A novel live-cell imaging assay reveals regulation of endosome maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

Assessment of intestinal macromolecular absorption in young piglets to pave the way to oral vaccination: preliminary results

The small intestine of the piglet has evolved to be permeable immediately after birth to facilitate the uptake of colostrum-derived immunoglobulins as well as other macromolecules, and cells. However, the precise timing of gut closure in today’s precocious pig is not known. We gavaged piglets immediately after birth and at 1-h after birth with Cy5-labeled Ovalbumin (Cy5-Ova) then harvested their small intestine’s 6–7 h later. To assess localization of Cy5-Ova in the small intestinal epithelial cells, we performed immunohistochemistry using a basolateral surface marker and a recycling endosome marker called pIgR, the late endosomal marker Rab7, and the lysosomal marker LAMP-1. Cy5-Ova co-localized with Rab7 and LAMP-1 in the duodenum and jejunum of 0-h old and 1-h old gavaged piglets, but only in the ileum of 0-h gavaged piglets. These data suggest that movement of Cy5-Ova through the late endosomes to the lysosomes was much reduced in the ileum of 1-h gavaged piglets. Cy5-Ova was largely present in epithelial cell digestive and transport vacuoles, but it did not colocalize with pIgR-positive endosomes in 0-h and 1-h gavaged piglets. Differences in macromolecular uptake across the different regions of the small intestine after only 1-h may be due to prior processing of colostral macromolecules, changes in the intestine due to initiation of colonization by microflora and/or the initiation of gut-closure. Understanding the relationship between the localization of Cy5-Ova and small intestinal permeability may contribute to establishing whether oral vaccination in the newborn can capitalize on the transient permeability before gut closure to promote immune protection.

EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions

The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.

Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

Tight-junction (TJ) proteins are essential for establishing the barrier function between neighbor epithelial cells, but also for recognition of pathogens or cell migration. Establishing the expression pattern and localization of different TJ proteins will help to understand the development and physiology of the airway. Here we identify that the junctional adhesion molecule 3 (Jam3) expression is restricted to multiciliated cells (MCCs) in the airway epithelium. In vitro, Jam3 expression varies along airway basal stem cell (BSC) differentiation and upon DAPT treatment or IL6 exposure. However, Jam3 is not required for BSC differentiation to specific cell types. In addition, we found that MCC lacking Jam3 display normal cilia morphology and cilia beating frequency with a delay in BB assembly/positioning in MCCs during differentiation. Remarkably, Jam3 in MCC is mostly localized to subapical organelles, which are negative for the apical recycling endosome marker Rab11 and positive for EEA1. Our data show that Jam3 expression is connected to mature MCC in the airway epithelium and suggest a Jam3 role unrelated to its known barrier function.

The Effects of CD82 Palmitoylation on the Metabolic Pathways of EGFR and c-Met in Breast Cancer Cells and their Molecular Mechanisms

Background: As a tumor metastasis suppressor, tetraspanin CD82 is reduced in many malignant tumors and often affects the composition of tumor microenvironment by changing the heterogeneity of cell membrane. EGFR or c-Met signaling pathway can regulate the metastasis ability of tumor cells and participate in the formation of tetraspanin web. The study of CD82 palmitoylation modification and metabolic pathway of tumor related molecules in tumor cells is still incomplete. This article focuses on studying the expression and distribution of EGFR and c-Met in cancer cells as well as related metabolic pathways and their molecular mechanisms after studying different palmitoylation site mutations.Methods: Western blot and immunofluorescence methods were used to detect the distribution of EGFR in breast cancer MDA-MB-231 cells after different CD82 palmitoylation site mutations. Then use the immunoprecipitation method to determine the interaction relationship between the molecules and the molecular mechanism.Results: We found that when CD82 combined with palmitoylation mutation at Cys5+Cys74 can enhance the internalization of EGFR, but has no effect on the expression and location of c-Met. When CD82 is combined with palmitoylation mutation at the Cys5+Cys74 site, with the assistance of tubulin, EGFR is internalized and strengthened by direct binding to CD82 and a large number of localizations on the recycling endosome. By forming the EGFR/CD82/Rab11a/FIP2 complex, it is metabolized through the circulation pathway, and re-expression of EGFR and CD82 on the cell membrane.Conclusions: From our results, we can demonatrate that CD82 palmitoylation mutation can change the distribution of EGFR in breast cancer cells, which may provide new ideas for breast cancer treatment.

The Rab11-Family Interacting Proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

After invasion of mammalian cells, the parasite Toxoplasma gondii multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that intravacuolar Toxoplasma interacts with many host cell organelles, especially recycling endosomes, and further manipulates the host endocytic recycling through the sequestration of Rab11 vesicles into the PV. Mammalian Rab-PV interactions are likely mediated by Toxoplasma and host proteins that remain to be identified. In this context, we have examined the specificity of host Rab vesicle interaction with the PV by monitoring the recruitment of subtypes of Rab11 vesicles differing in their composition in Rab11-Family Interacting Proteins (FIPs). We found that vesicles with FIPs from Class I (FIP1C, FIP2, FIP5) or Class II (FIP3, FIP4) are distributed at the PV and detected to varying degrees inside the PV. The PV delivery of vesicles with FIPs from Class I, but not Class II, is Rab11-dependent. In addition to Rab11, FIP3 binds to Arf6, and vesicles associated with FIP3-Arf6 complexes are observed within the PV. Binding of FIP3 to either Rab11 or Arf6 significantly increases the internalization of vesicles into the PV. These data point to a selective process of host recycling endosome recognition and scavenging mediated by Toxoplasma.

A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.

The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium

In emerging epithelial tissues, cells undergo dramatic rearrangements to promote tissue shape changes. Dividing cells remain interconnected via transient cytokinetic bridges. Bridges are cleaved during abscission and currently, the consequences of disrupting abscission in developing epithelia are not well understood. We show that the Rab GTPase Rab25 localizes near cytokinetic midbodies and likely coordinates abscission through endomembrane trafficking in the epithelium of the zebrafish gastrula during epiboly. In maternal-zygotic Rab25a and Rab25b mutant embryos, morphogenic activity tears open persistent apical cytokinetic bridges that failed to undergo timely abscission. Cytokinesis defects result in anisotropic cell morphologies that are associated with a reduction of contractile actomyosin networks. This slows cell rearrangements and alters the viscoelastic responses of the tissue, all of which likely contribute to delayed epiboly. We present a model in which Rab25 trafficking coordinates cytokinetic bridge abscission and cortical actin density, impacting local cell shape changes and tissue-scale forces.