Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/ß-catenin pathway, with significant downregulation of both Ser 675-phosphorylated ß-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and ß-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/ß-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer's patients' iPSCs in senescence-related conditions.

In addition to increasing ß-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.

PKM2 controls the translation of TFE3 to maintain the integrity of the Golgi apparatus for the survival of HeLa and ME-180 cervical cancer cells.

The M2 isoform of pyruvate kinase (PKM2) is abundantly expressed in various cancer cells and associated with tumorigenesis, tumour proliferation and tumour progression. However, the role of PKM2 in these oncological processes is not fully understood. In the present study, we depleted PKM2 expression using RNA interference (RNAi), which induced apoptotic cell death and was accompanied by the downregulation of GM130, giantin, and p115 in HeLa and ME-180 cervical cancer cells. The decreased expression of these proteins caused structural and functional disturbances in the Golgi apparatus, which manifested as the dispersion of the Golgi apparatus and delayed anterograde trafficking from the ER to the Golgi. The transcription factor, TFE3, which functions in the Golgi stress response, was responsible for the expression of GM130, giantin, and p115 that maintained the integrity of the organelle under normal growth conditions. In PKM2-knockdown cells, the translation of TFE3 was markedly reduced. Knockdown of TFE3 by RNAi resulted in the downregulation of GM130, giantin, and p115, dispersion of the Golgi apparatus, and apoptotic cell death, similar to those observed following PKM2 knockdown. Conversely, the exogenous expression of TFE3 in PKM2 knockdown cells partially mitigated the aforementioned effects. We also demonstrated that PKM2 bound to the 5' UTR on TFE3 mRNA and promoted translation. This study is the first to identify a new function for PKM2, which activates the basal Golgi stress response to maintain the integrity of the Golgi apparatus through the translation of TFE3 and promote cancer cell survival.

Microscopic image-based covariation network analysis for actin scaffold-modified insulin signaling.

To infer a "live" protein network in single cells, we developed a novel Protein Localization and Modification-based Covariation Network (PLOM-CON) analysis method using a large set of quantitative data on the abundance (quantity), post-translational modification state (quality), and localization/morphological information of target proteins from microscope immunostained images. The generated network exhibited synchronized time-dependent behaviors of the target proteins to visualize how a live protein network develops or changes in cells under specific experimental conditions. As a proof of concept for PLOM-CON analysis, we applied this method to elucidate the role of actin scaffolds, in which actin fibers and signaling molecules accumulate and form membrane-associated protein condensates, in insulin signaling in rat hepatoma cells. We found that the actin scaffold in cells may function as a platform for glycogenesis and protein synthesis upon insulin stimulation.

Phosphatidylinositol-3-phosphate-mediated actin domain formation linked to DNA synthesis upon insulin treatment in rat hepatoma-derived H4IIEC3 cells.

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.

LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight.

Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a "cell-resealing technique" that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.

Establishment and phenotyping of disease model cells created by cell-resealing technique.

Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called "normal" cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing technique, which reversibly permeabilizes the plasma membrane, to develop diabetic (Db) model hepatocytes into which cytosol from diabetic mouse liver had been introduced. Db model hepatocytes showed several disease-specific phenotypes, namely disturbance of insulin-induced repression of gluconeogenic gene expression and glucose secretion. Quantitative image analysis and principal component analysis revealed that the ratio of phosphorylated Akt (pAkt) to Akt was the best index to describe the difference between wild-type and Db model hepatocytes. By performing image-based drug screening, we found pioglitazone, a PPARγ agonist, increased the pAkt/Akt ratio, which in turn ameliorated the insulin-induced transcriptional repression of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1. The disease-specific model cells coupled with image-based quantitative analysis should be useful for drug development, enabling the reconstitution of disease conditions at the cellular level and the discovery of disease-specific markers.

Pyruvate kinase M1 interacts with A-Raf and inhibits endoplasmic reticulum stress-induced apoptosis by activating MEK1/ERK pathway in mouse insulinoma cells.

Apoptotic death of pancreatic ß cells is a major cause of type 2 diabetes mellitus (T2D) progression. Two isoforms of pyruvate kinase, PKM1 and PKM2, have been reported to participate in cell death in several cell types; however, little is known about their causal pathways in pancreatic ß-cell death. We examined whether the suppression of PKM1 or PKM2 affects endoplasmic reticulum (ER) stress-induced apoptosis in a pancreatic ß-cell line, MIN6, and Beta-TC-6 and found that knockdown of PKM1, but not of PKM2, leads to the induction of ER stress-induced apoptosis in these cells. We also investigated the mechanism by which PKM1 inhibits ER stress-induced apoptosis. We confirmed that PKM1 interacts with A-Raf, an upstream regulator of the MEK/ERK pathway, and that this interaction contributes to MEK1 phosphorylation by A-Raf. PKM1 knockdown suppresses the phosphorylation of MEK, ERK, and caspase-9 (Thr125), which is phosphorylated by the MEK/ERK pathway, thereby inhibiting the cleavage and activation of caspase-9. Thus, PKM1 knockdown activates the caspase-9/caspase-3 pathway under ER stress conditions and leads to apoptosis.

Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response.

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

Translocation of forkhead box O1 to the nuclear periphery induces histone modifications that regulate transcriptional repression of PCK1 in HepG2 cells.

Forkhead box O1 (FOXO1) is an important target for insulin. It is widely accepted that insulin-induced phosphorylation of FOXO1 by Akt leads to its nuclear exclusion and results in the inhibition of FOXO1-mediated transcription of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1 (PCK1) in hepatocytes. However, many results that contradict this model have accumulated. Here, we provide a new mechanism for insulin-dependent repression of FOXO1-mediated transcription. We showed insulin-induced translocation of endogenous Ser256-phosphorylated FOXO1, which is essential for regulation of FOXO1-mediated transcription, from nuclear speckles to the nuclear periphery. This insulin-dependent translocation of FOXO1 regulated transcriptional repression of PCK1 concomitant with the formation of the FOXO1-euchromatic histone-lysine N-methyltransferase2 (EHMT2) complex and histone modifications of the PCK1 promoter region. Notably, our results suggest that FOXO1 uses nucleoporin 98 kDa NUP98 for this transcriptional regulation. These results provide a new insight into various FOXO1-mediated transcriptional regulation and FOXO1-mediated essential biological pathways.

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic ß-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

Rab2A is a pivotal switch protein that promotes either secretion or ER-associated degradation of (pro)insulin in insulin-secreting cells.

Rab2A, a small GTPase localizing to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), regulates COPI-dependent vesicular transport from the ERGIC. Rab2A knockdown inhibited glucose-stimulated insulin secretion and concomitantly enlarged the ERGIC in insulin-secreting cells. Large aggregates of polyubiquitinated proinsulin accumulated in the cytoplasmic vicinity of a unique large spheroidal ERGIC, designated the LUb-ERGIC. Well-known components of ER-associated degradation (ERAD) also accumulated at the LUb-ERGIC, creating a suitable site for ERAD-mediated protein quality control. Moreover, chronically high glucose levels, which induced the enlargement of the LUb-ERGIC and ubiquitinated protein aggregates, impaired Rab2A activity by promoting dissociation from its effector, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in response to poly (ADP-ribosyl)ation of GAPDH. The inactivation of Rab2A relieved glucose-induced ER stress and inhibited ER stress-induced apoptosis. Collectively, these results suggest that Rab2A is a pivotal switch that controls whether insulin should be secreted or degraded at the LUb-ERGIC and Rab2A inactivation ensures alleviation of ER stress and cell survival under chronic glucotoxicity.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.

PPARγ-induced PARylation promotes local DNA demethylation by production of 5-hydroxymethylcytosine.

Recent studies have shown that DNA demethylation goes through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by Tet proteins. However, it is still unclear how the target regions for demethylation are distinguished within their genomic context. Here we show that the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has the ability to direct local demethylation around its binding sites, the PPAR response elements (PPREs), during adipocyte differentiation. PPARγ is a key regulator of the differentiation process that forms a PPARγ co-activator complex on PPREs and activates the expression of adipocyte-specific genes. The complex is poly(ADP-ribosyl)ated (PARylated) on PPREs, and Tet proteins catalyse the conversion of 5mC to 5hmC locally by their ability to bind to the PAR polymer, thereby inducing region-specific demethylation. Our study demonstrates that a sequence-dependent transcription factor complex can, through its post-translational modification, serve for Tet proteins as a landmark to identify sites of DNA demethylation.

A resealed-cell system for analyzing pathogenic intracellular events: perturbation of endocytic pathways under diabetic conditions.

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus.

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.

Hydrogen peroxide depletes phosphatidylinositol-3-phosphate from endosomes in a p38 MAPK-dependent manner and perturbs endocytosis.

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

An orally applicable Shiga toxin neutralizer functions in the intestine to inhibit the intracellular transport of the toxin.

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.

Yip1A regulates the COPI-independent retrograde transport from the Golgi complex to the ER.

Yip1A, a mammalian homologue of yeast Yip1p, is a multi-spanning membrane protein that is considered to be involved in transport between the endoplasmic reticulum (ER) and the Golgi. However, the precise role of Yip1A in mammalian cells remains unclear. We show here that endogenous Yip1A is localized to the ER-Golgi intermediate compartment (ERGIC). Knockdown of Yip1A by RNAi did not induce morphological changes in the Golgi, ER, or ERGIC. By analyzing a number of intracellular transport pathways, we found that Yip1A knockdown delayed the transport of Shiga toxin from the Golgi to the ER, but did not affect the anterograde transport of VSVGts045. We also found that a recombinant protein that corresponded to the N-terminal domain of Yip1A inhibited the COPI-independent retrograde transport of GFP-tagged galactosyltransferase, GT-GFP, but not the COPI-dependent retrograde transport of p58/ERGIC53. Furthermore, we found that Yip1A knockdown resulted in the dissociation of Rab6 from the membranes. These results suggested that Yip1A has a role in COPI-independent retrograde transport from the Golgi to the ER and regulates the membrane recruitment of Rab6.

NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.