Synopsis Zn is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular Zn is controlled by Zn-chelating proteins and by Zn transporters. The recent identification of Zn permeability of the lysosomal ion channel TRPML1, and the evidence of abnormal Zn levels in cells deficient in TRPML1, suggested a role for TRPML1 in Zn transport. Here we provide new evidence for such a role and identify additional cellular components responsible for it. In agreement with the previously published data, an acute siRNA-driven TRPML1 knockdown (KD) leads to the buildup of large cytoplasmic vesicles positive for Lysotracker and Zn staining, when cells are exposed to high concentrations of Zn. We now show that lysosomal enlargement and Zn buildup in TRPML1-KD cells exposed to Zn are ameliorated by KD of the Zn-sensitive transcription factor MTF-1 or Zn transporter ZnT4. TRPML1 KD is associated with a buildup of cytoplasmic Zn and with enhanced transcriptional response of mRNA for metallothionein 2a (MT2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay Zn leak from the lysosomes into the cytoplasm. These data underscore a role for TRPML1 in Zn metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate Zn translocation between the cytoplasm and lysosomes.

Zn2+ is an essential micronutrient and an important ionic signal whose excess, as well as scarcity, is detrimental to cells.

BackgroundNearly all cells have a primary cilia on their surface, which functions as a cellular antennae. Primary cilia assembly begins intracellularly and eventually emerges extracellularly. However, current ciliogenesis assays, which detect cilia length and number, do not monitor ciliary stages.MethodsWe developed a new assay that detects antibody access to a fluorescently tagged ciliary transmembrane protein, which revealed three ciliary states: classified as ‘inside,’ ‘outside,’ or ‘partial’ cilia.ResultsStrikingly, most cilia in RPE cells only partially emerged and many others were long and intracellular, which would be indistinguishable by conventional assays. Importantly, these states switch with starvation-induced ciliogenesis and the cilia can emerge both on the dorsal and ventral surface of the cell. Our assay further allows new molecular and functional studies of the ‘ciliary pocket,’ a deep plasma membrane invagination whose function is unclear. Molecularly, we show colocalization of EHD1, Septin 9 and glutamylated tubulin with the ciliary pocket.ConclusionsTogether, the IN/OUT assay is not only a new tool for easy and quantifiable visualization of different ciliary stages, but also allows molecular characterization of intermediate ciliary states.Electronic supplementary materialThe online version of this article (doi:10.1186/s13630-016-0044-2) contains supplementary material, which is available to authorized users.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

The critical processes driving successful research translation remain understudied. We describe a mixed-method case study protocol for analyzing translational research that has led to the successful development and implementation of innovative health interventions. An overarching goal of these case studies is to describe systematically the chain of events between basic, fundamental scientific discoveries and the adoption of evidence-based health applications, including description of varied, long-term impacts. The case study approach isolates many of the key factors that enable the successful translation of research into practice and provides compelling evidence connecting the intervention to measurable changes in health and medical practice, public health outcomes, and other broader societal impacts. The goal of disseminating this protocol is to systematize a rigorous approach, which can enhance reproducibility, promote the development of a large collection of comparable studies, and enable cross-case analyses. This approach, an application of the “science of translational science,” will lead to a better understanding of key research process markers, timelines, and potential points of leverage for intervention that may help facilitate decisions, processes, and policies to speed the sustainable translational process. Case studies are effective communication vehicles to demonstrate both accountability and the impacts of the public’s investment in research.