The KDEL receptor couples to Gαq/11 to activate Src kinases and regulate transport through the Golgi.

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gα(q/11) which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.

Enhancing Partnerships of Institutions and Journals to Address Concerns About Research Misconduct: Recommendations From a Working Group of Institutional Research Integrity Officers and Journal Editors and Publishers.

Importance: Institutions and journals strive to promote and protect the integrity of the research record, and both groups are equally committed to ensuring the reliability of all published data. Observations: Three US universities coordinated a series of virtual meetings from June 2021 to March 2022 for a working group composed of senior, experienced US research integrity officers (RIOs), journal editors, and publishing staff who are familiar with managing issues of research integrity and publication ethics. The goal of the working group was to improve the collaboration and transparency between institutions and journals to ensure that research misconduct and publication ethics are managed properly and efficiently. Recommendations address the following: identifying proper contacts at institutions and journals, specifying information to share between institutions and journals, correcting the research record, reconsideration of some fundamental research misconduct concepts, and journal policy changes. The working group identified 3 key recommendations to be adopted and implemented to change the status quo for better collaboration between institutions and journals: (1) reconsideration and broadening of the interpretation by institutions of the need-to-know criteria in federal regulations (ie, confidential or sensitive information and data are not disclosed unless there is a need for an individual to know the facts to perform specific jobs or functions), (2) uncoupling the evaluation of the accuracy and validity of research data from the determination of culpability and intent of the individuals involved, and (3) initiating a widespread change for the policies of journals and publishers regarding the timing and appropriateness for contacting institutions, either before or concurrently under certain conditions, when contacting the authors. Conclusions and Relevance: The working group recommends specific changes to the status quo to enable effective communication between institutions and journals. Using confidentiality clauses and agreements to impede sharing does not benefit the scientific community nor the integrity of the research record. However, a careful and informed framework for improving communications and sharing information between institutions and journals can foster better working relationships, trust, transparency, and most importantly, faster resolution to data integrity issues, especially in published literature.

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways.

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.

Group IV phospholipase A(2)alpha controls the formation of inter-cisternal continuities involved in intra-Golgi transport.

The organization of intra-Golgi trafficking and the nature of the transport intermediates involved (e.g., vesicles, tubules, or tubular continuities) remain incompletely understood. It was recently shown that successive cisternae in the Golgi stack are interconnected by membrane tubules that form during the arrival of transport carriers from the endoplasmic reticulum. Here, we examine the mechanisms of generation and the function of these tubules. In principle, tubule formation might depend on several protein- and/or lipid-based mechanisms. Among the latter, we have studied the phospholipase A(2) (PLA(2))-mediated generation of wedge-shaped lysolipids, with the resulting local positive membrane curvature. We show that the arrival of cargo at the Golgi complex induces the recruitment of Group IVA Ca(2+)-dependent, cytosolic PLA(2) (cPLA(2)alpha) onto the Golgi complex itself, and that this cPLA(2)alpha is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport. In contrast, silencing of cPLA(2)alpha has no inhibitory effects on peri-Golgi vesicles. These findings identify cPLA(2)alpha as the first component of the machinery that is responsible for the formation of intercisternal tubular continuities and support a role for these continuities in transport through the Golgi complex.

SRC-dependent signalling regulates actin ruffle formation induced by glycerophosphoinositol 4-phosphate.

The glycerophosphoinositols are diffusible phosphoinositide metabolites reported to modulate actin dynamics and tumour cell spreading. In particular, the membrane permeant glycerophosphoinositol 4-phosphate (GroPIns4P) has been shown to act at the level of the small GTPase Rac1, to induce the rapid formation of membrane ruffles. Here, we have investigated the signalling cascade involved in this process, and show that it is initiated by the activation of Src kinase. In NIH3T3 cells, exogenous addition of GroPIns4P induces activation and translocation of Rac1 and its exchange factor TIAM1 to the plasma membrane; in addition, in in-vitro assays, GroPIns4P favours the formation of a protein complex that includes Rac1 and TIAM1. Neither of these processes involves direct actions of GroPIns4P on these proteins. Thus, through the use of specific inhibitors of tyrosine kinases and phospholipase C (and by direct evaluation of kinase activities and inositol 1,4,5-trisphosphate production), we show that GroPIns4P activates Src, and as a consequence, phospholipase Cgamma and Ca(2+)/calmodulin kinase II, the last of which directly phosphorylates TIAM1 and leads to TIAM1/Rac1-dependent ruffle formation.

The new face of JEM.

We are excited to introduce a new design for JEM.

A time of change.

As Co-Chairs and Executive Editor of The Journal of Experimental Medicine, we write to share some changes that will help us provide JEM authors with the best service we can.

MEF promotes stemness in the pathogenesis of gliomas.

High-grade gliomas are aggressive and uniformly fatal tumors, composed of a heterogeneous population of cells that include many with stem-cell-like properties. The acquisition of stem-like traits might contribute to glioma initiation, growth, and recurrence. Here we investigated the role of the transcription factor myeloid Elf-1 like factor (MEF, also known as ELF4) in gliomas. We found that MEF is highly expressed in both human and mouse glioblastomas and its absence impairs gliomagenesis in a PDGF-driven glioma mouse model. We show that modulation of MEF levels in both mouse neural stem cells and human glioblastoma cells has a significant impact on neurosphere formation. Moreover, we identify Sox2 as a direct downstream target of MEF. Taken together, our studies implicate MEF as a previously unrecognized gatekeeper gene in gliomagenesis that promotes stem cell characteristics through Sox2 activation.

Dishevelled 2 signaling promotes self-renewal and tumorigenicity in human gliomas.

Glioblastoma multiforme is the most common glioma variant in adults and is highly malignant. Tumors are thought to harbor a subpopulation of stem-like cancer cells, with the bulk resembling neural progenitor-like cells that are unable to fully differentiate. Although multiple pathways are known to be involved in glioma tumorigenesis, the role of Wnt signaling has been poorly described. Here, we show that Dishevelled 2 (Dvl2), a key component of the Wnt signaling pathway, is overexpressed in human gliomas. RNA interference-mediated depletion of Dvl2 blocked proliferation and promoted the differentiation of cultured human glioma cell lines and primary, patient-derived glioma cells. In addition, Dvl2 depletion inhibited tumor formation after intracranial injection of glioblastoma cells in immunodeficient mice. Inhibition of canonical Wnt/ß-catenin signaling also blocked proliferation, but unlike Dvl2 depletion, did not induce differentiation. Finally, Wnt5a, a noncanonical Wnt ligand, was also required for glioma cell proliferation. The data therefore suggest that both canonical and noncanonical Wnt signaling pathways downstream of Dvl2 cooperate to maintain the proliferative capacity of human glioblastomas.

A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway.

As with other complex cellular functions, intracellular membrane transport involves the coordinated engagement of a series of organelles and machineries; however, the molecular basis of this coordination is unknown. Here we describe a Golgi-based signalling system that is activated by traffic and is involved in monitoring and balancing trafficking rates into and out of the Golgi complex. We provide evidence that the traffic signal is due to protein chaperones that leave the endoplasmic reticulum and reach the Golgi complex where they bind to the KDEL receptor. This initiates a signalling reaction that includes the activation of a Golgi pool of Src kinases and a phosphorylation cascade that in turn activates intra-Golgi trafficking, thereby maintaining the dynamic equilibrium of the Golgi complex. The concepts emerging from this study should help to understand the control circuits that coordinate high-order cellular functions.

The physiology of membrane transport and endomembrane-based signalling.

Some of the important open questions concerning the physiology of the secretory pathway relate to its homeostasis. Secretion involves a number of separate compartments for which their transport activities should be precisely cross-coordinated to avoid gross imbalances in the trafficking system. Moreover, the membrane fluxes across these compartments should be able to adapt to environmental 'requests' and to respond to extracellular signals. How is this regulation effected? Here, we consider evidence that endomembrane-based signalling cascades that are similar in organization to those used at the plasma membrane coordinate membrane traffic. If this is the case, this would also represent a model for a more general inter-organelle signalling network for functionally interconnecting different intracellular activities, a necessity for the maintenance of cellular homeostasis and to express harmonic global cellular responses.

Multilayer primary epithelial cell culture from bovine conjunctiva as a model for in vitro toxicity tests.

OBJECTIVE: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies. METHODS: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell filters (1.13 cm(2) area). One group of plates was maintained in immersion and another was cultured under air-lifted conditions. Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence. The cell layer was examined after histological processing of the Transwell filter. Ultrastructural analysis was carried out by scanning electron microscopy (SEM). The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures. Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances. RESULTS: Morphological studies showed pure and homogeneous cell cultures. In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e. resembling goblet cells. Air-lifted cultures also showed a tissue-like cellular organization (8-9 layers). Immersion cultures reached a maximum TEER value of around 2.95 kOmega x cm(2) 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 komega x cm(2) 11 days after plating. With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo. CONCLUSIONS: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity.