Intracellular Membrane Structures Research Articles

Poly(lactic-co-glycolic) acid nanoparticles localize in vesicles after diffusing into cells and are retained by intracellular traffic modulators.

Aim: We investigated our previous finding of increased retention of poly(lactic-co-glycolic)acidnanoparticles (PLGA-NPs) with metabolic inhibitors (MI) and studied the effect of some small molecule inhibitors on PLGA-NP assimilation. Materials & methods: Intracellular PLGA-NP colocalization in the presence ofMIwas investigated by confocal microscopy. Intracellular retention of PLGA-NPs by some small molecules was estimated by fluorescence microscopy and flow cytometry after Pulse/Chase experiments. Results: MI caused PLGA-NP colocalization in intracellular membranous structures, mainly endosomes and lysosomes. Some small molecule inhibitors demonstrated increased intracellular PLGA-NP accumulation. Conclusion: This study elucidates the movement of PLGA-NP in cells and suggests that clinically used small molecules can reduce their extrusion by enhancing their stay within intracellular vesicles, with possible clinically beneficial consequences.

Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice.

The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.

PITPNC1 promotes the thermogenesis of brown adipose tissue under acute cold exposure

Brown adipose tissue (BAT) plays an essential role in non-shivering thermogenesis. The phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) is identified as a lipid transporter that reciprocally transfers phospholipids between intracellular membrane structures. However, the physiological significance of PITPNC1 and its regulatory mechanism remain unclear. Here, we demonstrate that PITPNC1 is a key player in thermogenesis of BAT. While Pitpnc1−/− mice do not differ with wildtype mice in body weight and insulin sensitivity on either chow or high-fat diet, they develop hypothermia when subjected to acute cold exposure at 4°C. The Pitpnc1−/− brown adipocytes exhibit defective β-oxidation and abnormal thermogenesis-related metabolism pathways in mitochondria. The deficiency of lipid mobilization in Pitpnc1−/− brown adipocytes might be the result of excessive accumulation of phosphatidylcholine and a reduction of phosphatidic acid. Our findings have uncovered significant roles of PITPNC1 in mitochondrial phospholipid homeostasis and BAT thermogenesis.

Membrane-Associated Flavivirus Replication Complex-Its Organization and Regulation.

Flavivirus consists of a large number of arthropod-borne viruses, many of which cause life-threatening diseases in humans. A characteristic feature of flavivirus infection is to induce the rearrangement of intracellular membrane structure in the cytoplasm. This unique membranous structure called replication organelle is considered as a microenvironment that provides factors required for the activity of the flaviviral replication complex. The replication organelle serves as a place to coordinate viral RNA amplification, protein translation, and virion assembly and also to protect the viral replication complex from the cellular immune defense system. In this review, we summarize the current understanding of how the formation and function of membrane-associated flaviviral replication organelle are regulated by cellular factors.

Boosting endoplasmic reticulum folding capacity reduces unfolded protein response activation and intracellular accumulation of human kidney anion exchanger 1 in Saccharomyces cerevisiae.

Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid-base balance along with the proton pump v-H+ -ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells.

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Micro-structure Proteins Characterization in Tissue Sections and Clinical Biopsy Samples by a Novel Spatial Proteomic Method Named AutoSTOMP

Abstract We aim at structure determining immunological machinery using a novel spatial selective proteomic tool, autoSTOMP, developed in Ewald lab. AutoSTOMP comprises of UV initiated biotin tagging by confocal microscopy, affinity isolation, and peptide identification by the mass spectrometer, rendering a precise characterization of micro-structures of interest (mSOIs) at intracellular organelle scale excluding surrounding area with the decent signal-to-noise ratio. Previously, autoSTOMP was employed to investigate the parasitophorous vacuole membrane, a unique intracellular membrane structure in apicomplexan infected cells. We broaden the application of autoSTOMP to respond to the calls from the research or clinical laboratory for a reliable, repeatable, and re-constructible protocol to explore the mSOIs of an animal specimen or a patient biopsy. We demonstrated, in the cardiac infarcted tissue in ischemic injured rat, the mSOIs stained against the macrophage/monocyte marker, CD68, are enriched in lysosomal proteins, and characteristic proteins involved in the Fcγ receptor-activated macrophage deformation, migration, and phagocytosis. Furthermore, in eosinophilic esophagitis (EoE) biopsy, we identify the lgG4+ cell-cluster structures are enriched in eosinophil-derived granule proteins and lgE receptor FcɛRI signaling associated proteins as well as food allergens to build up the correlation between patient’s diet with EoE progression.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non‐selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP‐tagged PQL proteins localized to intracellular membrane structures, both when transiently over‐expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non‐stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

Glutathione and the intracellular labile heme pool

One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and that this pool is important for the intracellular transfer of heme. Here we describe a study designed to characterise conjugates that form between heme and glutathione. In contrast to hydrated iron(II), heme reacts with glutathione, under aerobic conditions, to form the stable hematin–glutathione complex, which contains iron(III). Thus, glutathione is clearly not the cytosolic ligand for heme, indeed we demonstrate that the rate of heme degradation is enhanced in the presence of glutathione. We suggest that the concentration of heme in the cytosol is extremely low and that intracellular heme transfer occurs via intracellular membrane structures. Should any heme inadvertently escape into the cytosol, it would be rapidly conjugated to glutathione thereby protecting the cell from the toxic effects of heme.

Visualization of cytoplasmic organelles via in-resin CLEM using an osmium-resistant far-red protein

Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.

Fluorescence Probes Exhibit Photoinduced Structural Planarization: Sensing In Vitro and In Vivo Microscopic Dynamics of Viscosity Free from Polarity Interference.

We demonstrate the construction of wavelength λ-ratiometric images that allow visualizing the distribution of microscopic dynamics within living cells and tissues by using the newly developed principle of fluorescence response. The bent-to-planar motion in the excited state of incorporated fluorescence probes leads to elongation of the π-delocalization, resulting in microviscosity-dependent but polarity-insensitive interplay between well-separated blue and red bands in emission spectra. This allows constructing the exceptionally contrasted images of cellular dynamics. Moreover, the application of probes with increased affinity toward biological membranes allowed detecting the differences in dynamics between the plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Chapter 2a: Virology

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6

BackgroundCystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli.ResultsUsing transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles.ConclusionsOur results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

Fluorescence Probes Exhibit Photoinduced Structural Planarization: Sensing <i>in vitro</i> and <i>in vivo</i> Microscopic Dynamics of Viscosity Free from Polarity Interference

We demonstrate the construction of wavelength λ-ratiometric images that allow visualizing the distribution of microscopic dynamics within living cells and tissues by using the newly developed principle of fluorescence response. The bent-to-planar motion in the excited state of incorporated fluorescence probes leads to elongation of the π-delocalization, resulting in microviscosity-dependent but polarity-insensitive interplay between well-separated blue and red bands in emission spectra. This allows constructing the exceptionally contrasted images of cellular dynamics. Moreover, the application of probes with increased affinity towards biological membranes allowed detecting the differences in dynamics between plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

Chapter 2a: Virology

• TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. • Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). Additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalaya subtype (Himalayan and “178-79” group; TBEV-HIM). • TBEV virions are small enveloped spherical particles about 50 nm in diameter. • The TBEV genome consists of a single-stranded positive sense RNA molecule. • The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). • The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. • The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). • TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Methods to Measure Autophagy in Cancer Metabolism.

Autophagy, a dynamic pathway in which intracellular membrane structures sequester portions of the cytosol for degradation, plays multiple roles in physiological and pathological processes. Autophagy may have suppressive and promotive roles in the formation and progression of cancer. A growing number of methods to identify, quantify, and manipulate autophagy have been developed. Because most of these methods are semiquantitative and have significant limitations, it is important to emphasize that a combination of these assays is recommended for the analysis of autophagy. Here, I briefly discuss the autophagic process, its role in disease, and I summarize some of the best-known and most widely used methods to study autophagy in vitro in the context of cancer, including transmission electron microscopy (TEM), detection and quantification of the autophagy protein LC3 by western blot, and the use of GFP-LC3 to quantify puncta by fluorescence microscopy and tandem labeled RFP/mCherry-GFP-LC3 fluorescence microscopy to measure autophagic flux.