Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.     

The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions

During development, all cells make the decision to live or die. Although the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C.?elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C.?elegans divisions that produce apoptotic cells [3]. We identified CNT-2, an Arf GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is essential for its function in these divisions. The N terminus of CNT-2, which contains?a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2's function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions.     

Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice

Diesel exhaust particles (DEP) are a major source of air-borne pollution and are linked to increased risk of disease including lung cancer. Here we investigated effects of exposure to DEP on the frequency of DNA deletions, levels of oxidative DNA damage and DNA adduct formation during embryonic development in mice. Pregnant dams were orally exposed to various doses of DEP (500, 250, 125, 62.5, 31.25 mg/kg/day) at embryonic days 10.5–15.5. We determined the frequency of 70 kb DNA deletions spanning exons 6–18 at the p^un allele that results in black-pigmented spots in the unpigmented retinal pigment epithelium in the eyes of p^un/p^un offspring mice. DEP caused a significant increase in the frequency of DNA deletions. Levels of 8-OH deoxyguanosine indicating oxidative DNA damage were within the limits of the unexposed mouse embryos. ³³P post-labeling analysis revealed very low levels of DNA adducts in the embryo tissue. Thus, transplacental exposure to DEP resulted in a significant increase in the frequency of DNA deletions in the mouse fetus and such genetic alterations in the offspring may have pathological consequences later in life.     

Synthesis, X-ray crystal structure, DNA binding, antioxidant and cytotoxicity studies of Ni(ii) and Pd(ii) thiosemicarbazone complexes

New complexes, [Ni(HL)(PPh(3))]Cl (1), [Pd(L)(PPh(3))](2), and [Pd(L)(AsPh(3))](3), were synthesized from the reactions of 4-chloro-5-methyl-salicylaldehyde thiosemicarbazone [H(2)L] with [NiCl(2)(PPh(3))(2)], [PdCl(2)(PPh(3))(2)] and [PdCl(2)(AsPh(3))(2)]. They were characterized by IR, electronic, (1)H-NMR spectral data. Further, the structures of the complexes have been determined by single crystal X-ray diffraction. While the thiosemicarbazone coordinated as binegative tridentate (ONS) in complexes 2 and 3, it is coordinated as mono negative tridentate (ONS) in 1. The interactions of the new complexes with calf thymus DNA was examined by absorption and emission spectra, and viscosity measurements. Moreover, the antioxidant properties of the new complexes have also been tested against DPPH radical in which complex 1 exhibited better activity than that of the other two complexes 2 and 3. The in vitro cytotoxicity of complexes 1-3 against A549 and HepG2 cell lines was assayed, and the new complexes exhibited higher cytotoxic activity with lower IC(50) values indicating their efficiency in killing the cancer cells even at very low concentrations.     

Development of a Prototype System for Archiving Integrative/Hybrid Structure Models of Biological Macromolecules

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Phylogenetic Analysis of the NEEP21/Calcyon/P19 Family of Endocytic Proteins: Evidence for Functional Evolution in the Vertebrate CNS

Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.     

Physiological history may mask the inherent inducible desiccation tolerance strategy of the desert moss Crossidium crassinerve

Shoots of bryophytes collected in the desiccated state from the field are likely to be hardened to desiccation tolerance (DT) to varying degrees. To account for this, most studies on DT include a relatively short deacclimation period. However, no study has experimentally determined the appropriate deacclimation time for any bryophyte species. Our purposes are to (i) determine if ‘field effects’ are biologically relevant to DT studies and how long a deacclimation period is required to remove them; and (ii) utilise field versus cultured shoot responses within the context of a deacclimation period to elucidate the ecological strategy of DT. Our hypothesis (based on an extensive literature on DT) is that a deacclimation period from 24 to 72 h should be sufficient to eliminate historical stress effects on the physiology of the shoots and allow an accurate determination of the inherent ecological DT strategy (constitutive or inducible). We determined, however, using chlorophyll fluorescence and visual estimates of shoot damage, that field‐collected shoots of the desert moss Crossidium crassinerve required an experimental deacclimation period of >7 days before field effects were removed, and revealed an ecological DT strategy of inducible DT. If the deacclimation period was <6 days, the shoot response conformed to an ecological strategy of constitutive protection. Thus the presence of field effects can obscure the ecological strategy of desiccation tolerance exhibited by the species, and this translates into a need to re‐evaluate previous mechanistic and ecological studies of desiccation tolerance in plants.     

Modularity of Protein Folds as a Tool for Template-Free Modeling of Structures

Predicting the three-dimensional structure of proteins from their amino acid sequences remains a challenging problem in molecular biology. While the current structural coverage of proteins is almost exclusively provided by template-based techniques, the modeling of the rest of the protein sequences increasingly require template-free methods. However, template-free modeling methods are much less reliable and are usually applicable for smaller proteins, leaving much space for improvement. We present here a novel computational method that uses a library of supersecondary structure fragments, known as Smotifs, to model protein structures. The library of Smotifs has saturated over time, providing a theoretical foundation for efficient modeling. The method relies on weak sequence signals from remotely related protein structures to create a library of Smotif fragments specific to the target protein sequence. This Smotif library is exploited in a fragment assembly protocol to sample decoys, which are assessed by a composite scoring function. Since the Smotif fragments are larger in size compared to the ones used in other fragment-based methods, the proposed modeling algorithm, SmotifTF, can employ an exhaustive sampling during decoy assembly. SmotifTF successfully predicts the overall fold of the target proteins in about 50% of the test cases and performs competitively when compared to other state of the art prediction methods, especially when sequence signal to remote homologs is diminishing. Smotif-based modeling is complementary to current prediction methods and provides a promising direction in addressing the structure prediction problem, especially when targeting larger proteins for modeling.