Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation.

Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.

Liposome-coated lipoplex-based carrier for antisense oligonucleotides.

The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL­2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

Novel effects of Brefeldin A (BFA) in signaling through the insulin receptor (IR) pathway and regulating FoxO1-mediated transcription.

Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.

Identification of a functional domain within the p115 tethering factor that is required for Golgi ribbon assembly and membrane trafficking.

The tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C. elegans causes accumulation of the 170 kD soluble yolk protein (YP170) in the body cavity and retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes. Structure-function analyses of p115 have identified two homology regions (H1 and H2) within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a new C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants that lack the fourth CC domain (CC4) act in a dominant-negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi of p115 and the subsequent transfection with p115 deletion mutants, we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115. p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function, and suggest that both the CC1 and the CC4 SNARE-binding motifs participate in p115-mediated membrane tethering.

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells.

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations--one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP--with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes.

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.