Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (also called autocrine motility factor or AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized via a PI3K- and dynamin-dependent raft endocytic pathway to the smooth endoplasmic reticulum (ER), however the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent, inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and therefore distinct from Cdc42 and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, that is reduced by dynasore treatment and absent in Gp78-knockdown cells and therefore requires Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, Mitofusin 1 and 2 in a dynamin, Rac1 and PI3K-dependent manner. Gp78 induces mitochondrial clustering and fission in a ubiquitin ligase-dependent manner that is also reversed by AMF. The raft-dependent endocytosis of AMF therefore promotes Rac1/PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics.

Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with ER-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I(-) transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I(-) transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the δ-amino of R124 in the transporter's maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na(+)/galactose symporter, we propose an interaction between the δ-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.

In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the amino-terminal MAPK binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with.

Cellular architectures require regulated mechanisms to correctly localize the appropriate plasma membrane lipids and proteins. Microvilli are dynamic, filamentous-actin based protrusions of the plasma membrane in the apical membrane of epithelial cells. However, it remains poorly understood how their formation is regulated. In the present study, we found that sphingomyelin clustering underlies the formation of microvilli. Clustering of sphingomyelin is required for the co-clustering of a sialomucin membrane protein podocalyxin-1 at microvilli. Podocalyxin-1 recruits ERM-binding phosphoprotein-50 (also known as NHERF1), which recruits Ezrin/Radixin/Moesin proteins (ERM) and phosphatidylinositol 4-phosphate 5-kinase beta (PIP5Kbeta). Thus, clustering of PIP5Kbeta leads to local accumulation of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], which enhances the accumulation of ERM family proteins and induces the formation of microvilli. The present study revealed novel interactions between sphingomyelin and cytoskeletal proteins by which microvilli are formed, and it clarified the physiological importance of the chemical properties of sphingomyelin that facilitate cluster formation.

In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a sub-nuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex. However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging we show that the dynamic localization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochomatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and ChIP analyses revealed that in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used Peptide Nucleic Acids (PNA) to down-regulate PfSec13 and show that is essential for parasite proliferation in human erythrocytes.

The SNARE proteins VAMP/synaptobrevin, SNAP-25 and Syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine, derived from experiments performed with botulinum neurotoxins, which led to the identification of one arginine in SNAP-25 and one aspartate in Syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions among SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and Syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

Plakoglobin (γ-catenin), a constituent of the adherens junction and desmosomes, has signaling capabilities typically associated with tumor/metastasis suppression through mechanisms that remain undefined. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We detected several p53-target genes whose levels were altered upon plakoglobin expression. In this study, we identified the p53 regulated tumor suppressor 14-3-3σ as a direct plakoglobin-p53 target gene. Coimmunoprecipitation experiments revealed that plakoglobin and p53 interact while chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that plakoglobin and p53 associate with the 14-3-3σ promoter. Furthermore, luciferase reporter assays showed that p53 transcriptional activity is increased in the presence of plakoglobin. Finally, knockdown of plakoglobin in MCF-7 cells followed by luciferase assays confirmed that p53 transcriptional activity is enhanced in the presence of plakoglobin. Our data suggest that plakoglobin regulates gene expression in conjunction with p53 and that plakoglobin may regulate p53 transcriptional activity, which may account, in part, for the tumor/metastasis suppressor activity of plakoglobin.

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. While it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

Chemotherapy of advanced pancreatic cancer has mainly been gemcitabine-based for the past fifteen years, with only limited effect. Recently, combination therapy that also targets checkpoint kinase 1 (CHK1) has become an attractive option. The central role of CHK1 in many DNA damage response pathways, however, may result in undesired cytotoxicity in normal cells causing side effects. We were searching for other target molecules of similar function that may be more specific and thus better suited for combination therapy. To this end a negative selection RNAi screen was performed in cell lines with small hairpin RNA molecules targeting over 10,000 genes. Genes that were found to be synthetically lethal with gemcitabine and whose proteins are acting upstream of CHK1 were characterised in more detail. In particular, the inhibition of RAD17 potentiated gemcitabine cytotoxicity in the pancreatic cancer cell lines BxPC-3, MiaPaca-2 and the primary cell line JoPaca-1 that closely resembles primary tumour tissue. Further analysis showed that the synergistic effect of RAD17 knockdown and gemcitabine leads to forced mitotic entry of cells arrested in S-phase by gemcitabine treatment, resulting in asymmetric DNA distribution during anaphase followed by DNA fragmentation and finally cell death by mitotic catastrophe. Our data suggest RAD17 as a novel target for gemcitabine combination therapy supplementing or complementing inhibition of checkpoint kinase 1. As opposed to CHK1, RAD17 knockdown by itself does not lead to abnormal DNA segregation, suggesting a more specific action.

Family members of the cationic TRP channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within the TRPC subfamily, not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRPL are expressed together in photoreceptors where they generate the light induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro was reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL did not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.