the-tomatogfp-flpfrt-method-for-live-imaging-mosaic-adult-drosophila

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult <em>Drosophila</em> Photoreceptor Cells

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Ecole Normale Supérieure de Lyon

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.

Regulation of R7 and R8 differentiation by the spalt genes.

The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. Here, we show that the spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, we find that the spalt genes are specifically required in R3 for the establishment of correct ommatidial polarity. In addition, we show that spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. We show that spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and that seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, we show that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate.

Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye.

How a pool of equipotent cells acquires a multitude of distinct fates is a major question in developmental biology. The study of photoreceptor (PR) cell differentiation in Drosophila has been used to address this question. PR differentiation is a process that extends over a period of 5 days: It begins in the larval eye imaginal disc when PRs are recruited and commit to particular PR fates, and it culminates in the pupal eye disc with the morphogenesis of the rhabdomeres and the initiation of rhodopsin expression. Several models for PR specification agree that the Ras and Notch signaling pathways are important for the specification of different PR subtypes (Freeman [1997] Development 124:261-270; Cooper and Bray [2000] Curr. Biol. 10:1507-1510; Tomlinson and Struhl [2001] Mol. Cell. 7:487-495). In the first part of this review, we briefly describe the different signaling pathways and transcription factors required for the specification and differentiation of the different PR subtypes in the larval eye disc. In the second part, we review the roles of several transcription factors, which are required for the terminal photoreceptor differentiation and rhodopsin expression.

Photoreceptor differentiation in Drosophila: from immature neurons to functional photoreceptors.

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina.

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.

ER stress protects from retinal degeneration.

Cell death: what can we learn from flies? Editorial for the special review issue on Drosophila apoptosis.

Wolbachia is an intracellular bacterium generally described as being a facultative reproductive parasite. However, Wolbachia is necessary for oogenesis completion in the wasp Asobara tabida. This dependence has evolved recently as a result of interference with apoptosis during oogenesis. Through comparative transcriptomics between symbiotic and aposymbiotic individuals, we observed a differential expression of ferritin, which forms a complex involved in iron storage. Iron is an essential element that is in limited supply in the cell. However, it is also a highly toxic precursor of Reactive Oxygen Species (ROS). Ferritin has also been shown to play a key role in host-pathogen interactions. Measuring ferritin by quantitative RT-PCR, we confirmed that ferritin was upregulated in aposymbiotic compared to symbiotic individuals. Manipulating the iron content in the diet, we showed that iron overload markedly affected wasp development and induced apoptotic processes during oogenesis in A. tabida, suggesting that the regulation of iron homeostasis may also be related to the obligate dependence of the wasp. Finally, we demonstrated that iron metabolism is influenced by the presence of Wolbachia not only in the obligate mutualism with A. tabida, but also in facultative parasitism involving Drosophila simulans and in Aedes aegypti cells. In these latter cases, the expression of Wolbachia bacterioferritin was also increased in the presence of iron, showing that Wolbachia responds to the concentration of iron. Our results indicate that Wolbachia may generally interfere with iron metabolism. The high affinity of Wolbachia for iron might be due to physiological requirement of the bacterium, but it could also be what allows the symbiont to persist in the organism by reducing the labile iron concentration, thus protecting the cell from oxidative stress and apoptosis. These findings also reinforce the idea that pathogenic, parasitic and mutualistic intracellular bacteria all use the same molecular mechanisms to survive and replicate within host cells. By impacting the general physiology of the host, the presence of a symbiont may select for host compensatory mechanisms, which extends the possible consequences of persistent endosymbiont on the evolution of their hosts.

Wolbachia interferes with ferritin expression and iron metabolism in insects.

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP, plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.

Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants.

We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The "Tomato/GFP-FLP/FRT" method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.

Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.

ER stress inhibits neuronal death by promoting autophagy.

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

2008 &#x5E74;&#x6211;&#x4EEC;&#x53D1;&#x8868;&#x4E86;&#x7B2C;&#x4E00;&#x5957;&#x6307;&#x5F15;&#xFF0C;&#x89C4;&#x8303;&#x81EA;&#x566C;&#x7684;&#x7814;&#x7A76;&#x3002;&#x81EA;&#x90A3;&#x65F6;&#x4EE5;&#x6765;&#xFF0C;&#x5173;&#x4E8E;&#x8FD9;&#x4E00;&#x4E3B;&#x9898;&#x7684;&#x7814;&#x7A76;&#x5DF2;&#x7EE7;&#x7EED;&#x52A0;&#x901F;&#xFF0C;&#x548C;&#x8BB8;&#x591A;&#x65B0;&#x79D1;&#x5B66;&#x5BB6;&#x5DF2;&#x8F93;&#x5165;&#x8BE5;&#x5B57;&#x6BB5;&#x3002;&#x6B64;&#x5916;&#x6269;&#x5927;&#x6211;&#x4EEC;&#x7684;&#x77E5;&#x8BC6;&#x5E93;&#x548C;&#x76F8;&#x5173;&#x65B0;&#x6280;&#x672F;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5B83;&#x662F;&#x91CD;&#x8981;&#x7684;&#x662F;&#x8981;&#x66F4;&#x65B0;&#x8FD9;&#x4E9B;&#x51C6;&#x5219;&#xFF0C;&#x4EE5;&#x4FBF;&#x76D1;&#x6D4B;&#x4E0D;&#x540C;&#x751F;&#x7269;&#x4F53;&#x4E2D;&#x7684;&#x81EA;&#x566C;&#x3002;&#x5404;&#x9879;&#x5BA1;&#x67E5;&#x6240;&#x63CF;&#x8FF0;&#x5DF2;&#x7528;&#x4E8E;&#x6B64;&#x76EE;&#x7684;&#x7684;&#x68C0;&#x6D4B;&#x7684;&#x8303;&#x56F4;&#x3002;&#x5C3D;&#x7BA1;&#x5982;&#x6B64;&#xFF0C;&#x7EE7;&#x7EED;&#x5173;&#x4E8E;&#x53EF;&#x63A5;&#x53D7;&#x7684;&#x65B9;&#x6CD5;&#x6765;&#x8861;&#x91CF;&#x81EA;&#x566C;&#xFF0C;&#x5C24;&#x5176;&#x662F;&#x5728;&#x591A;&#x7EC6;&#x80DE;&#x771F;&#x6838;&#x751F;&#x7269;&#x7684;&#x6DF7;&#x4E71;&#x3002;&#x9700;&#x8981;&#x5F3A;&#x8C03;&#x6307;&#x51FA;&#x7684;&#x5173;&#x952E;&#x70B9;&#x5C31;&#x662F;&#x76D1;&#x6D4B;&#x6570;&#x5B57;&#x6216;&#x5377;&#x7684;&#x81EA;&#x566C;&#x6027;&#x5143;&#x7D20; &#xFF08;&#x4F8B;&#x5982;&#xFF0C;autophagosomes &#x6216; autolysosomes&#xFF09; &#x7684;&#x6D4B;&#x91CF;&#x7ED3;&#x679C;&#x4E4B;&#x95F4;&#x7684;&#x5DEE;&#x5F02;&#x4E0E;&#x90A3;&#x4E9B;&#x81EA;&#x566C;&#x8FC7;&#x7A0B;&#x7684;&#x4EFB;&#x4F55;&#x9636;&#x6BB5;&#xFF0C;&#x68C0;&#x6D4B;&#x6D41;&#x91CF;&#x901A;&#x8FC7;&#x81EA;&#x566C;&#x9014;&#x5F84; &#xFF08;&#x5373;&#xFF0C;&#x5B8C;&#x6574;&#x7684;&#x8FDB;&#x7A0B;&#xFF09; &#xFF1B;&#x56E0;&#x6B64;&#xFF0C;macroautophagy autophagosome &#x79EF;&#x7D2F;&#x7684;&#x7ED3;&#x679C;&#x9700;&#x8981;&#x52A0;&#x4EE5;&#x533A;&#x5206;&#x4ECE;&#x81EA;&#x566C;&#x6027;&#x6D3B;&#x52A8;&#x589E;&#x52A0;&#xFF0C;&#x4F1A;&#x5BFC;&#x81F4;&#x7684;&#x523A;&#x6FC0;&#x4E2D;&#x7684;&#x5757;&#x5B9A;&#x4E49;&#x4E3A;&#x589E;&#x52A0;&#x81EA;&#x566C;&#x8BF1;&#x5BFC;&#x52A0;&#x4E0A;&#x589E;&#x52A0;&#x7684;&#x4EA4;&#x4ED8;&#x548C;&#x6EB6;&#x9176;&#x4F53; &#xFF08;&#x5728;&#x5927;&#x591A;&#x6570;&#x9AD8;&#x7B49;&#x771F;&#x6838;&#x751F;&#x7269;&#x548C;&#x76D8;&#x57FA;&#x7F51;&#x67C4;&#x7B49;&#x4E00;&#x4E9B;&#x751F;&#x7269;&#x4F53;&#xFF09; &#x6216; &#xFF08;&#x5728;&#x690D;&#x7269;&#x548C;&#x771F;&#x83CC;&#xFF09; &#x6CE1;&#x5185;&#x7684;&#x964D;&#x89E3;&#x3002;&#x6362;&#x53E5;&#x8BDD;&#x8BF4;&#xFF0C;&#x5B83;&#x662F;&#x5C24;&#x5176;&#x91CD;&#x8981;&#x7684;&#x662F;&#x65B0;&#x7684;&#x9886;&#x57DF;&#x7684;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#x4E86;&#x89E3;&#x66F4;&#x591A; autophagosomes &#x7684;&#x5916;&#x89C2;&#x4E0D;&#x4E00;&#x5B9A;&#x7B49;&#x540C;&#x66F4;&#x591A;&#x81EA;&#x566C;&#x3002;&#x4E8B;&#x5B9E;&#x4E0A;&#xFF0C;&#x5728;&#x8BB8;&#x591A;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;autophagosomes &#x79EF;&#x7D2F;&#x7531;&#x4E8E;&#x8D29;&#x8FD0;&#x5230;&#x6EB6;&#x9176;&#x4F53;&#x4E0D;&#x968F;&#x4E4B;&#x6539;&#x53D8;&#x7684;&#x751F;&#x7269;&#x53D1;&#x751F; autophagosome&#xFF0C;&#x4E00;&#x4E2A;&#x5757;&#x800C;&#x589E;&#x52A0; autolysosomes &#x53EF;&#x964D;&#x89E3;&#x6D3B;&#x52A8;&#x4E2D;&#x53CD;&#x6620;&#x51CF;&#x5C11;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x5957;&#x51C6;&#x5219;&#x7684;&#x9009;&#x62E9;&#x548C;&#x4F7F;&#x7528;&#x65B9;&#x6CD5;&#x7684;&#x89E3;&#x91CA;&#x7531;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#xFF0C;&#x4ED6;&#x4EEC;&#x7684;&#x76EE;&#x7684;&#x662F;&#x68C0;&#x67E5; macroautophagy &#x548C;&#x76F8;&#x5173;&#x7684;&#x8FDB;&#x7A0B;&#xFF0C;&#x4EE5;&#x53CA;&#x5BA1;&#x9605;&#x8005;&#x9700;&#x8981;&#x63D0;&#x4F9B;&#x8BBA;&#x6587;&#x4FA7;&#x91CD;&#x4E8E;&#x8FD9;&#x4E9B;&#x8FDB;&#x7A0B;&#x7684;&#x73B0;&#x5B9E;&#x548C;&#x5408;&#x7406;&#x7684;&#x6279;&#x8BC4;&#x3002;&#x8FD9;&#x4E9B;&#x51C6;&#x5219;&#x4E0D;&#x662F;&#x8981;&#x5957;&#x516C;&#x5F0F;&#x5316;&#x7684;&#x89C4;&#x5219;&#xFF0C;&#x56E0;&#x4E3A;&#x9002;&#x5F53;&#x7684;&#x68C0;&#x6D4B;&#x90E8;&#x5206;&#x53D6;&#x51B3;&#x4E8E;&#x7684;&#x8D28;&#x8BE2;&#x548C;&#x6B63;&#x5728;&#x4F7F;&#x7528;&#x7684;&#x7CFB;&#x7EDF;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x5F3A;&#x8C03;&#x4FDD;&#x8BC1;&#x6CA1;&#x6709;&#x4E2A;&#x522B;&#x6D4B;&#x5B9A;&#x90FD;&#x662F;&#x5728;&#x6BCF;&#x4E2A;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x4E00;&#x4E2A;&#x6700;&#x5408;&#x9002;&#xFF0C;&#x6211;&#x4EEC;&#x5F3A;&#x70C8;&#x5EFA;&#x8BAE;&#x4F7F;&#x7528;&#x591A;&#x4E2A;&#x5316;&#x9A8C;&#x76D1;&#x6D4B;&#x81EA;&#x566C;&#x3002;&#x5728;&#x672C;&#x6307;&#x5357;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x8003;&#x8651;&#x8FD9;&#x4E9B;&#x4E0D;&#x540C;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x8BC4;&#x4F30;&#x81EA;&#x566C;&#x548C;&#x54EA;&#x4E9B;&#x4FE1;&#x606F;&#x53EF;&#x4EE5;&#xFF0C;&#x6216;&#x4E0D;&#x80FD;&#xFF0C;&#x4ECE;&#x5B83;&#x4EEC;&#x83B7;&#x5F97;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x901A;&#x8FC7;&#x8BA8;&#x8BBA;&#x6848;&#x60C5;&#x548C;&#x7279;&#x5B9A;&#x7684;&#x81EA;&#x566C;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x7684;&#x9650;&#x5236;&#xFF0C;&#x6211;&#x4EEC;&#x5E0C;&#x671B;&#x9F13;&#x52B1;&#x6280;&#x672F;&#x521B;&#x65B0;&#x7684;&#x9886;&#x57DF;&#x3002;

&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x76D1;&#x6D4B;&#x81EA;&#x566C;&#x7684;&#x89E3;&#x91CA;&#x548C;&#x4F7F;&#x7528;&#x7684;&#x6307;&#x5357;&#x3002;

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy.

En 2008, nous avons publi&eacute; le premier ensemble de lignes directrices pour la normalisation des recherches dans l&#39;autophagie. Depuis lors, les recherches sur ce sujet a continu&eacute; d&#39;acc&eacute;l&eacute;rer, et beaucoup de scientifiques nouveaux entr&eacute;s dans le champ. Notre base de connaissances et les nouvelles technologies ont &eacute;galement &eacute;tendu. En cons&eacute;quence, il est important de mettre &agrave; jour ces lignes directrices pour la surveillance de l&#39;autophagie dans diff&eacute;rents organismes. Diff&eacute;rents examens ont d&eacute;crit les divers essais qui ont &eacute;t&eacute; utilis&eacute;s &agrave; cet effet. N&eacute;anmoins, il continue d&#39;y avoir confusion au sujet des m&eacute;thodes acceptables pour mesurer l&#39;autophagie, surtout chez les eucaryotes multicellulaires. Un point essentiel qui doit &ecirc;tre soulign&eacute;, c&#39;est qu&#39;il y a une diff&eacute;rence entre les diff&eacute;rentes mesures qui surveillent les chiffres ou le volume des &eacute;l&eacute;ments autophagiques (autophagosomes ou autolysosomes) &agrave; tout stade du processus autophagique vs ceux ce flux de mesure par la voie de l&#39;autophagie (c.-&agrave;-d. le processus complet) ; ainsi, un bloc en macroautophagy, ce qui entra&icirc;ne l&#39;accumulation autophagosome doit &ecirc;tre diff&eacute;renci&eacute;s des stimuli qui r&eacute;sultent dans l&#39;augmentation de l&#39;activit&eacute; autophagique, d&eacute;finie comme augmentation autophagie induction coupl&eacute;e avec livraison accrue &agrave; et d&eacute;gradation dans les lysosomes (dans la plupart des eucaryotes sup&eacute;rieurs et certains protistes comme Dictyostelium) ou la vacuole (chez les plantes et les champignons). En d&#39;autres termes, il est particuli&egrave;rement important que les chercheurs &agrave; nouveau dans le domaine comprennent que l&#39;apparence des autophagosomes plus ne correspond pas n&eacute;cessairement avec autophagie plus. En effet, dans de nombreux cas, autophagosomes s&#39;accumulent en raison d&#39;un bloc dans le trafic vers les lysosomes sans changement concomitant autophagosome biogen&egrave;se, alors qu&#39;une augmentation d&#39;autolysosomes peut refl&eacute;ter une r&eacute;duction dans l&#39;activit&eacute; de d&eacute;gradation. Nous pr&eacute;sentons ici une s&eacute;rie de lignes directrices pour la s&eacute;lection et l&#39;interpr&eacute;tation des m&eacute;thodes pour l&#39;utilisation par les enqu&ecirc;teurs, qui visent &agrave; examiner les macroautophagy et les processus connexes, ainsi que pour les examinateurs qui doivent fournir des critiques r&eacute;alistes et raisonnables des papiers qui mettent l&#39;accent sur ces processus. Ces lignes directrices ne visent pas &agrave; &ecirc;tre un ensemble de formule de r&egrave;gles, parce que les dosages appropri&eacute;s d&eacute;pendent en partie de la question pos&eacute;e et le syst&egrave;me utilis&eacute;. En outre, nous soulignons qu&#39;aucun dosage individuel n&#39;est garanti d&#39;&ecirc;tre le plus appropri&eacute; dans chaque situation, et nous recommandons fortement l&#39;utilisation de multiples essais pour surveiller l&#39;autophagie. Dans ces lignes directrices, nous consid&eacute;rons ces diverses m&eacute;thodes d&#39;&eacute;valuation d&#39;autophagie et quels renseignements peuvent, ou ne peut &ecirc;tre obtenu d&#39;eux. Enfin, en discutant les avantages et les limites des analyses particuli&egrave;res autophagie, nous esp&eacute;rons encourager l&#39;innovation technique dans le domaine.

Lignes directrices pour l&#39;utilisation et l&#39;interpr&eacute;tation d&#39;essais pour la surveillance d&#39;autophagie.

Im Jahr 2008 haben wir den ersten Satz von Richtlinien f&uuml;r die Standardisierung der Forschung in Autophagy ver&ouml;ffentlicht. Seitdem Forschung zu diesem Thema hat sich weiter beschleunigen, und viele neue Wissenschaftler haben das Spielfeld betreten. Unsere Wissensdatenbank und relevante neue Technologien haben auch erweitert. Dementsprechend ist es wichtig, diese Leitlinien f&uuml;r die &Uuml;berwachung Autophagy in verschiedenen Organismen zu aktualisieren. Verschiedene Meinungen haben die Proben beschrieben, die f&uuml;r diesen Zweck verwendet wurden. Dennoch weiterhin besteht Verwirrung in Bezug auf akzeptierte, Autophagy, vor allem bei mehrzelligen Eukaryoten zu messen. Ein wesentlicher Punkt, der betont werden muss ist, dass es ein Unterschied zwischen Messungen, die &Uuml;berwachung der Zahlen oder Volume autophagic Elemente (z. B. Autophagosomes oder Autolysosomes) in jedem Stadium des Prozesses gegen die autophagic dieser Ma&szlig;nahme-Fluss durch den Autophagy Pathway (d. h. der vollst&auml;ndige Proze&szlig;); so ein Block in Macroautophagy, Ergebnisse in Autophagosomen Anh&auml;ufung von Reize unterschieden werden, die erh&ouml;hte autophagic Aktivit&auml;t, f&uuml;hren die definiert als erh&ouml;hte Autophagy Induktion gepaart mit erh&ouml;hter Lieferung an und Verschlechterung in Lysosomen (in den meisten h&ouml;heren Eukaryotes und einigen Einzellern wie Dictyostelium) oder die Vakuole (in Pflanzen und Pilzen). Mit anderen Worten, ist es besonders wichtig, dass die Ermittler in das Feld neue verstehen, dass die Darstellung des mehr Autophagosomes nicht unbedingt mit mehr Autophagy gleichzusetzen ist. In der Tat sammeln in vielen F&auml;llen Autophagosomes wegen einen Block in den Handel zu Lysosomen ohne eine damit einhergehende &Auml;nderung in Autophagosomen Biogenese, w&auml;hrend eine Zunahme der Autolysosomes eine Verringerung in Degradations-Aktivit&auml;t widerspiegeln kann. Hier pr&auml;sentieren wir eine Reihe von Leitlinien f&uuml;r die Auswahl und Interpretation der Methoden f&uuml;r die Verwendung von Ermittlern, die Macroautophagy und die damit verbundenen Prozesse untersuchen wollen, sowie f&uuml;r Pr&uuml;fer, die realistische und vern&uuml;nftige Kritiken von Papier bereitstellen, die auf diese Prozesse gerichtet werden m&uuml;ssen. Diese Richtlinien sollen kein formelhaften Satz von Regeln, weil die entsprechenden Assays teilweise auf die Frage und das System verwendet wird. Dar&uuml;ber hinaus betonen wir, dass keine einzelne Assay ist garantiert das passende Produkt in jeder Situation, und wir empfehlen die Verwendung von mehreren Proben Autophagy &uuml;berwachen. In diesen Leitlinien halten wir diese verschiedenen Methoden zur Bewertung Autophagy und welche Informationen kann, oder nicht, von ihnen abgerufen werden. Abschlie&szlig;end diskutieren die Vorz&uuml;ge und Grenzen von bestimmten Autophagy Assays, hoffen wir auf technische Innovationen im Bereich zu f&ouml;rdern.

Richtlinien f&uuml;r die Verwendung und Interpretation von Assays zur &Uuml;berwachung Autophagy in Verbindung.

Nel 2008 abbiamo pubblicato la prima serie di linee guida per la standardizzazione della ricerca in autophagy. Da allora, ricerca su questo argomento ha continuato ad accelerare, e molti nuovi scienziati hanno entrato in campo. Hanno anche ampliato la nostra knowledge base e le nuove tecnologie pertinenti. Di conseguenza, &egrave; importante aggiornare queste linee guida per il monitoraggio autophagy in diversi organismi. Varie recensioni hanno descritto la gamma di dosaggi che sono stati utilizzati per questo scopo. Tuttavia, ci continua a essere confusione per quanto riguarda i metodi accettabili per misurare autophagy, specialmente negli eucarioti pluricellulari. Un punto chiave che deve essere sottolineato &egrave; che c&#39;&egrave; una differenza tra le misure che monitorare il volume di autophagic elementi (ad esempio, autophagosomes o autolysosomes) o numeri in qualsiasi fase del processo autophagic vs quelli che misura flusso attraverso il pathway autophagy (cio&egrave;, il processo completo); cos&igrave;, un blocco in macroautophagy che risultati in accumulo di autophagosome deve essere differenziato da stimoli che si traducono in maggiore attivit&agrave; autophagic, definita come aumento autophagy induzione accoppiato con consegna aumentata e la degradazione nei lisosomi (nella maggior parte dei eucarioti superiori e alcuni protisti come Dictyostelium) o il vacuolo (in piante e funghi). In altre parole, &egrave; particolarmente importante che gli investigatori di nuovi al campo capiscano che l&#39;aspetto di pi&ugrave; autophagosomes non necessariamente identificare con pi&ugrave; autophagy. Infatti, in molti casi, autophagosomes si accumulano a causa di un blocco nel traffico ai lisosomi senza un cambiamento concomitante di autophagosome biogenesi, considerando che un aumento di autolysosomes pu&ograve; riflettere una riduzione nell&#39;attivit&agrave; degradanti. Qui, vi presentiamo una serie di linee guida per la selezione e l&#39;interpretazione dei metodi per l&#39;uso dagli investigatori che mirano a esaminare macroautophagy e dei processi correlati, nonch&eacute; per i revisori che hanno bisogno di fornire critiche realistiche e ragionevole delle carte che sono focalizzate su questi processi. Queste linee guida non sono destinate ad essere una serie stereotipata di regole, perch&eacute; i dosaggi appropriati dipendono in parte la domanda e il sistema in uso. Inoltre, sottolineiamo che nessun dosaggio individuale &egrave; garantito per essere quello pi&ugrave; appropriato in ogni situazione, e si consiglia vivamente l&#39;uso di dosaggi multipli di monitorare autophagy. In queste linee guida, consideriamo questi vari metodi di valutazione autophagy e quali informazioni possono, o non pu&ograve; essere ottenuto da loro. Infine, da discutere i meriti e i limiti delle analisi particolare autophagy, speriamo di incoraggiare l&#39;innovazione tecnica nel campo.

Linee guida per l&#39;uso e l&#39;interpretazione delle analisi per il monitoraggio autophagy.

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1 &#x3064;&#x306B;&#x500B;&#x3005; &#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x4FDD;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x304A;&#x3088;&#x3073;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x8907;&#x6570;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x4F7F;&#x7528;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x306F;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3068;&#x3069;&#x306E;&#x3088;&#x3046;&#x306A;&#x60C5;&#x5831;&#x3092;&#x3067;&#x304D;&#x308B;&#x3001;&#x8A55;&#x4FA1;&#x306E;&#x3053;&#x308C;&#x3089;&#x306E;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x65B9;&#x6CD5;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x307E;&#x305F;&#x306F;&#x305D;&#x308C;&#x3089;&#x304B;&#x3089;&#x53D6;&#x5F97;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x30E1;&#x30EA;&#x30C3;&#x30C8;&#x3068;&#x7279;&#x5B9A;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x9650;&#x754C;&#x3092;&#x8AD6;&#x8B70;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3067;&#x306E;&#x6280;&#x8853;&#x9769;&#x65B0;&#x3092;&#x5968;&#x52B1;&#x3059;&#x308B;&#x9858;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x4F7F;&#x7528;&#x3068;&#x89E3;&#x91C8;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3002;

2008 &#xB144; &#xC6B0;&#xB9AC; &#xCD9C;&#xD310; &#xC5F0;&#xAD6C; autophagy&#xC758; &#xD45C;&#xC900;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xCCAB; &#xC138;&#xD2B8;. &#xADF8; &#xC774;&#xD6C4;&#xB85C;, &#xAC00;&#xC18D;,&#xC774; &#xC8FC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC5F0;&#xAD6C;&#xB294; &#xACC4;&#xC18D; &#xD558; &#xACE0; &#xB9CE;&#xC740; &#xC0C8;&#xB85C;&#xC6B4; &#xACFC;&#xD559;&#xC790; &#xD544;&#xB4DC;&#xB97C; &#xC785;&#xB825; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xAE30;&#xC220; &#xC790;&#xB8CC; &#xBC0F; &#xAD00;&#xB828; &#xC2E0;&#xAE30;&#xC220;&#xB3C4; &#xD655;&#xB300; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xB530;&#xB77C;, &#xADF8;&#xAC83;&#xC740; &#xB2E4;&#xB978; &#xC720;&#xAE30; &#xCCB4;&#xC5D0;&#xC11C; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xC5D0; &#xB300; &#xD55C;&#xC774; &#xC9C0;&#xCE68;&#xC744; &#xC5C5;&#xB370;&#xC774;&#xD2B8; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC591; &#xD55C; &#xB9AC;&#xBDF0;&#xB294;&#xC774; &#xBAA9;&#xC801;&#xC744; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB41C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xBC94;&#xC704;&#xB97C; &#xC124;&#xBA85; &#xD588;&#xB2E4;. &#xADF8;&#xB7FC;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0;, &#xAC70;&#xAE30;&#xC5D0; &#xD5C8;&#xC6A9; &#xD2B9;&#xD788; &#xB2E4;&#xC138;&#xD3EC; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; autophagy &#xCE21;&#xC815; &#xBC29;&#xBC95;&#xC5D0; &#xAD00;&#xD55C; &#xD63C;&#xB780; &#xB418; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC15;&#xC870; &#xD558;&#xB294; &#xD575;&#xC2EC; &#xD3EC;&#xC778;&#xD2B8;&#xAC00;&#xC785;&#xB2C8;&#xB2E4; &#xC22B;&#xC790; &#xB610;&#xB294; autophagic &#xC694;&#xC18C; (&#xC608;: autophagosomes &#xB610;&#xB294; autolysosomes)&#xC758; &#xBCFC;&#xB968;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xCE21;&#xC815;&#xC758; &#xCC28;&#xC774; &#xADF8; &#xB300; autophagic &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xBAA8;&#xB4E0; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; autophagy &#xD1B5;&#xB85C; (&#xC989;, &#xC804;&#xCCB4; &#xD504;&#xB85C;&#xC138;&#xC2A4;);&#xB97C; &#xD1B5;&#xD574; &#xADF8; &#xCE21;&#xC815; &#xC720;&#xB7C9; &#xB530;&#xB77C;&#xC11C;, &#xACB0;&#xACFC;: autophagosome &#xCD95;&#xC801; &#xC99D;&#xAC00; autophagic &#xD65C;&#xB3D9;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC790;&#xADF9;&#xC5D0;&#xC11C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; macroautophagy&#xC5D0;&#xC11C; &#xBE14;&#xB85D; &#xC99D;&#xAC00; autophagy &#xC720;&#xB3C4;, &#xC99D;&#xAC00; &#xC804;&#xB2EC;&#xACFC; &#xB9AC;&#xC18C;&#xC880; (&#xB300;&#xBD80;&#xBD84;&#xC758; &#xB192;&#xC740; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0; Dictyostelium &#xAC19;&#xC740; &#xC77C;&#xBD80; protists) &#xB610;&#xB294; (&#xC2DD;&#xBB3C;&#xACFC; &#xACF0; &#xD321;&#xC774;)&#xC5D0; &#xACF5;&#xD3EC; &#xB0B4; &#xC800;&#xD558;&#xC640; &#xACB0;&#xD569;&#xC73C;&#xB85C; &#xC815;&#xC758; &#xB429;&#xB2C8;&#xB2E4;. &#xB2E4;&#xB978; &#xB9D0;&#xB85C; &#xD558;&#xBA74;, &#xADF8;&#xAC83;&#xC740; &#xD2B9;&#xD788; &#xC911;&#xC694; &#xD55C; &#xC218;&#xC0AC; &#xBD84;&#xC57C;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xB354; &#xB9CE;&#xC740; autophagosomes&#xC758; &#xC678;&#xAD00;&#xC744; &#xBC18;&#xB4DC;&#xC2DC; &#xB354; autophagy&#xC640; &#xB3D9;&#xC77C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xB294;&#xB2E4; &#xC774;&#xD574;. &#xC0AC;&#xC2E4;, &#xB300;&#xBD80;&#xBD84;&#xC758; &#xACBD;&#xC6B0;&#xC5D0;&#xC11C; autophagosomes &#xCD95;&#xC801; autophagosome &#xC18D;&#xC5D0; &#xC218; &#xBC18;&#xD558;&#xB294; &#xBCC0;&#xD654; &#xC5C6;&#xC774; &#xB9AC;&#xC18C;&#xC880;&#xC744; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xBE14;&#xB85D; &#xB54C;&#xBB38;&#xC5D0; &#xBC18;&#xBA74;&#xC5D0; autolysosomes &#xC99D;&#xAC00; degradative &#xD65C;&#xB3D9;&#xC758; &#xAC10;&#xC18C;&#xB97C; &#xBC18;&#xC601; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; &#xC120;&#xD0DD; &#xC601;&#xC5ED;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xC124;&#xC815; &#xBC0F; &#xC0AC;&#xC6A9; &#xBC29;&#xBC95;&#xC758; &#xD574;&#xC11D; &#xC774;&#xB7EC;&#xD55C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xC9D1;&#xC911; &#xB418;&#xB294; &#xB17C;&#xBB38;&#xC758; &#xD604;&#xC2E4;&#xC801;&#xC774; &#xACE0; &#xD569;&#xB9AC;&#xC801;&#xC778; &#xBE44;&#xD310;&#xC744; &#xC81C;&#xACF5; &#xD574;&#xC57C; &#xD558;&#xB294; &#xAC80;&#xD1A0;&#xC790;&#xC5D0; &#xB300; &#xD55C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; macroautophagy &#xBC0F; &#xAD00;&#xB828;&#xB41C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xAC83;&#xC744; &#xBAA9;&#xD45C;&#xB85C; &#xD558;&#xB294; &#xC870;&#xC0AC;&#xC5D0; &#xC758;&#xD574;. &#xC801;&#xC808; &#xD55C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; &#xBD80;&#xBD84;&#xC5D0;&#xC11C; &#xC694;&#xCCAD;&#xC744; &#xBC1B;&#xACE0; &#xC9C8;&#xBB38;&#xC744; &#xD558; &#xACE0; &#xC0AC;&#xC6A9; &#xC911;&#xC778; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68; &#xADDC;&#xCE59;, formulaic &#xC9D1;&#xD569;&#xC73C;&#xB85C; &#xC758;&#xBBF8;&#xAC00; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC544;&#xBB34; &#xAC1C;&#xBCC4; &#xBD84;&#xC11D; &#xACB0;&#xACFC;, &#xBAA8;&#xB4E0; &#xC0C1;&#xD669;&#xC5D0;&#xC11C; &#xAC00;&#xC7A5; &#xC801;&#xD569; &#xD55C; &#xBCF4;&#xC7A5; &#xD558; &#xACE0; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xD560; &#xC5EC;&#xB7EC; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xAC15;&#xC870;. &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68;&#xC5D0; &#xC6B0;&#xB9AC; autophagy&#xC640; &#xC815;&#xBCF4; &#xC218; &#xC788;&#xB294; &#xD3C9;&#xAC00;&#xC758; &#xC774;&#xB7EC;&#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBC29;&#xBC95;&#xC744; &#xACE0;&#xB824; &#xADF8;&#xB4E4; &#xB85C;&#xBD80;&#xD130; &#xC5BB;&#xC744; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;, &#xB610;&#xB294;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC7A5;&#xC810; &#xBC0F; &#xD2B9;&#xC815; autophagy &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD55C;&#xACC4;&#xB97C; &#xB17C;&#xC758; &#xD558; &#xC5EC; &#xD604;&#xC7A5;&#xC5D0;&#xC11C; &#xAE30;&#xC220; &#xD601;&#xC2E0;&#xC744; &#xC7A5;&#xB824; &#xD558;&#xACA0;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC0AC;&#xC6A9; &#xBC0F; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD574;&#xC11D;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;.

En 2008 public&oacute; el primer conjunto de pautas para estandarizar la investigaci&oacute;n en la autofagia. Desde entonces, ha continuado la investigaci&oacute;n sobre este tema acelerar, y muchos nuevos cient&iacute;ficos han entrado en el campo. Tambi&eacute;n han estado ampliando nuestra base de conocimientos y tecnolog&iacute;as relevantes. Por consiguiente, es importante actualizar estas directrices para la supervisi&oacute;n de autofagia en diferentes organismos. Varios comentarios han descrito la gama de ensayos que se han utilizado para este prop&oacute;sito. Sin embargo, contin&uacute;an confusi&oacute;n con respecto a los m&eacute;todos aceptables para medir la autofagia, especialmente en eucariotas pluricelulares. Un punto clave que debe destacarse es que hay una diferencia entre las medidas que monitorean los n&uacute;meros o volumen de autofagia elementos (por ejemplo, autophagosomes o autolysosomes) en cualquier etapa del proceso de autofagia vs los ese flujo medida a trav&eacute;s de la v&iacute;a de la autofagia (es decir, el proceso completo); as&iacute;, un bloque en macroautophagy que resulta en acumulaci&oacute;n de autophagosome debe ser distinguido de est&iacute;mulos que resultan en un aumento de la actividad autofagia, definido como inducci&oacute;n autofagia creciente juntada con mayor entrega y degradaci&oacute;n dentro, lisosomas (en la mayor&iacute;a de eucariotas superiores y algunos protistas como Dictyostelium) o la vacuola (en plantas y hongos). En otras palabras, es especialmente importante que los investigadores de nuevos al campo entiendan que la aparici&oacute;n de m&aacute;s autophagosomes no corresponde necesariamente con autofagia m&aacute;s. De hecho, en muchos casos, autophagosomes se acumulan debido a un bloque en el tr&aacute;fico a los lisosomas sin un cambio concomitante en la biog&eacute;nesis de autophagosome, mientras que un aumento en la autolysosomes puede reflejar una reducci&oacute;n en la actividad de degradaci&oacute;n. Aqu&iacute;, presentamos un conjunto de directrices para la selecci&oacute;n e interpretaci&oacute;n de los m&eacute;todos para su uso por los investigadores que tienen como objetivo examinar macroautophagy y procesos relacionados, as&iacute; como para los revisores que deban proporcionar cr&iacute;ticas realistas y razonables de papeles que se centran en estos procesos. Estas directrices no pretenden ser un formulista conjunto de reglas, porque los ensayos adecuados dependen en parte de la pregunta y el sistema utilizado. Adem&aacute;s, hacemos hincapi&eacute; en que ning&uacute;n ensayo individual est&aacute; garantizada para ser el m&aacute;s adecuado en cada situaci&oacute;n, y le recomendamos el uso de m&uacute;ltiples ensayos para monitorear la autofagia. En estas directrices, consideramos estos diversos m&eacute;todos de evaluaci&oacute;n de autofagia y qu&eacute; informaci&oacute;n puede, o no puede obtenerse de ellos. Finalmente, discutiendo los m&eacute;ritos y los l&iacute;mites de an&aacute;lisis particular de la autofagia, queremos fomentar la innovaci&oacute;n t&eacute;cnica en el campo.

Directrices para el uso e interpretaci&oacute;n de ensayos para el control de la autofagia.

Establishing links between endoplasmic reticulum-mediated hormesis and cancer.

Metals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also used to traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved in the homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells.

Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster.

How morphogen gradients are shaped is a major question in developmental biology, but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to Hh N-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.

Bertrand Mollereau

Laboratory of Molecular Biology of the Cell

Ecole Normale Supérieure de Lyon

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

Bertrand Mollereau

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Molecular Biology of the Cell

Ecole Normale Supérieure de Lyon

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

Laboratory of Molecular Biology of the Cell