A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis.

Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e. ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g. Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g. Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.

Interactions of pharmacologically active snake venom sPLA2 with different cell lines.

Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity - hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits - a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential.

Ciliary length variations impact cilia-mediated signaling and biological responses.

Primary cilia are thin hair-like organelles that protrude from the surface of most mammalian cells. They act as specialized cell antennas that can vary widely in response to specific stimuli. However, the effect of changes in cilia length on cellular signaling and behavior remains unclear. Therefore, we aimed to characterize the elongated primary cilia induced by different chemical agents, lithium chloride (LiCl), cobalt chloride (CoCl2), and rotenone, using human retinal pigmented epithelial 1 (hRPE1) cells expressing ciliary G protein-coupled receptor (GPCR), melanin-concentrating hormone (MCH) receptor 1 (MCHR1). MCH induces cilia shortening mainly via MCHR1-mediated Akt phosphorylation. Therefore, we verified the proper functioning of the MCH-MCHR1 axis in elongated cilia. Although MCH shortened cilia that were elongated by LiCl and rotenone, it did not shorten CoCl2-induced elongated cilia, which exhibited lesser Akt phosphorylation. Furthermore, serum readdition was found to delay cilia shortening in CoCl2-induced elongated cilia. In contrast, rotenone-induced elongated cilia rapidly shortened via a chopping mechanism at the tip of the cilia. Conclusively, we found that each chemical exerted different effects on ciliary GPCR signaling and serum-mediated ciliary structure dynamics in cells with elongated cilia. These results provide a basis for understanding the functional consequences of changes in ciliary length.

The N-terminal Leu-Pro-Gln sequence of Rab34 is required for ciliogenesis in hTERT-RPE1 cells.

We have previously shown that Rab34 is an important regulator of ciliogenesis and that its unique long N-terminal region (amino acids 1-49) is essential for ciliogenesis in certain cultured mammalian cells. In the present study, we performed an in-depth deletion analysis of the N-terminal region of Rab34 together with Ala-based site-directed mutagenesis to identify the essential amino acids that are required for serum-starvation-induced ciliogenesis in hTERT-RPE1 cells. The results showed that a Rab34 mutant lacking an N-terminal 18 amino acids and a Rab34 mutant carrying an LPQ-to-AAA mutation (amino acids 16-18) failed to rescue a Rab34-KO phenotype (i.e., defect in ciliogenesis). Our findings suggest that the LPQ sequence of Rab34 is crucial for ciliogenesis in hTERT-RPE1 cells.Abbreviations: AA, amino acid(s); ac-Tub, acetylated tubulin; bsr, blasticidin S-resistant gene; HRP, horseradish peroxidase; hTERT-RPE1, human telomerase reverse transcriptase retinal pigment epithelium 1; KO, knockout; NS, not significant; PBS, phosphate-buffered saline; puro, puromycin-resistant gene.

ALMS1 Regulates TGF-ß Signaling and Morphology of Primary Cilia.

In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-ß/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFß-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome.

On-section correlative light and electron microscopy of large cellular volumes using STEM tomography.

The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5µm.

Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines.

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.