Macrophage polarization contributes to the efficacy of an oncolytic HSV-1 targeting human uveal melanoma in a murine xenograft model.

Uveal melanoma (UM), the most common primary malignant tumor of the eye in adults, is difficult-to-treat. UM has a relatively high mortality secondary to distant metastasis and poor overall survival with existing therapies. The oncolytic virus herpes simplex virus type-1 (HSV-1) has been approved for clinical use in melanoma. This double-stranded DNA virus was suspected to directly activate lysis specifically in neoplastic cells. We tested the antitumor efficacy of recombinant oncolytic HSV-1-EGFP (oHSV-EGFP) in UM and characterized the local and systemic antitumor innate immune response in a murine xenograft model. We first determined the efficacy of the oncolytic virus in 92.1, MUM2B and MP41 cell lines. In murine xenograft models, oHSV-EGFP reduced intraocular tumors as well as systemic subcutaneous tumors. A significant change in cytokines was observed in viral infected cells, especially a rise in IFNγ. In vivo analyses showed increased anti-tumorigenic M1 macrophages and decreased pro-tumorigenic M2 macrophages in peripheral blood, and intraocular and distant tumors after intravitreal viral treatment. Increased infiltration of natural killer cells and mature dendritic cells was also detected after viral treatment. In addition, no virus was detected in major organs after the treatment. Our data support that oHSV-EGFP is effective, neoplasm specific, immune active and safe, providing possible clinical translatable options to treat ocular and metastatic UM.

Virus Injection to the Pituitary via Transsphenoidal Approach and the Innervation of Anterior and Posterior Pituitary of Rat.

The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV.

A reporter cell line for real-time imaging of autophagy and apoptosis.

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

The C. elegans heterochronic gene lin-28 coordinates the timing of hypodermal and somatic gonadal programs for hermaphrodite reproductive system morphogenesis.

C. elegans heterochronic genes determine the timing of expression of specific cell fates in particular stages of developing larvae. However, their broader roles in coordinating developmental events across diverse tissues have been less well investigated. Here, we show that loss of lin-28, a central heterochronic regulator of hypodermal development, causes reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes that act downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, presumably by cell non-autonomous signal(s). This article has an associated 'The people behind the papers' interview.

Intracellular Motility of Intermediate Filaments.

SUMMARYThe establishment and continuous cell type-specific adaptation of cytoplasmic intermediate filament (IF) networks are linked to various types of IF motility. Motor protein-driven active transport, linkage to other cellular structures, diffusion of small soluble subunits, and intrinsic network elasticity all contribute to the motile behavior of IFs. These processes are subject to regulation by multiple signaling pathways. IF motility is thereby connected to and involved in many basic cellular processes guarding the maintenance of cell and tissue integrity. Disturbances of IF motility are linked to diseases that are characterized by cytoplasmic aggregates containing IF proteins together with other cellular components.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells.

Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.

A novel technique of delivering viral vectors to trigeminal ganglia in rats.

The objective of this study was to develop a viable and reliable technique of delivering viral vectors to rat trigeminal ganglia. Adult Sprague-Dawley rats (200-300 g) were used, and lentiviral vectors containing enhanced green fluorescence protein and calcitonin gene-related peptide short hairpin RNA (shRNA) were generated. Following general anesthesia, viral vectors were delivered to rat trigeminal ganglia using the technique described in this study. Both X-ray and micro-computed tomography (micro-CT) were employed to verify the position of the needles when injecting the vectors. In vivo fluorescence imaging and immunostaining against enhanced green fluorescence protein were performed to determine the success of viral transduction.The levels of calcitonin gene-related peptide in trigeminal ganglia were determined using real-time PCR, and pain levels following injections were evaluated using the Rat Grimace Scale. Our results show that injection needles can be advanced precisely at the trigeminal fossa and that viral vectors can successfully transduce trigeminal ganglia. Moreover, the levels of calcitonin gene-related peptide at trigeminal ganglia were down-regulated on day 7 after viral transduction. Pain levels returned to baseline by day 7 following injection. Therefore, we suggest that our trigeminal ganglion-targeting technique could be used for delivering genes or drugs to rat trigeminal ganglia.

Lifespan extension and increased resistance to environmental stressors by N-acetyl-L-cysteine in Caenorhabditis elegans.

OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV), was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors.

A cell-based functional assay using a green fluorescent protein-based calcium indicator dCys-GCaMP.

Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.

Cholinergic neurons excite cortically projecting basal forebrain GABAergic neurons.

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 µm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

Local regulation of neurofilament transport by myelinating cells.

Axons in the vertebrate nervous system only expand beyond ∼ 1 µm in diameter if they become myelinated. This expansion is due in large part to the accumulation of space-filling cytoskeletal polymers called neurofilaments, which are cargoes of axonal transport. One possible mechanism for this accumulation is a decrease in the rate of neurofilament transport. To test this hypothesis, we used a fluorescence photoactivation pulse-escape technique to compare the kinetics of neurofilament transport in contiguous myelinated and unmyelinated segments of axons in long-term myelinating cocultures established from the dorsal root ganglia of embryonic rats. The myelinated segments contained more neurofilaments and had a larger cross-sectional area than the contiguous unmyelinated segments, and this correlated with a local slowing of neurofilament transport. By computational modeling of the pulse-escape kinetics, we found that this slowing of neurofilament transport could be explained by an increase in the proportion of the time that the neurofilaments spent pausing and that this increase in pausing was sufficient to explain the observed neurofilament accumulation. Thus we propose that myelinating cells can regulate the neurofilament content and morphology of axons locally by modulating the kinetics of neurofilament transport.

Biochemical analysis of a rhodopsin photoactivatable GFP fusion as a model of G-protein coupled receptor transport.

Rhodopsin is trafficked to the rod outer segment of vertebrate rod cells with high fidelity. When rhodopsin transport is disrupted retinal photoreceptors apoptose, resulting in the blinding disease autosomal dominant retinitis pigmentosa. Herein, we introduce rhodopsin-photoactivatable GFP-1D4 (rhodopsin-paGFP-1D4) for the purposes of monitoring rhodopsin transport in living cells. Rhodopsin-paGFP-1D4 contains photoactivatable GFP (paGFP) fused to rhodopsin's C-terminus and the last eight amino acids of rhodopsin (1D4) appended to the C-terminus of paGFP. The fusion protein binds the chromophore 11-cis retinal and photoisomerizes upon light activation similarly to rhodopsin. It activates the G-protein transducin with similar kinetics as does rhodopsin. Rhodopsin-paGFP-1D4 localizes to the same compartments, the primary cilium in cultured IMCD cells and the outer segment of rod cells, as rhodopsin in vitro and in vivo. This enables its use as a model of rhodopsin transport and details the importance of a free rhodopsin C-terminus in rod cell localization and health.

Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri and preliminary investigation of its effects on virulence.

Edwardsiella ictaluri is the etiologic agent of enteric septicemia of catfish, which causes substantial losses in catfish aquaculture. To determine pathogen-host interactions, previous studies have used the green fluorescence protein (GFP) gene. Here, the pEI2 plasmid of E. ictaluri isolate I49 was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of open reading frame 2 and appeared to be at a neutral site in the plasmid. Purification of the pEI2::GFPKan plasmid and mobilization into E. coli resulted in GFP expression. The isolated pEI2::GFPkan plasmid was used to retransform the wild type I49 isolate (ensuring a single Tn10-GFP-kan insertion) and an independent E. ictaluri isolate, S97-73-3. The wild type and the green fluorescent-tagged strains were compared for modulation of pathogenicity in channel catfish Ictalurus punctatus by immersion challenge. A significant reduction in mortalities occurred for the I49GFPkan strain as compared to its isogenic parent, but no difference was observed between the S97-73-3GFPkan strain and the S97-73-3 wild type. This GFP-tagged plasmid will be useful for determining the effects that the pEI2::GFPkan plasmid has on virulence and host-pathogen interactions between E. ictaluri isolates.

Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein.

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride­proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.

Survival, migration and differentiation of mouse tau-GFP embryonic stem cells transplanted into the rat auditory nerve.

Stem cells have been investigated as treatment for a variety of diagnoses such as Parkinson's disease, Alzheimer's disease and spinal cord injuries. Here, we investigated the possibility of using stem cells as a replacement therapy for lesions of the auditory nerve (AN). We transplanted tau-GFP mouse embryonic stem cells into the AN either by the internal auditory meatus or via the modiolus in rats that had been previously deafened by application of ß-bungarotoxin to the round window niche. We investigated the effect of brain derived neurotrophic factor (BDNF) on cell transplant survival and differentiation. Additionally chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core chondroitin sulfate proteoglycans, was used in order to promote possible migration of cells and axons through the transitional zone. A bioactive isoleucine-lysine-valine-alanine-valine (IKVAV) peptide amphiphile (PA) nanofiber gel was applied around the cell injection site. This nanofiber gel has been shown to promote neural differentiation and other similar gels have been used to encapsulate and release proteins. Three weeks after injection, transplanted cells were found in the scala tympani, the modiolus, the AN trunk and the brain stem. As compared to cell transplantation and gel only, BDNF content in the PA gel increased cell survival and neuronal differentiation. In the animals treated with ChABC we observed extensive migration of cells through the transitional zone to or from the CNS.

A multifunctional teal-fluorescent Rosa26 reporter mouse line for Cre- and Flp-mediated recombination.

Reporters of Cre and/or Flp activity are important for defining the spatial and temporal extent of Cre/Flp-mediated recombination. Here, we describe R26-CAG-LF-mTFP1, a multifunctional fluorescent reporter mouse that strongly expresses mTFP1 (bright teal fluorescent protein) after Cre- and Flp-mediated recombination. To meet the need for single recombinase-mediated reporter expression, we generated derivatives of R26-CAG-LF-mTFP1. The germline excision of the Frt-flanked stop cassette in R26-CAG-LF-mTFP1 generated a Cre-dependent reporter (R26-CAG-LoxP-mTFP1). Similarly, R26-CAG-FRT-mTFP1, in which the loxP-flanked stop cassette was excised in the germline, requires only Flp to activate mTFP1 expression.

Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein.

It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.