Background Nearly 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. Methods We 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. Results Strikingly, 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. Conclusions Together, 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.

Zn(2+) is an essential micronutrient and an important ionic signal whose excess, as well as scarcity, is detrimental to cells. Free cytoplasmic Zn(2+) is controlled by a network of Zn(2+) transporters and chelating proteins. Recently, lysosomes became the focus of studies in Zn(2+) transport, as they were shown to play a role in Zn(2+)-induced toxicity by serving as Zn(2+) sinks that absorb Zn(2+) from the cytoplasm. Here, we investigated the impact of the lysosomal Zn(2+) sink on the net cellular Zn(2+) distribution and its role in cell death. We found that lysosomes played a cytoprotective role during exposure to extracellular Zn(2+). Such a role required lysosomal acidification and exocytosis. Specifically, we found that the inhibition of lysosomal acidification using Bafilomycin A1 (Baf) led to a redistribution of Zn(2+) pools and increased apoptosis. Additionally, the inhibition of lysosomal exocytosis through knockdown (KD) of the lysosomal SNARE proteins VAMP7 and synaptotagmin VII (SYT7) suppressed Zn(2+) secretion and VAMP7 KD cells had increased apoptosis. These data show that lysosomes play a central role in Zn(2+) handling, suggesting that there is a new Zn(2+) detoxification pathway.

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.

Zinc is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular zinc is controlled by zinc-chelating proteins and by zinc transporters. The recent identification of zinc permeability of the lysosomal ion channel TRPML1 (transient receptor potential mucolipin 1), and the evidence of abnormal zinc levels in cells deficient in TRPML1, suggested a role for TRPML1 in zinc transport. In the present study 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 (small interfering RNA)-driven TRPML1 KD (knockdown) leads to the build-up of large cytoplasmic vesicles positive for LysoTracker™ and zinc staining, when cells are exposed to high concentrations of zinc. We now show that lysosomal enlargement and zinc build-up in TRPML1-KD cells exposed to zinc are ameliorated by KD of the zinc-sensitive transcription factor MTF-1 (metal-regulatory-element-binding transcription factor-1) or the zinc transporter ZnT4. TRPML1 KD is associated with a build-up of cytoplasmic zinc and with enhanced transcriptional response of mRNA for MT2a (metallothionein 2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay zinc leak from the lysosomes into the cytoplasm. These results underscore a role for TRPML1 in zinc metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate zinc translocation between the cytoplasm and lysosomes.