevent hypertrophy and subsequent calcification and vascularisation. We propose that MEF2C and SOX9 may show similar cooperative activity in other tissues, and across a range of adult murine tissues we found co-expression of both transcription factors in cartilage, trachea, brain, eyes and heart. Strikingly, all of these tissues are prone to calcification and further study of MEF2C/SOX9 cooperativity in these organs will be revealing.

cell biology↗

bioRxiv2018

A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which give rise to micronucleated daughter cells.

cell biology↗

bioRxiv2018

Chimeric antigen receptors that trigger phagocytosis

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity, and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcR{gamma} robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing macrophages reduce cancer cell number in co-culture by over 40%.\n\nSummaryWe report the first Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that promote engulfment of antigen-coated particles and cancer cells.

cell biology↗

bioRxiv2018

Fission yeast cytokinesis requires a putative mechanosensitive channel Pkd2p

Force plays a central role in separating daughter cells during cytokinesis, the last stage of cell division. However, the mechanism of force-sensing during cytokinesis remains unknown. Here we discovered that Pkd2p, a putative force-sensing TRP channel, localizes to the cleavage furrow during cytokinesis of the fission yeast, Schizosaccharomyces pombe. Pkd2p, whose human homologues are associated with Autosomal Polycystic Kidney Disease, is an essential protein whose localization depends on the contractile ring and the secretory pathway. We identified and characterized a novel pkd2 mutant pkd2-81KD. The pkd2 mutant cells show signs of osmotic stress, including temporary shrinking, paused turnover of the cytoskeletal structures and hyper-activated MAPK signaling. During cytokinesis, although the contractile ring constricts more rapidly in the pkd2 mutant than the wild-type cells (50% higher), the cell separation in the mutant is slower and often incomplete. These cytokinesis defects are also consistent with mis-regulated turgor pressure. Lastly, the pkd2 mutant exhibits strong genetic interactions with two mutants of the SIN pathway, a signaling cascade essential for cytokinesis. We propose that Pkd2p modulates osmotic homeostasis and is potentially a novel regulator of cytokinesis.\n\nHighlight summary for TOCFission yeast TRP channel Pkd2p is the homologue of human polycystins. The pkd2 mutant exhibits defects in the contractile ring closure and cell separation during cytokinesis. This essential protein localizes to the cleavage furrow where it likely regulates osmotic homeostasis during cytokinesis.

cell biology↗

bioRxiv2018

A Composition-Dependent Molecular Clutch Between T Cell Signaling Clusters and Actin

Biomolecular condensates play important roles in eukaryotic cells by concentrating molecules into foci without a surrounding membrane. During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of the adaptor protein LAT and its binding partners Grb2, Sos1, SLP-76, Nck and WASP. These condensates move radially at the IS, traversing a radially-oriented and then a concentric actin network. To understand the persistent radial movement, we biochemically reconstituted LAT condensates with mobile actomyosin filaments. We found that basic regions of Nck and N-WASP promote strong association and co-movement of LAT condensates with actin. Condensates lacking these components were instead propelled by steric interactions. In cells, LAT condensates lost Nck while traversing the boundary between the two actin networks, and condensates engineered to constitutively bind actin moved aberrantly. We propose that Nck and WASP form a clutch between LAT condensates and actin, and changes in composition enable condensate movement by distinct actin networks in different regions of the IS.

cell biology↗

bioRxiv2018

Automated Tracking of Biopolymer Growth and Network Deformation with TSOAX

Studies of how individual semi-flexible biopolymers and their network assemblies change over time reveal dynamical and mechanical properties important to the understanding of their function in tissues and living cells. Automatic tracking of biopolymer networks from fluorescence microscopy time-lapse sequences facilitates such quantitative studies. We present an open source software tool that combines a global and local correspondence algorithm to track biopolymer networks in 2D and 3D, using stretching open active contours. We demonstrate its application in fully automated tracking of elongating and intersecting actin filaments, detection of loop formation and constriction of tilted contractile rings in live cells, and tracking of network deformation under shear deformation.

cell biology↗

bioRxiv2018

Three-photon light-sheet fluorescence microscopy

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 {micro}m diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

cell biology↗

bioRxiv2018

A positive feedback loop drives centrosome maturation in flies

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn form an extensive scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If Spd-2 cannot recruit Polo to the expanding scaffold, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. We conclude that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic centrosome in flies.

cell biology↗

bioRxiv2026

Proteolytic Remodeling of Cargo Receptor Networks by RHBDL4 Tunes Secretory Pathway Flux

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

cell biology↗

bioRxiv2026

Red and blue light cues drive contrasting remodeling of lipophilic metabolites and photophysiology in natural benthic diatom biofilms

Intertidal mudflats are low hydrodynamic energy environments hosting microphytobenthic communities that experience strong spatiotemporal variability in light regimes, including changes in spectral quality and light intensity that can lead to cellular photooxidative stress. To cope with these fluctuations, autotrophs exhibit diverse and highly plastic adaptations that are often species-dependent and shaped by their ecological niches. This study investigates photophysiological responses and metabolic remodeling in a diatom assemblage originating from a natural winter microphytobenthic biofilm under contrasting red and blue light intensities. To this end, photosynthetic parameters were monitored alongside changes in lipophilic metabolites, including untargeted lipids and lipophilic pigments. While few metabolites showed temporal remodeling, rapid and contrasting changes were observed within 30 minutes in response to both spectral quality and light intensity. Red light treatments induced broader remodeling of lipophilic metabolites than blue light, whereas blue light appeared to have a greater impact on photosynthetic parameters. Moreover, red light induced xanthophyll-cycle responses comparable to those observed under blue light at equivalent incident intensity. We discuss these metabolic responses in relation to diatom photoadaptive strategies, placing these findings within the intertidal environmental framework. This work further underlines the importance of understanding rapid metabolic plasticity in coping with light fluctuations, providing new insights into the photoregulatory strategies of natural microphytobenthic communities.

cell biology↗