Gerrit van Meer

Summary: Journals and research institutions have common interests regarding the trustworthiness of research publications but their specific roles and responsibilities differ. These draft recommendations aim to address issues surrounding cooperation and liaison between journals and institutions about possible and actual problems with reported research. The proposals will be discussed at various meetings including the World Conference on Research Integrity in May 2017. We will also consider comments and suggestions posted on this preprint. The main recommendations are that: National registers of individuals or departments responsible for research integrity at institutions should be created. Institutions should develop mechanisms for assessing the validity of research reports that are independent from processes to determine whether individual researchers have committed misconduct. Essential research data and peer review records should be retained for at least 10 years. While journals should normally raise concerns with authors in the first instance, they also need criteria to determine when to contact the institution before, or at the same time as, alerting the authors in cases of suspected data fabrication or falsification to prevent the destruction of evidence. Anonymous or pseudonymous allegations made to journals or institutions should be judged on their merit and not dismissed automatically. Institutions should release relevant sections of reports of research trustworthiness or misconduct investigations to all journals that have published research that was the subject of the investigation.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans‐Golgi network and lysosomes of wild‐type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar‐type H⁺‐translocating ATPase (V‐ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K_i for inhibition of the V‐ATPase by concanamycin A and archazolid A, which share a common binding site on the c‐ring, was lower in glycosphingolipid‐deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V‐ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

Cells have thousands of different lipids. In the plasma membrane, and in membranes of the late secretory and endocytotic pathways, these lipids are not evenly distributed over the two leaflets of the lipid bilayer. The basis for this transmembrane lipid asymmetry lies in the fact that glycerolipids are primarily synthesized on the cytosolic and sphingolipids on the noncytosolic surface of cellular membranes, that cholesterol has a higher affinity for sphingolipids than for glycerolipids. In addition, P4-ATPases, “flippases,” actively translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine to the cytosolic surface. ABC transporters translocate lipids in the opposite direction but they generally act as exporters rather than “floppases.” The steady state asymmetry of the lipids can be disrupted within seconds by the activation of phospholipases and scramblases. The asymmetric lipid distribution has multiple implications for physiological events at the membrane surface. Moreover, the active translocation also contributes to the generation of curvature in the budding of transport vesicles.

Homo‐FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo‐FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo‐FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo‐FRET signals in protein‐ and lipid‐clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K‐ras, as well as for EGF receptor clustering in the plasma membrane.

The Keystone Symposium on the Molecular Basis for Biological Membrane Organization and Dynamics held in January this year offered new insights into the molecular machines at work in cells. Topics included the machinery responsible for the dynamic shape of organelles, the budding and fusion of vesicular carriers, and the intricate sorting systems that ensure the correct delivery of cellular components.