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

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

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Insulin controls target cells by binding to its cell surface receptor. The further intracellular transmission of the insulin signal involves phosphorylation of the receptor as well as other proteins, in particular the insulin receptor substrate (IRS), 1 on specific tyrosine residues. After tyrosine phosphorylation IRS is recognized by Src homology 2 domain-containing proteins for metabolic and glucose transport control, or activation of the mitogen-activated protein kinase (MAP kinase) pathway and mitogenic control (1-4). In type 2 diabetes target cells of the hormone are not fully responsive, which is compensated temporarily by enhanced insulin secretion. The pathogenic mechanisms for this insulin resistance are not known, but an important common feature appears to be a reduced activation/ tyrosine phosphorylation of IRS-1 (5).The insulin receptors are sequestered in the caveolae microdomains of the plasma membrane in adipocytes, and caveolae appear to be critical for insulin control (6). By thin-section electron microscopy, caveolae appear as omega-shaped invaginations of 50 -100 nm diameter in the plasma membrane (7). Caveolae invaginations are found in the plasma membrane of many cell types, but are particularly abundant in adipocytes where they increase in number in conjunction with the differentiation of 3T3-L1 fibroblasts to mature adipocytes (8 -10). Caveolae are involved in receptor-mediated uptake of solutes into the cytosol (11) and in transcytosis (12). A number of proteins, in addition to the insulin receptor, involved in signal transduction are localized to caveolae, which suggests that they may be involved in cellular signaling and control (reviewed in Refs. 13-16).Caveolae are rich in cholesterol and sphingolipids. Caveolae may indeed form from cholesterol-and sphingolipid-rich rafts in the membrane in a process requiring the caveolae-specific structural protein caveolin. Caveolin is found in the plasma membrane and intracellularly, but in the plasma membrane is confined to caveolae; it is therefore used as a marker for these structures. The function of caveolae is dependent on a sufficient level of cholesterol in the plasma membrane and caveolae (12,17). We have also demonstrated a critical dependence of the insulin receptor signal transduction on cholesterol; depletion of cholesterol from the plasma membrane of rat adipocytes reversibly inhibited insulin stimulation of glucose transport and metabolic protein phosphorylation control (6). The importance of caveolae for insulin receptor signaling is further indicated by a consensus binding site for interaction with caveolin (18), and coprecipitation of the receptor with caveolin (4) indicates that the interaction may be physiological. Moreover, the insulin receptor appears to phosphorylate caveolin (19), whereas caveolin was shown to activate the isolated receptor, although the physiological relevance of this is not known (20).Herein we examine in detail the dependence of the insulin receptor on caveolae for signal transduction: the effects of cho...

A type-1 protein phosphatase (protein phosphatase-1 G) was purified to homogeneity from the glycogenprotein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1, had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1 : 1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1,) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels.Protein phosphatase-lG was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-lc, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-lG was inactivated very rapidly by inhibitor-1. Protein phosphatase-1 interacted with inhibitor-2 (1-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3.The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-lG by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.The name protein phosphatase-1 was originally introduced to describe an enzyme activity in rabbit skeletal muscle that dephosphorylated the j-subunit of phosphorylase kinase at least 20-fold more rapidly than the a-subunit, and which was potently inhibited by two thermostable proteins, termed inhibitor-1 and inhibitor-2 [l]. These properties distinguished it from other protein phosphatases which dephosphorylated the a-subunit of phosphorylase kinase preferentially and were unaffected by inhibitors-1 and 2 [2]. However, the term 'protein phosphatase-l' encompasses a number of phosphatase preparations whose apparent molecular masses range from 30 kDa to 260 kDa (discussed in [3]), raising the question of whether each of these type-1 protein phosphatases contain the same catalytic subunit.A 37-kDa type-1 catalytic subunit, termed protein phosphatase-lc, has been purified to homogeneity from rabbit skeletal muscle extracts by a procedure involving treatment with 80% ethanol at room temperature [4], while an inactive