p62/SQSTM1: A Missing Link between Protein Aggregates and the Autophagy Machinery

In eukaryotic cells short-lived proteins are degraded in a specific process by the ubiquitin-proteasome system (UPS), whereas long-lived proteins and damaged organelles are degraded by macroautophagy (hereafter referred to as autophagy). A growing body of evidence now suggests that autophagy is important for clearance of protein aggregates that form in cells as a consequence of ageing, oxidative stress, alterations that elevate the amounts of certain aggregation-prone proteins or expression of aggregating mutant variants of specific proteins. Autophagy is generally considered to be a non-specific, bulk degradation process. However, a recent study suggests that p62/SQSTM1 may link the recognition of polyubiquitinated protein aggregates to the autophagy machinery.1 This protein is able to polymerize via its N-terminal PB1 domain and to recognize polyubiquitin via its C-terminal UBA domain. It can also recruit the autophagosomal protein LC3 and co-localizes with many types of polyubiquitinated protein aggregates.1 Here we discuss possible implications of these findings and raise some questions for further investigation.     

The Role of Autophagy During Development in Higher Eukaryotes

Autophagy is a lysosome‐mediated degradation pathway used by eukaryotes to recycle cytosolic components in both basal and stress conditions. Several genes have been described as regulators of autophagy, many of them being evolutionarily conserved from yeast to mammals. The study of autophagy‐defective model systems has made it possible to highlight the importance of correctly functioning autophagic machinery in the development of invertebrates as, for example, during the complex events of fly and worm metamorphosis. In vertebrates, on the other hand, autophagy defects can be lethal for the animal if the mutated gene is involved in the early stages of development, or can lead to severe phenotypes if the mutation affects later stages. However, in both lower and higher eukaryotes, autophagy seems to be crucial during embryogenesis by acting in tissue remodeling in parallel with apoptosis. An increase of autophagic cells is, in fact, observed in the embryonic stages characterized by massive cell elimination. Moreover, autophagic processes probably protect cells during metabolic stress and nutrient paucity that occur during tissue remodeling. In light of such evidence, it can be concluded that there is a close interplay between autophagy and the processes of cell death, proliferation and differentiation that determine the development of higher eukaryotes.     

LYST Affects Lysosome Size and Quantity,but not Trafficking or Degradation Through Autophagy or Endocytosis

Mutations in the large BEACH domain‐containing protein LYST causes Chediak–Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome‐related organelles in various cell types. Dysfunctional secretion of enlarged lysosome‐related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA‐depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein‐3 (AP‐3) and cation‐independent mannose‐6‐phosphate receptor (CI‐MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events.      

Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation

The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho‐proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. Here, we show that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. We show that neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.     

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

小盐芥受精作用和胚发育的解剖学研究

用石蜡切片法对小盐芥(Thellungiella halophila(C.A.Mey.)O.E.Schulz)的受精作用及胚和胚乳的发育过程进行了解剖学观察,结果显示:花盛开前后,是受精作用的核融合时期;受精前,2个极核融合成次生核;受精作用属有丝分裂前型;胚乳发育为核型;胚发育属柳叶菜型,子叶背倚胚根;成熟种子无胚乳.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

The Balanced Regulation of Hsc70 by DNJ-13 and UNC-23 Is Required for Muscle Functionality

The molecular chaperone Hsc70 assists in the folding of non-native proteins together with its J domain- and BAG domain-containing cofactors. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Using reporter strains, we find that the full-length UNC-23, in contrast to C-terminal fragments, localizes specifically to the muscular attachment sites. C-terminal fragments of UNC-23 instead perform all Hsc70-related functions, like ATPase stimulation and regulation of folding activity, albeit with lower affinity than BAG-1. Interestingly, overexpression of CFP-Hsc70 can induce muscular defects in wild-type nematodes that phenocopy the knockout of its cofactor UNC-23. Strikingly, the motility dysfunction in the unc-23 mutated strain can be cured specifically by down-regulation of the antagonistic Hsc70 cochaperone DNJ-13, implying that the severe phenotype is caused by misregulation of the Hsc70 cycle. These findings point out that the balanced action of cofactors in the ATP-driven cycle of Hsc70 is crucial for the contribution of Hsc70 to muscle functionality.     

新疆2个主栽早实核桃品种无融合生殖及胚细胞发育研究

以新疆‘新新2’、‘温185’2个主栽早实核桃品种为材料,通过对雌花采取套硫酸纸袋、聚乙烯醇封柱头、切柱头等3种无融合生殖处理,以自然授粉为对照,观察分析了无融合生殖胚乳和胚发育过程,结果显示:(1)‘新新2’的自然座果率比‘温185’要高,但其无融合生殖率却略低于‘温185’。(2)无融合生殖形成的果实较正常自然生长形成的果实略小。(3)2个核桃品种胚囊均属蓼型,历经10d左右发育成熟。(4)2个品种无融合生殖胚均由卵细胞分裂发育而来,经原胚期、球形期、心形期、鱼雷形期、成熟期发育完成,历经10d左右,属孤雌生殖类型。(5)2个核桃品种均属核型胚乳,8d左右形成胚乳细胞。     

Development and evaluation of a genome‐wide Coffee 8.5K SNP array and its application for high‐density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.     

New Insights into an Old Organelle: Meeting Report on Biology of Cilia and Flagella

The rising interest of the scientific community in cilia biology was evident from the fact that registration for the third FASEB conference on ‘The Biology of Cilia and Flagella’ closed out before the early bird deadline. Cilia and flagella are organelles of profound medical importance; defects in their structure or function result in a plethora of human diseases called ciliopathies. 240 clinicians and basic scientists from around the world gathered from 23 June 2013 to 28 June 2013 at Sheraton at the Falls, Niagara Falls, NY to present and discuss their research on this intensely studied subcellular structure. The meeting was organized by Gregory Pazour (University of Massachusetts Medical School), Bradley Yoder (University of Alabama‐Birmingham), and Maureen Barr (Rutgers University) and was sponsored by the Federation of American Societies for Experimental Biology (FASEB). Here, we report highlights, points of discussion, and emerging themes from this exciting meeting.