Rab11 regulates exocytosis of recycling vesicles at the plasma membrane

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.102913 Senye Takahashi, Keiji Kubo, Satoshi Waguri, Atsuko Yabashi, Hye-Won Shin, Yohei Katoh and Kazuhisa Nakayama*?*Corresponding author: Kazuhisa Nakayama, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Phone: +81-75-753-4527; Fax: +81-75-753-4557; E-mail: kazunaka{at}pharm.kyoto-u.ac.jp. Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We here show that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of a component of the exocyst tethering complex, Sec15 or Exo70, the former which interacts with Rab11, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

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APPL1 regulates basal NF-{kappa}B activity by stabilizing NIK

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.105171 APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-?B under basal but not TNFa-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate the canonical NF-?B signaling. APPL1 synergized with TRAF2 to induce NF-?B activation and both proteins were necessary for this process by functioning upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-?B pathway. Importantly, in the canonical pathway APPL1 appeared to regulate the proper spatial distribution of p65 in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. Analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-?B target genes encoding cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-?B pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-?B activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-?B activation.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Toxofilin upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.103648 Violaine Delorme-Walker, Marie Abrivard, Vanessa Lagal, Karen Anderson, Audrey Perazzi, Virginie Gonzalez, Christopher Page, Juliette Chauvet, Wendy Ochoa, Niels Volkmann, Dorit Hanein and Isabelle Tardieux*?5 Correspondence should be addressed to I. T. (email : isabelle.tardieux{at}inserm.fr) Toxoplasma, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, progressively propels itself through the junction inside a newly formed vacuole that encloses the entering parasite. Litle is known how a few micron-large diameter parasite overcome the host cell cortical actin barrier to support these remarkably rapid process of internalization (< few seconds). Correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis indicate that toxofilin an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy in cells expressing toxofilin indicates that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, are impaired in cortical actin disassembly and exhibit delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that, in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

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Autophagy and cancer - issues we need to digest

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.093708 May 15, 2012 J Cell Sci 125, 2349-2358. Emma Y. Liu and Kevin M. Ryan*

Tumour Cell Death Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK ?* Author for correspondence (k.ryan{at}beatson.gla.ac.uk) Autophagy is an evolutionarily conserved catabolic pathway that has multiple roles in carcinogenesis and cancer therapy. It can inhibit the initiation of tumorigenesis through limiting cytoplasmic damage, genomic instability and inflammation, and the loss of certain autophagy genes can lead to cancer. Conversely, autophagy can also assist cells in dealing with stressful metabolic environments, thereby promoting cancer cell survival. In fact, some cancers rely on autophagy to survive and progress. Furthermore, tumour cells can exploit autophagy to cope with the cytotoxicity of certain anticancer drugs. By contrast, it appears that certain therapeutics require autophagy for the effective killing of cancer cells. Despite these dichotomies, it is clear that autophagy has an important, if complex, role in cancer. This is further exemplified by the fact that autophagy is connected with major cancer networks, including those driven by p53, mammalian target of rapamycin (mTOR), RAS and glutamine metabolism. In this Commentary, we highlight recent advances in our understanding of the role that autophagy has in cancer and discuss current strategies for targeting autophagy for therapeutic gain.

Key words This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Ubiquitin-like proteins and autophagy at a glance’ by Tomer Shpilka et al. (J. Cell Sci. 125, 2343-2348) and ‘Autophagy and cell growth – the yin and yang of nutrient responses’ by Thomas Neufeld (J. Cell Sci. 125, 2359-2368).

Funding

Work in the Tumour Cell Death Laboratory is supported by Cancer Research UK and the Association for International Cancer Research.

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SNX9, SNX18 and SNX33 are required for progression through and completion of mitosis

Advance Online Publication June 20, 2012 doi: 10.1242/?jcs.105981 Maggie P.C. Ma and Megan Chircop?Address correspondence to:
Dr Megan Chircop, Children's Medical Research Institute, The University of Sydney, Locked Bag 23, Wentworthville, NSW, 2145, Australia, Phone: +61-2-9687 2800, Fax: +61-2-9687 2120, Email: mchircop{at}cmri.org.auMitosis involves considerable membrane remodelling and vesicular trafficking to generate two independent cells. Consequently, endocytosis and endocytic proteins are required for efficient mitotic progression and completion. Several endocytic proteins also participate in mitosis in an endocytosis-independent manner. Here, we report that the sorting nexin (SNX) 9 subfamily members – SNX9, SNX18 and SNX33 – are required for progression and completion of mitosis. Depletion of any one of these proteins using siRNA induces multinucleation, an indicator of cytokinesis failure, as well as an accumulation of cytokinetic cells. Time-lapse microscopy on siRNA-treated cells reveals a role for SNX9 subfamily members in progression through the ingression and abscission stages of cytokinesis. Depletion of these three proteins disrupted MRLCS19 localization during ingression and recruitment of Rab11-positive recycling endosomes to the intracellular bridge between nascent daughter cells. SNX9 depletion also disrupted the localization of Golgi during cytokinesis. Endocytosis of transferrin (Tfn) was blocked during cytokinesis by depletion of the SNX9 subfamily members, suggesting that these proteins participate in cytokinesis in an endocytosis-dependent manner. In contrast, depletion of SNX9 did not block Tfn uptake during metaphase but did delay chromosome alignment and segregation, suggesting that SNX9 plays an additional non-endocytic role at early mitotic stages. We conclude that SNX9 subfamily members are required for mitosis through both endocytosis-dependent and -independent processes.

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Ubiquitin-like proteins and autophagy at a glance

doi: 10.1242/?jcs.093757 May 15, 2012 J Cell Sci 125, 2343-2348. Tomer Shpilka1, Noboru Mizushima2 and Zvulun Elazar1,*

1Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel

2Department of Physiology and Cell Biology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan ?* Author for correspondence (zvulun.elazar{at}weizmann.ac.il) Autophagy is a major catabolic process that is predominantly controlled by amino acids and hormones, and delivers cytoplasmic components to lysosomes or the vacuole for degradation and recycling (Weidberg et al., 2011a). This process is essential for maintaining cell homeostasis by providing energy and clearing damaged or unnecessary substrates from the cytoplasm through lysosomes and the vacuole. Autophagy also has important roles in numerous physiological and pathophysiological processes, such as cell development, programmed cell death, cancer, defense mechanisms against pathogen infection, neurodegenerative disorders and myopathies (Ravikumar et al., 2010a). Several forms of autophagy have been described, including macroautophagy, microautophagy and chaperone-mediated autophagy (Mijaljica et al., 2011; Orenstein and Cuervo, 2010; Weidberg et al., 2011a).

Macroautophagy (hereafter referred to as autophagy) – the best characterized form of autophagy – refers to the formation of autophagic vesicles through the nucleation, assembly and elongation of membrane structures (Rubinsztein et al., 2012). It is initiated by the formation of a cup-shaped membrane called the phagophore, which engulfs parts of the cytoplasm. The phagophore subsequently elongates and becomes sealed, forming a unique double-membrane vesicle termed the autophagosome, which fuses with the lysosome and thereby leads to degradation of the inner membrane and the cargo of the autophagosome. In Saccharomyces cerevisiae, autophagosome formation takes place in a unique site termed the pre-autophagosomal structure (PAS) (Suzuki et al., 2001).

Genetic screens in yeast have led to the identification of 35 autophagy-related (ATG) genes, many of which are evolutionarily conserved in other species, and the genes identified in these screens have paved the way for elucidating the molecular machinery that drives autophagosome formation at the PAS (Thumm et al., 1994; Tsukada and Ohsumi, 1993). Atg proteins consist of several functional units: the Atg1–unc-51-like kinase (ULK) complex, the class III phosphoinositide 3-kinase (PI3K) complex, the Atg12 conjugation machinery, the Atg8 conjugation machinery, as well as a number of other proteins that are essential for autophagosome formation (Mizushima et al., 2011). The order in which these proteins are recruited to the PAS and participate in its organization has been established (Suzuki et al., 2007); however, the precise roles of each of the different Atgs is not yet fully understood.

Two of the proteins involved in the autophagic process, Atg12 and Atg8, are structurally related to ubiquitin (Paz et al., 2000; Suzuki et al., 2005). These molecules participate in the early stages of autophagosome biogenesis, where Atg12 is covalently attached (conjugated) to Atg5 and Atg8 becomes conjugated to phosphatidylethanolamine (PtdEth) (Ichimura et al., 2000; Mizushima et al., 1998a). In this Cell Science at a Glance article, we focus on the roles of these two proteins and their conjugation machineries in both yeast and mammals.

Atg12 was the first ubiquitin-like protein (Ubl) identified as an essential component for autophagy. Although Atg12 does not have a similar sequence to ubiquitin, its crystal structure reveals a ubiquitin fold (Suzuki et al., 2005). Atg12 is covalently conjugated to Atg5 through the Ubl-conjugation machinery. First, the Atg12 C-terminal Gly186 is activated by the E1-like enzyme Atg7 through the formation of a thioester bond with Cys507 of Atg7 (Tanida et al., 1999). Subsequently, Atg12 is transferred to the E2-like enzyme Atg10 (Shintani et al., 1999), which is followed by Atg12 conjugation to Lys149 on its target protein Atg5 (Mizushima et al., 1998b). The Atg12–Atg5 conjugate can further interact with the coiled-coil protein Atg16 (following its oligomerization) to form a dimeric complex (Fujioka et al., 2010; Kuma et al., 2002).

No deconjugation or E3-like enzyme that associates with the Atg12–Atg5 complex has been detected so far, and it seems that formation of the Atg12–Atg5 conjugate occurs constitutively. Atg5, which also has no substantial sequence similarity with ubiquitin, comprises two ubiquitin-like domains flanking a helix-rich domain (Matsushita et al., 2007). Binding of Atg5 to Atg16 and to Atg12 occurs on opposite sides of the protein. Atg16 in S. cerevisiae comprises a coiled-coil domain through which it forms homo-oligomers, and an N-terminal Atg5-binding domain (Fujioka et al., 2010). In addition, the mammalian Atg16 (ATG16L) has seven WD repeats in its C-terminus and forms a larger ATG12–ATG5–ATG16L complex (Mizushima et al., 2003). This complex is essential for the formation and elongation of the autophagosome (Mizushima et al., 2001) and orthologs of this system, with the respective functionality, are found in yeast and mammals (Ohsumi, 2001).

Small fractions of the cytosolic ATG12–ATG5–ATG16L complex associate with the outer membrane of the phagophore and dissociate from it on or near completion of the double-membrane autophagosome (Mizushima et al., 2001; Mizushima et al., 2003). Atg5 and Atg16 depend on each other for their membrane targeting, whereas Atg12 is dispensable for Atg5–Atg16 membrane association (Mizushima et al., 2003; Suzuki et al., 2001). The targeting of this complex to the membranes also depends on the formation of phosphatidylinositol 3-phosphate [PtdIns(3)P], but no known PtdIns(3)P-binding domain is found within the Atg12–Atg5–Atg16 complex. However, during the process of xenophagy (the degradation of pathogens by autophagy; for further details, see below), recruitment of the ATG12–ATG5–ATG16L complex appears to be independent of PtdIns(3)P and other upstream ATG proteins (Kageyama et al., 2011).

The second ubiquitin-like conjugation system involves the conjugation of Atg8 proteins to the lipid PtdEth (Ichimura et al., 2000). Yeast have one ATG8 gene, whereas mammalians possess several genes encoding Atg8-like proteins that are divided into three subfamilies: microtubule-associated protein 1 light chain 3 (LC3), ?-aminobutyric acid (GABA) receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE16, also known as GABARAPL2) (Shpilka et al., 2011). Mammalian ATG8 proteins were originally characterized as factors that participate in membrane trafficking processes (Kuznetsov and Gelfand, 1987; Mann and Hammarback, 1994), and it was only discovered later that they are indispensable for autophagy (Lang et al., 1998; Scott et al., 1996).

Atg8 proteins are synthesized as precursors that are immediately cleaved in their C-terminus by the specific cysteine protease Atg4, which leads to the exposure of a glycine residue (Kabeya et al., 2000; Kirisako et al., 2000). The carboxy group of the exposed glycine is then activated by Atg7, the same E1-like enzyme that acts on Atg12, by forming a thioester bond between Atg8 and Atg7. The glycine residue of Atg8 is subsequently transferred to the specific E2-like enzyme Atg3, and this interaction leads to formation of an amide bond between PtdEth and the exposed glycine residue of Atg8 (Ichimura et al., 2000). The conjugation of Atg8 to PtdEth is reversible, as Atg4 can also function as a deconjugating enzyme (Kirisako et al., 2000), and Atg8 proteins that are conjugated to PtdEth reside on both sides of the phagophore membrane as well as on the inner and outer membranes of complete autophagosomes.

Three-dimensional structure analysis has revealed that all Atg8 proteins are structurally similar to ubiquitin (Coyle et al., 2002; Kouno et al., 2005; Kumeta et al., 2010; Paz et al., 2000). Atg8s comprise a C-terminal ubiquitin core that is decorated with two additional N-terminal a-helices. The three-dimensional structure of GABARAP has been solved and has revealed two different conformations of the protein, in which the a-helices are projected either towards the ubiquitin core (closed conformation) or away from it (open conformation) (Coyle et al., 2002). It has been suggested that Atg8 is found in an open conformation following conjugation to PtdEth, implying that there is a function for the Atg8 N-terminus in autophagy (Ichimura et al., 2004). Furthermore, this unique region differs in the different mammalian ATG8 subfamilies: the first a-helix of the mammalian LC3 subfamily is basic, whereas in the GABARAP and GATE16 subfamilies this region is acidic (Sugawara et al., 2004).

Atg12–Atg5–Atg16 as an E3-like enzyme The Atg12–Atg5–Atg16 complex has an important regulatory role in Atg8 conjugation. Thus, Atg5 deficiency or Box 1. Additional cellular roles for Atg12–Atg5–Atg16

The ATG12–ATG3 conjugate and mitochondrial quality control Disruption of the ATG12–ATG3 conjugate by mutagenesis does not seem to affect nonselective autophagy. Instead, it leads to an increase in mitochondrial mass, fragmentation of the mitochondrial network and resistance to mitochondrial cell death. The mechanism by which the ATG12–ATG3 conjugate regulates mitochondrial homeostasis is not yet understood, but it is known that following disruption of the conjugate, the basal levels of the anti-apoptotic protein Bcl-XL increase, which is correlated to the resistance of these cells to mitochondria-mediated apoptosis (Radoshevich et al., 2010).

ATG16L and Crohn's disease

Genetic studies in humans have shown that a single nucleotide polymorphism in the C-terminal WD repeats of the ATG16L1 protein – which results in substitution of alanine for threonine at amino acid 300 – leads to an increased susceptibility to Crohn's disease (Cadwell et al., 2008). The pathogenesis of this inflammatory disease of the intestine is poorly understood (Cadwell et al., 2009). Dendritic cells taken from patients with the ATG16L1 (T300A) variant are defective in the induction of autophagy, bacterial trafficking and antigen presentation (Cooney et al., 2010). This polymorphism has been further shown to impair autophagy and clearance of several pathogens (Kuballa et al., 2008; Lapaquette et al., 2010). Additionally, macrophages derived from mice with a deletion of the coiled-coil domain of ATG16L1 show an enhanced inflammatory response to Toll-like receptor ligands (Saitoh et al., 2008). Paneth cells of mice that are hypomorphic for ATG16L1 protein expression show defects in handling antimicrobial granules and exhibit the same defects as Paneth cells taken from Crohn's disease patients that are homozygous for the ATG16L1 risk allele (Cadwell et al., 2008). Taken together, these studies suggest that ATG16L1 and autophagy influence the immune response and the biology of Paneth cells. The role of the WD repeats in ATG16L1 is unknown, but this region is known to participate in protein–protein interactions. Revealing the proteins that interact with the WD region of ATG16L1 might therefore prove useful in understanding the pathogenesis of Crohn's disease.

mutations that hamper the formation of the Atg12–Atg5 complex almost completely abolish the conjugation of Atg8 to PtdEth (Hanada and Ohsumi, 2005; Mizushima et al., 2001). Furthermore, in vitro experiments have demonstrated that the Atg12–Atg5 conjugate strongly enhances the formation of Atg8–PtdEth by interacting with and stimulating the activity of Atg3 (Hanada et al., 2007). Atg16 is required for the localization of Atg8 to the pre-autophagosomal structure in yeast (Suzuki et al., 2001), and mammalian ATG16L determines the site of LC3 lipidation (Fujita et al., 2008). Taken together, these findings suggest that the Atg12–Atg5–Atg16 complex has an E3-like enzyme activity, which determines the site of Atg8 conjugation to PtdEth. A recent study has shown that ATG12 is covalently conjugated to ATG3 through a reaction that is mediated by ATG7 and the autocatalytic activity of ATG3. The ATG12–ATG3 conjugate participates in mitochondrial quality control (Box 1), and blocking this interaction does not affect the conjugation of mammalian ATG8 proteins to PtdEth. Evidently, the interaction between ATG12–ATG5 and ATG3, and their effect on ATG8 lipidation, requires further study (Radoshevich et al., 2010). The autophagic pathway was initially considered to be nonselective, but it is now clear that both yeast and mammalian ATG8 proteins participate in selective cargo recruitment to the autophagosome (Weidberg et al., 2011a). In yeast, Atg8 participates in the selective recruitment of prApe1 (for precursor Ape1) to the vacuole by interacting with Atg19 (Lynch-Day and Klionsky, 2010), whereas in mammals the two scaffold proteins p62 (also known as sequestosome-1, SQSTM1) and neighbour of BRCA1 gene 1 (NBR1) are recruited to the autophagosome by mammalian ATG8 proteins through their Atg8-family interacting motif (AIM) [also known as LC3-interacting region (LIR)]. The AIM motif is composed of acidic residues followed by the sequence W/YxxL/I/V (Ichimura et al., 2008; Noda et al., 2010). This motif is found in numerous proteins and enables them to interact directly with Atg8 family members (Noda et al., 2010). Importantly, different mammalian ATG8 proteins appear to differ in their selectivity towards cargo molecules. Thus, p62 is recruited to the autophagosomes by LC3 but not by GATE16, although it is able to interact with both proteins in their soluble form (Shvets et al., 2011).

Autophagy also participates in the selective delivery of protein aggregates (aggrephagy), organelles, such as peroxisomes (pexophagy) and mitochondria (mitophagy), bacteria and viruses (xenophagy) and ribosomes (ribophagy) to the lysosome and/or vacuole (Johansen and Lamark, 2011). In the following sections we exemplify the role of Atg8 proteins in two of these processes, mitophagy and xenophagy.

The term ‘mitophagy’ refers to the selective clearance of mitochondria by autophagy. In yeast, mitophagy involves Atg32, an outer mitochondrial membrane (OMM)-spanning protein, which interacts with the cargo protein Atg11 (Kanki et al., 2009; Okamoto et al., 2009). The interaction is mediated by Atg32 phosphorylation and is thought to be the initial step in mitophagy (Aoki et al., 2011). In addition, Atg32 contains AIMs in its cytosolic domain and is thus able to interact with Atg8. Mutations in the Atg32 AIM result in partial defects in mitophagy and hamper the Atg32–Atg8 interaction in yeast two-hybrid assays (Okamoto et al., 2009). In mammals, NIX (also known as BNIP3-like protein, BNIP3L), an OMM protein, contains the AIM in its cytosolic domain. As in yeast, this AIM mediates direct binding to mammalian ATG8 proteins, and NIX can recruit mitochondria to the autophagosomes. Ablation of the interaction between NIX and mammalian ATG8 proteins hinders clearance of mitochondria (Novak et al., 2010).

In addition, mitophagy can also occur in a NIX-independent manner (reviewed by Youle and Narendra, 2011). For example, the E3 ubiquitin ligase Parkin has been shown to mediate mitophagy. Although p62 has been reported to function as an adaptor protein for Parkin-mediated mitophagy, this idea remains controversial (Narendra et al., 2010; Okatsu et al., 2010).

As described above, xenophagy refers to the selective autophagy of pathogens. In this process, adaptor proteins such as p62, NBR1, nuclear dot protein 52 (NDP52) and optineurin (OPTN), which all contain AIMs, bind both ubiquitylated pathogens and mammalian ATG8 proteins, thereby targeting the pathogens to autophagosomes. In addition, LC3, through a pathway involving p62, targets cytosolic proteins to the lysosome, where they are processed into anti-microbial peptides (Ponpuak et al., 2010). A recent study has shown that TANK-binding kinase 1 (TBK1) phosphorylates the autophagic adaptor OPTN, which is a ubiquitin-binding protein. This phosphorylation, which occurs at a site within the OPTN AIM motif, increases the affinity of OPTN binding to LC3 and thereby promotes selective autophagy of ubiquitylated Salmonella enterica (Wild et al., 2011). The regulation of cargo recruitment to mammalian ATG8 proteins by phosphorylation is of great importance in the elucidation of signaling events that target cargo proteins for degradation.

A key question in autophagy focuses on the mechanism of autophagosome biogenesis. Atg8 proteins have certain characteristics that could be important for this process, and they are thought to have a role in autophagosome biogenesis. However, their mode of action is not fully understood. Early studies have shown that Atg8 proteins promote elongation of the autophagosomal membrane and determine the size of the autophagosome (Abeliovich et al., 2000; Xie et al., 2008). It has been suggested that the yeast Atg8, following lipidation, mediates membrane tethering and hemifusion, which drive the growth of autophagosomal membranes (Nakatogawa et al., 2007). A recent in vitro study has indeed shown that mammalian ATG8 proteins, through their N-terminus, mediate fusion processes that are required for autophagosome biogenesis (Weidberg et al., 2011b). Furthermore, preventing the lipidation of all mammalian ATG8 proteins blocks autophagosome biogenesis (Fujita et al., 2008; Sou et al., 2008), and the mammalian ATG8 subfamilies LC3, GABARAP and GATE16 are essential for autophagosome biogenesis and have been suggested to act at different stages of this process (Weidberg et al., 2010).

The role of yeast Atg8 in autophagosome biogenesis is expanded by its ability to recruit essential factors that participate in the process. For example, it has been intriguing to find that Atg8 interacts with the cell division control protein 48 (Cdc48) cofactor Shp1 (for suppressor of high-copy PP1 protein 1). Cdc48 belongs to the AAA ATPase superfamily and participates in numerous cellular functions, including homotypic membrane fusion, endoplasmic reticulum (ER)-associated protein degradation (ERAD) and protein degradation through the ubiquitin–proteasome system (Dargemont and Ossareh-Nazari, 2011). Cdc48 and Shp1 mediate Golgi reassembly by extracting a monoubiquitylated protein regulator (Wang et al., 2004). A recent study has revealed that Cdc48 and its cofactor Shp1 have a role in autophagosome biogenesis (Krick et al., 2010), and the authors of this study suggest that this role resembles their function in Golgi reassembly, where the ubiquitin-like protein Atg8 acts as the essential monoubiquitylated protein that is extracted by Cdc48 and Shp1 (Krick et al., 2010).

Numerous Rab proteins, Sec proteins and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) have been implicated in autophagosome biogenesis and some have been shown to interact with Atg proteins (Chua et al., 2011; Longatti and Tooze, 2009; Moreau et al., 2011; Nair et al., 2011; Ohashi and Munro, 2010). RAB33A and RAB33B interact directly and in a GTP-dependent manner with the coiled-coil domain of ATG16L (Itoh et al., 2008), and it has been suggested that interaction modulates autophagosome formation (Itoh et al., 2008). Ornithine aminotransferase-like 1 (OATL1, also known as TBC1D25), a putative Rab GTP-activating protein (GAP), has recently been shown to bind mammalian ATG8 proteins (Itoh et al., 2011). This protein, through its interaction with mammalian ATG8 proteins, is recruited to isolation membranes and to autophagosomes, where it participates in autophagosome maturation. Furthermore, RAB33B is a target of the OATL1 GAP activity (Itoh et al., 2011). Thus, the interaction between Atg8 and the Atg12–Atg5–Atg16 complex might serve as a scaffold that brings RAB33 and OATL1 into close proximity. In summary, the participation of Atg8 proteins in autophagosome biogenesis might occur through their ability to promote vesicle fusion, as well as by their ability to recruit essential factors to the phagophore and/or to work together with these factors.

The NSF protein Sec18, which mediates the disassembly of SNARE proteins, has been implicated as an essential component of autophagosome biogenesis in yeast (Nair et al., 2011). Interestingly, mammalian NSF associates with both the GATE16 and the GABARAP subfamilies (Kittler et al., 2001; Sagiv et al., 2000). However, a role for this interaction in autophagosome biogenesis has not yet been established. It has been suggested GATE16 that acts as an intermediary between NSF activity and SNARE activation (Sagiv et al., 2000). If so, GATE16 could act in concert with NSF and SNARE proteins to mediate autophagosome biogenesis. Recent studies have revealed that vesicles containing the ATG5–ATG12–ATG16L complex, derived from the plasma membrane, undergo SNARE-mediated homotypic fusion, and that this process is important for autophagosome formation (Moreau et al., 2011; Ravikumar et al., 2010b).

To sum up these recent findings, both the Atg12 and Atg8 conjugation systems act at early stages of autophagosome formation by recruiting the protein machinery involved in this process and by participating in membrane remodeling.

Numerous studies have recently established that the Atg12 and Atg8 conjugation systems have a dual role in early stages of autophagosome formation. These systems are needed for the elongation and, possibly, sealing of the autophagosomal membrane and, at the same time, act as part of the machinery for selective cargo recruitment into autophagosomes. Nevertheless, there are still many questions regarding these systems that need to be addressed.

Until recently, Atg5 was thought to be the sole target of Atg12. New research has revealed, however, that Atg12 is also conjugated to Atg3. This conjugation, and its surprising role in mitochondrial quality control, raises the exciting possibility that both Atg12 and Atg3 have additional interacting partners with important regulatory functions, and this is a fertile area for future research. Another major question in the autophagy field has to do with the biogenesis of autophagosomes. This process requires numerous factors, including Atg proteins and SNAREs. However, the details of the interplay between these proteins and their contribution to autophagosome biogenesis are still vague. Studying autophagosome biogenesis is extremely difficult because of the large number of factors and organelles that participate in this process. Isolation of phagophores might be a crucial step towards understanding the machinery involved and the necessary steps in autophagosome biogenesis. Moreover, the finding that the Atg12–Atg5–Atg16 complex possesses E3-like activity for Atg8 lipidation but does not reside on the phagophore inner membrane raises an important question: does the Atg12–Atg5–Atg16 complex direct Atg8 conjugation on both sides of the phagophore or only on its outer membrane? It is unclear, moreover, how the Atg12–Atg5–Atg16 complex associates with the phagophore and dissociates from it. Moreover, it will be essential to determine the exact localization of Atg8 proteins to understand their role in autophagosome biogenesis. Furthermore, the crosstalk between Atg8 proteins and other factors, such as NSF and CDC48, is in need of elucidation. These are among the questions to be addressed in future studies.

This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Autophagy and cancer – issues we need to digest’ by Emma Liu and Kevin Ryan (J. Cell Sci. 125, 2349-2358) and ‘Autophagy and cell growth – the yin and yang of nutrient responses’ by Thomas Neufeld (J. Cell Sci. 125, 2359-2368).

Funding

Z.E. is the incumbent of the Harold Korda Chair of Biology. We are grateful for funding from the Legacy Heritage Fund, the Israeli Science Foundation ISF, the German Israeli Foundation GIF, and the Louis Brause Philanthropic Fund (all to Z.E.).

A high-resolution version of the poster is available for downloading in the online version of this article at jcs.biologists.org. Individual poster panels are available as JPEG files at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.093757/-/DC1.

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Opposing microtubule motors drive robust nuclear dynamics in developing muscle cells

Advance Online Publication May 23, 2012 doi: 10.1242/?jcs.108688 Meredith H. Wilson and Erika L. F. Holzbaur*?*Corresponding author: Erika Holzbaur, Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, D400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. T: (215) 573-3257, F: (215) 573-5851, Email: holzbaur{at}mail.med.upenn.eduDynamic interactions with the cytoskeleton drive the movement and positioning of nuclei in many cell types. During muscle cell development, myoblasts fuse to form syncytial myofibers with nuclei positioned regularly along the length of the cell. Nuclear translocation in developing myotubes requires microtubules, but the mechanisms involved have not been elucidated. We find that as nuclei actively translocate through the cell, they rotate in three-dimensions. The nuclear envelope, nucleoli, and chromocenters within the nucleus rotate together as a unit. Both translocation and rotation require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around nuclei. The plus- and minus-end directed microtubule motor proteins, kinesin-1 and dynein, localize to the nuclear envelope in myotubes. Kinesin-1 localization is mediated at least in part by interaction with klarsicht/ANC-1/Syne homology (KASH) proteins. Depletion of kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei at the midline of the myotube. Dynein depletion also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. Thus, oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube likely result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton that is also continuously remodeled as the nuclei move. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

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Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.108118 Telomeres play critical roles in the maintenance of genome integrity and control of cellular senescence. Most eukaryotic telomeres can be transcribed to generate a telomeric repeat-containing RNA (TERRA) that persists as a heterogeneous nuclear RNA and can be developmentally regulated. However, the precise function and regulation of TERRA in normal and cancer cell development remains poorly understood. Here, we show that TERRA accumulates in highly proliferating normal and cancer cells, and forms large nuclear foci, which are distinct from previously characterized markers of DNA damage or replication stress. Using a mouse model for medulloblastoma driven by chronic Sonic hedgehog (SHH) signaling, TERRA RNA was detected in tumor, but not adjacent normal cells using both RNA FISH and Northern blotting. RNA-FISH revealed the formation of TERRA foci (TERFs) in the nuclear regions of rapidly proliferating tumor cells. In the normal developing cerebellum, TERRA aggregates could also be detected in highly proliferating zones of progenitor neurons. SHH could enhance TERRA expression in purified granule progenitor cells in vitro, suggesting that proliferation signals contribute to TERRA expression in responsive tissue. TERFs did not colocalize with ?H2AX foci, PML, or Cajal bodies in mouse tumor tissue. We also provide evidence that TERRA is elevated in a variety of human cancers. These findings suggest that elevated TERRA levels reflect a novel early form of telomere regulation during replication stress and cancer cell evolution, and the TERRA RNA aggregates may form a novel nuclear body in highly proliferating mammalian cells.

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Regulation of multiple target genes by miR-1/miR-206 is pivotal for C2C12 myoblast differentiation

Advance Online Publication May 17, 2012 doi: 10.1242/?jcs.101758 Katarzyna Goljanek-Whysall, Helio Pais, Tina Rathjen, Dylan Sweetman, Tamas Dalmay and Andrea Münsterberg*?*Author for correspondence: Andrea Münsterberg, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK, phone: +44(0)1603 592232. Fax: +44(0)1603 592250; E-mail: a.munsterberg{at}uea.ac.ukmicroRNAs are short non-coding RNAs involved in post-transcriptional regulation of multiple messenger RNA targets. The miR-1/miR-206 family is expressed during skeletal muscle differentiation and is an integral component of myogenesis. To better understand miR-1/miR-206 function during myoblast differentiation we identified novel target mRNAs by microarray and characterized their function in C2C12 myoblasts. Candidate targets from the screen were experimentally validated together with target genes that were predicted by three different algorithms. Some targets characterized have a known function in skeletal muscle development and/or differentiation and include Meox2, RARB, Fzd7, MAP4K3, CLCN3 and NFAT5, others are potentially novel regulators of myogenesis, such as the chromatin remodeling factors Smarcd2 and Smarcb1 or the anti-apoptotic protein SH3BGRL3. The expression profiles of confirmed target genes were examined during C2C12 cell myogenesis. We found that inhibition of endogenous miR-1/miR-206 by antimiRs blocked the downregulation of most targets in differentiating cells, thus indicating that microRNA activity and target interaction is required for muscle differentiation. Finally, we show that sustained expression of validated miR-1/206 targets resulted in increased proliferation and inhibition of C2C12 cell myogenesis. In many cases the expression of genes related to non-muscle cell fates, such as chondrogenesis, was activated. This indicates that the concerted downregulation of multiple microRNA targets is not only crucial to the skeletal muscle differentiation program but also serves to prevent alternative cell fate choices.

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Inhibiting myostatin reverses muscle fibrosis through apoptosis

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.090365 Zhao Bo Li, Jiangyang Zhang and Kathryn R. Wagner*?*Communicating Author: Kathryn R. Wagner, M.D., Ph.D., Center for Genetic Muscle Disorders, The Kennedy Krieger Institute, The Johns Hopkins School of Medicine, 707 North Broadway, Baltimore, MD 21205, 443-923-9525 (t), 443-923-9545 (f), wagnerk{at}kennedykrieger.orgSkeletal muscle fibrosis is a defining feature of the muscular dystrophies in which contractile myofibers are replaced by fibroblasts, adipocytes and extracellular matrix. This maladaptive response of muscle to repetitive injury is progressive, self-perpetuating and thus far, has been considered irreversible. We have previously shown that myostatin, a known endogenous modulator of muscle growth, stimulates normal muscle fibroblasts to proliferate. Here, we demonstrate that myostatin also regulates the proliferation of dystrophic muscle fibroblasts, and increases resistance of fibroblasts to apoptosis through Smad and MAPK signaling. Inhibiting myostatin signaling pathways with a soluble activin IIB receptor (ActRIIB.Fc), reduces resistance of muscle fibroblasts to apoptosis in vitro. Systemic administration of ActRIIB.Fc in senescent mdx mice, a model of muscular dystrophy, significantly increases the number of muscle fibroblasts undergoing apoptosis. This leads to the reversal of pre-existed muscle fibrosis as determined by histological, biochemical and radiographical criteria. These results demonstrate that skeletal muscle fibrosis can be pharmacologically reversed through induction of fibroblast apoptosis.

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Cofilin Nuclear-Cytoplasmic Shuttling Affects Cofilin-Actin Rod Formation During Stress

Advance Online Publication May 23, 2012 doi: 10.1242/?jcs.097667 Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.

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Alternative translation initiation gives rise to two isoforms of orai1 with distinct plasma membrane mobilities

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.104919 Store-operated calcium entry is a nearly ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1 as well as expressed Orai1 exists in two forms in similar quantities: a longer form (Orai1a) of approximately 33 kDa, and a shorter form (Orai1ß) of approximately 23 kDa. The second Orai1ß form arises from alternative translation initiation from a methionine at position 64, and possibly also 71, in the longer, Orai1a form. In the sequence upstream of the initiation site of Orai1ß, there is a poly-arginine sequence previously suggested to be involved in interaction of Orai1 with plasma membrane phosphatidylinositol 4,5-bisphosphate. The loss of this phospholipid binding domain would be expected to influence the mobility of Orai1 protein in the plasma membrane. Indeed, experiments utilizing fluorescence recovery after photobleaching (FRAP) revealed that the recovery half-time for Orai1ß was significantly faster than for Orai1a. Since Orai1 must diffuse to sites of interaction with the Ca2+ sensor, STIM1, these two mobilities might provide for efficient recruitment of Orai1 subunits to sites of store-operated Ca2+ entry during agonist-induced Ca2+ signaling.

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ATF2 - at the crossroad of nuclear and cytosolic functions

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.095000 An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCe) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for ‘oncogene addiction’.

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The Nup155-mediated Organization of Inner Nuclear Membrane Proteins is Independent of Nup155 Anchoring to the Metazoan Nuclear Pore Complex

Advance Online Publication June 20, 2012 doi: 10.1242/?jcs.105809 The nuclear envelope (NE), an important barrier between the nucleus and the cytoplasm, is composed of three structures: The outer nuclear membrane (ONM), which is continuous with the ER, the inner nuclear membrane (INM), which interfaces with chromatin, and nuclear pore complexes (NPCs), which are essential for the exchange of macromolecules between the two compartments. The NPC protein Nup155 has an evolutionarily conserved role in the metazoan NE formation; but the in vivo analysis of Nup155 has been severely hampered by the essential function of this protein in cell viability. Here, we take advantage of the hypomorphicity of RNAi systems and use a combination of protein binding and rescue assays to map the interaction sites of two neigbouring NPC proteins Nup93 and Nup53 on Nup155, and to define the requirement of these interactions in INM protein organization. We show that different parts of Drosophila Nup155 have distinct functions: The Nup155 ß-propeller anchors the protein to the NPC, while the a-solenoid part of Nup155 is essential for the correct localization of INM proteins LBR and otefin. Using chromatin extracts from semi-synchronized cells, we also provide evidence that the Nup155 a-solenoid has a chromatin-binding activity that is stronger at the end of mitosis. Our results argue that the role of Nup155 in INM protein localization is not mediated through the NPC anchoring activity of the protein and suggest that regions other than Nup155 ß-propeller are necessary for the targeting of proteins to the INM.

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Advances in high-resolution imaging - techniques for three-dimensional imaging of cellular structures

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.090027 A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

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Autophagy and cell growth - the yin and yang of nutrient responses

Advance Online Publication May 30, 2012 doi: 10.1242/?jcs.103333 May 15, 2012 J Cell Sci 125, 2359-2368. Thomas P. Neufeld?

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA neufeld{at}med.umn.edu As a response to nutrient deprivation and other cell stresses, autophagy is often induced in the context of reduced or arrested cell growth. A plethora of signaling molecules and pathways have been shown to have opposing effects on cell growth and autophagy, and results of recent functional screens on a genomic scale support the idea that these processes might represent mutually exclusive cell fates. Understanding the ways in which autophagy and cell growth relate to one another is becoming increasingly important, as new roles for autophagy in tumorigenesis and other growth-related phenomena are uncovered. This Commentary highlights recent findings that link autophagy and cell growth, and explores the mechanisms underlying these connections and their implications for cell physiology and survival. Autophagy and cell growth can inhibit one another through a variety of direct and indirect mechanisms, and can be independently regulated by common signaling pathways. The central role of the mammalian target of rapamycin (mTOR) pathway in regulating both autophagy and cell growth exemplifies one such mechanism. In addition, mTOR-independent signaling and other more direct connections between autophagy and cell growth will also be discussed.

Key words This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Ubiquitin-like proteins and autophagy at a glance’ by Tomer Shpilka et al. (J. Cell Sci. 125, 2343-2348) and ‘Autophagy and cancer – issues we need to digest’ by Emma Liu and Kevin Ryan (J. Cell Sci. 125, 2349-2358).

Funding

The work of our laboratory is supported by the National Institutes of Health [grant number GM62509]. Deposited in PMC for release after 12 months.

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Perilipin family members preferentially sequester to either triacylglycerol- or cholesteryl ester-specific intracellular lipid storage droplets

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.104943 Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes encode 5 distinct Plin gene members and additional protein forms derive from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging, and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to lipid droplets, comprised distinctly of either triacylcerides or of cholesterol esters. Further, Plins with strong association preferences to TAG (or CE) droplets can re-direct the relative intracellular TAG/CE balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.

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Regulation of apoptosis by Bat3-enhanced YWK-II protein/APLP2 stability

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.086553 YWK-II protein/APLP2 is a member of an evolutionarily conserved protein family that includes amyloid precursor protein (APP) and amyloid precursor like protein-1 (APLP1). We have previously demonstrated that YWK-II/APLP2 functions as a novel G0-protein-coupled receptor for Müllerian inhibiting substance (MIS) in cell survival. However, factors regulating the stability and turnover of YWK-II/APLP2 have not been identified. Here we present evidence that human leukocyte antigen-B-associated transcript 3 (Bat3), an important regulator involved in apoptosis, can interact with YWK-II/APLP2 and enhance its stability by reducing its ubiquitination and degradation by the ubiquitin-proteasome system. Co-expression of different Bat3 domain deletion constructs with YWK-II/APLP2 reveals that the proline-rich domain of Bat3 is required for its binding to YWK-II/APLP2. In addition, we find that the protein levels of YWK-II/APLP2 could be enhanced by nuclear export of Bat3 under apoptotic stimulation. We also find elevated levels of Bat3 and YWK-II/APLP2 in human colorectal cancer with a positive correlation between the two. Taken together, these results have revealed a previously undefined mechanism regulating cell apoptosis and suggest that aberrant enhancement of YWK-II/APLP2 by nuclear export of Bat3 may play a role in cancer development by inhibiting cell apoptosis.

This work was supported by grants from the National program for the Important Research Plan (2011CB944302, 2012CB944902/903), National Key Technology Support Program (2012BAI31B08), State Key Laboratory Special Fund (2060204), 111 Project (B08007) from Ministry of Education and the Focused Investment Scheme of The Chinese University of Hong Kong.

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Heat shock induces rapid resorption of primary cilia

Advance Online Publication June 20, 2012 doi: 10.1242/?jcs.100545 Natalia V. Prodromou*, Clare Thompson*, Daniel P. S. Osborn, Kathryn F. Cogger, Rachel Ashworth, Martin M. Knight, Philip L. Beales and J. Paul Chapple?Correspondence: Dr Paul Chapple, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. Tel: +44 (0) 20 7882 6242; Fax +44 (0) 20 7882 6197; E-mail: j.p.chapple{at}qmul.ac.uk?*These authors contributed equally to this work

Primary cilia are involved in important developmental and disease pathways, such as regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. Here we investigate the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells cilia loss correlated with a reduction in hedgehog signalling. Heat shock dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in primary cilia maintenance. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in remaining cilia. We hypothesise ciliary resorption serves to attenuate cilia mediated signalling pathways in response to extracellular stress and that this mechanism is regulated in part by HDAC6 and Hsp90.

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Semaphorin 3A elevates endothelial cell permeability through PP2A inactivation

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.108282 Armelle Le Guelte, Eva-Maria Galan-Moya, Julie Dwyer, Lucas Treps, Garance Kettler, Jagoda K. Hebda, Sonia Dubois, Cedric Auffray, Herve Chneiweiss, Nicolas Bidere and Julie Gavard*?*Correspondence: Julie Gavard, Institut Cochin 22 rue Mechain Rm. 306, 75014 Paris, France. Ph: +33 1 4051 6424; Fax: +33 1 4051 6430; e-mail: julie.gavard{at}inserm.frVE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalization, cell-cell junction destabilization, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability.

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