Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of Endogenous MAP1LC3B.

Assays that monitor autophagic flux, or degradative completion of autophagy, are crucial for the assessment of the dynamic autophagy process in a variety of systems. Such assays help to distinguish between an increase in autophagosomes resulting from induced autophagic activity versus an increase in autophagosomes due to reduced lysosomal turnover. The majority of flux assays use autophagy protein MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3B, here referred to as LC3B) as a marker for autophagy, and most are based on the use of reporters. Here, we describe a method, suitable for monitoring flux in primary cells and/or when reporters are not available or desirable, that uses lysosomal inhibitors and the analysis of endogenous LC3B-II (the lipidated form of LC3B that is associated with autophagosomes) by western blotting. A common application of this method, detailed here, is to test whether a treatment of interest (e.g., chemotherapy drug) induces autophagic flux in the cells of interest. If it is found that there is no difference in LC3B-II levels between treatment with lysosomal inhibitor alone versus drug plus lysosomal inhibitor, then this suggests that the drug is not inducing autophagic flux. Elevated levels of LC3B-II in treatments with drug plus lysosomal inhibitor, compared with drug treatment alone and inhibitor treatment alone, indicate that the drug is probably leading to an increase in autophagic flux.

Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages.

Autophagy plays specific roles in host innate and adaptive immune responses to numerous intracellular pathogens, including Mycobacterium tuberculosis. The ESAT-6 and CFP-10 proteins are secreted by M. tuberculosis and play important roles in pathogenesis. We hypothesized that these two proteins may affect the autophagy function of host macrophages during infection with M. tuberculosis, thereby shaping the immune reaction toward the pathogen. Interestingly, we found that rapamycin-induced autophagy of macrophages infected with M. tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes and lysosomes. Ectopic expression of the ESAT-6/CFP-10 fusion in macrophages dramatically inhibited autophagosome formation, and M. tuberculosis survival inside infected macrophages was significantly affected as well. Further, M. tuberculosis viability was increased by the fusion protein. Expression levels of autophagy-related genes (ATG), especially atg8, also decreased (p<0.05). These results suggested that ESAT-6 and CFP-10 proteins play significant roles in autophagy formation in M. tuberculosis infection and that autophagosome formation is regulated through the expression of ATG.

Microliths in the parotid of ferret investigated by electron microscopy and microanalysis.

The present investigation is an attempt to determine the occurrence, elemental composition and formation of microliths in the parotid of ferret. Parotids from four normal ferrets were examined by electron microscopy and X-ray microanalysis. Crystalline microliths were found in phagosomes of acinar cells, which occasionally contained secretory material, and in phagosomes situated between mitochondria of striated ductal cells. Crystalline microliths and microliths that consisted of granular material either without crystals or mixed with a component of crystals were found in lumina, where they were often associated with cellular debris. The crystals contained calcium and phosphorus. Phagy and stagnation related to pockets of inefficient secretory activity have been previously found to be features of the parotid of ferret. Thus, possibly persistent degradation of redundant cellular material, particularly secretory granules, in phagosomes results in accumulation of calcium and leads to calcified microliths, whereas consolidation of stagnant debris extracellularly does not involve such accumulation and leads to non-calcified or mixed microliths.

Small molecule regulators of autophagy identified by an image-based high-throughput screen.

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Harboring of particulate allergens within secretory compartments by mast cells following IgE/FcepsilonRI-lipid raft-mediated phagocytosis.

Although much is known regarding the exocytic responses of mast cells following allergen/IgE-mediated activation, little is currently known of the fate of the activating allergens, many of which are particles. We have found that IgE-bound particulate allergens were phagocytosed by activated mast cells in a lipid raft-dependent manner. The nascent allergen-containing phagosomes were found to transform into granule compartments by acquiring VAMP7 and serotonin and exhibited the capacity to empty their contents upon mast cell activation. When allergen-harboring mast cells were stimulated, the intracellular allergens were expelled intact and shown to activate adjacent mast cells. This capacity of mast cells to phagocytose and retain whole and antigenically intact allergens could potentially contribute to the course of inflammatory diseases such as asthma.

Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload.

After a single subcutaneous dose of iron-dextran (600 mg of iron/kg), iron overload developed in C57BL/10ScSn mice. At 4, 24 and 78 wk liver nonheme iron concentrations were 67-, 42- and 21-fold higher than controls, respectively. Much of the iron was in macrophages, but hepatocytes were also strongly positive for Perls' stainable iron. One feature was the development of iron-positive nuclear inclusions in hepatocytes. After a delay of at least 8 wk when no stainable iron was evident, a maximum of 37% of periportal hepatocytes contained inclusions by 24 wk. Although this proportion remained constant for the remainder of the study, the size of the inclusions (which were not membrane-limited) increased to greater than 3 microns in diameter, occupying greater than 25% of the nuclear volume. The presence of iron in the inclusions was confirmed by energy dispersive x-ray microanalysis. Immunocytochemical studies showed that the iron was present as aggregates of ferritin. Quantitation of nonaggregated ferritin molecules by image analyses after electron microscopy demonstrated that within 4 wk ferritin levels in cytoplasm and nucleoplasm had greatly increased but that there was a concentration gradient of approximately one order of magnitude across the nuclear envelope. These findings are consistent with the hypothesis that in iron-loaded mouse hepatocytes there is a slow passage of ferritin-molecules through the nuclear pores; the gradient is maintained by the continual aggregation of ferritin within the nucleus. Intranuclear ferritin may provide a source of iron for catalyzing hydroxyl radical formation in nuclei during some toxic, carcinogenic and aging processes.