Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV—initial (AVi), intermediate (AVi/d), and degradative (AVd)—were defined by morphological criteria and immunogold labeling characteristics of marker enzymes.The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15–45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/ RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.

Using C. elegans genetics we identified a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains RasGAP-like and Vsp9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to C. elegans Rab5 in the GDP-bound conformation, and rme-6 mutants display phenotypes that indicate low Rab5 activity. Furthermore, rme-6 interacts genetically with the worm homologue of the known Rab5 exchange factor Rabex-5. However, unlike other Rab5 associated proteins, a rescuing GFP::RME-6 fusion protein primarily localizes to clathrincoated pits, physically interacts with α-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants transport from the plasma membrane to endosomes is defective, and small 100 nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.

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Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.

BackgroundIdentification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%–20% of patients.MethodsA multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray.ResultsThese approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 “quadruple-negative” (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%.ConclusionsConventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.