Lytic Organelles Research Articles

Evolutionary conservation and metabolic significance of autophagy in algae.

Autophagy is a highly conserved 'self-digesting' mechanism used in eukaryotes to degrade and recycle cellular components by enclosing them in a double membrane compartment and delivering them to lytic organelles (lysosomes or vacuoles). Extensive studies in plants have revealed how autophagy is intricately linked to essential aspects of metabolism and growth, in both normal and stress conditions, including cellular and organelle homeostasis, nutrient recycling, development, responses to biotic and abiotic stresses, senescence and cell death. However, knowledge regarding autophagic processes in other photosynthetic organisms remains limited. In this review, we attempt to summarize the current understanding of autophagy in algae from a metabolic, molecular and evolutionary perspective. We focus on the composition and conservation of the autophagy molecular machinery in eukaryotes and discuss the role of autophagy in metabolic regulation, cellular homeostasis and stress adaptation in algae. This article is part of the theme issue 'The evolution of plant metabolism'.

Autophagy in maternal tissues contributes to Arabidopsis seed development.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development, including autophagy, which facilitates cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy affects nutrient remobilization from mother plants and functions in the embryo. However, it is impossible to distinguish between the contribution of autophagy in the source (i.e. the mother plant) and the sink tissue (i.e. the embryo) when using autophagy knockout (atg mutant) plants. To address this, we employed an approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between wild type and atg mutant Arabidopsis (Arabidopsis thaliana) plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from altered seed coat development. Our study emphasizes the importance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It also sheds light on the tissue-specific functions of autophagy, offering potential for research into the underlying mechanisms governing seed development and crop yield.

Metabolism and autophagy in plants-a perfect match.

Autophagy is a eukaryotic cellular transport mechanism that delivers intracellular macromolecules, proteins, and even organelles to a lytic organelle (vacuole in yeast and plants/lysosome in animals) for degradation and nutrient recycling. The process is mediated by highly conserved autophagy-related (ATG) proteins. In plants, autophagy maintains cellular homeostasis under favorable conditions, guaranteeing normal plant growth and fitness. Severe stress such as nutrient starvation and plant senescence further induce it, thus ensuring plant survival under unfavorable conditions by providing nutrients through the removal of damaged or aged proteins, or organelles. In this article, we examine the interplay between metabolism and autophagy, focusing on the different aspects of this reciprocal relationship. We show that autophagy has a strong influence on a range of metabolic processes, whereas at the same time, even single metabolites can activate autophagy. We highlight the involvement of ATG genes in metabolism, examine the role of the macronutrients carbon and nitrogen, and various micronutrients, and take a closer look at how the interaction between autophagy and metabolism impacts on plant phenotypes and yield.

Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi.

Emerging evidence implicates the vital role of mitochondria in lipid consumption and storage, highlighting the intimate link between energy production and saving. Although formation of giant lipid droplets, which is the key hallmark of the oleaginous yeast Lipomyces starkeyi, appears to be regulated in response to changes in mitochondrial shape and metabolism, technical limitations of genetic manipulation have become an obstacle to uncover the mitochondrial behavior in this nonconventional yeast. Here, we established an L. starkeyi strain stably expressing a fluorescent marker for monitoring mitochondrial morphology and degradation and found that mitochondria are mostly fragmented in L. starkeyi cells under fermentable, nonfermentable, and nitrogen depletion conditions. Notably, a fraction of mitochondria-specific fluorescent signals was localized to the vacuole, a lytic organelle in yeast, indicating degradation of mitochondria in those cells. This possible catabolic event was more predominant in cells under nutrient-poor conditions than that in cells under nutrient-rich conditions, concomitantly with lipid droplet formation. Collectively, our studies provide a new tool to investigate mitochondrial dynamics in L. starkeyi and decipher the potential role of mitochondrial degradation in lipid metabolism.

Microautophagy – distinct molecular mechanisms handle cargoes of many sizes

Autophagy is fundamental for cell and organismal health. Two types of autophagy are conserved in eukaryotes: macroautophagy and microautophagy. During macroautophagy, autophagosomes deliver cytoplasmic constituents to endosomes or lysosomes, whereas during microautophagy lytic organelles take up cytoplasm directly. While macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become clear that microautophagy has a broad range of functions in biosynthetic transport, metabolic adaptation, organelle remodeling and quality control. This Review discusses the selective and non-selective microautophagic processes known in yeast, plants and animals. Based on the molecular mechanisms for the uptake of microautophagic cargo into lytic organelles, I propose to distinguish between fission-type microautophagy, which depends on ESCRT proteins, and fusion-type microautophagy, which requires the core autophagy machinery and SNARE proteins. Many questions remain to be explored, but the functional versatility and mechanistic diversity of microautophagy are beginning to emerge.

Aquaporin activity of barley tonoplast intrinsic proteins is involved in the delay of the coalescence of protein storage vacuoles in aleurone cells

The coalescence of protein storage vacuoles (PSVs) is one of the most prominent cellular changes occurring in cereal aleurone cells during germination. This structural change is highly coupled with the functional transition of this organelle from a storage compartment to a lytic section. Gibberellic acid (GA) promotes this process, whereas abscisic acid (ABA) prevents it. Previously, we demonstrated that ABA-inducible HvTIP3;1 plays a decisive role in ABA-mediated prevention of PSV fusion. In this follow-up study, we examined whether the aquaporin activity of tonoplast intrinsic protein (TIP) is related to its preventive effect on PSV fusion using various functional mutants. The defective forms of aquaporin (HvTIP3;1m and HvTIP3;1ΔNPA-GFPs for HvTIP3;1, and HvTIP1;2m for HvTIP1;2) were found to be less effective than the usual form in delaying the PSV fusion process occurring in GA-treated cells. In contrast, overexpression of HvTIP3;1m reduced the preventive effect of ABA on PSV fusion. Upon inhibition of aquaporin activity using mercury, PSV fusion occurred to a greater extent in ABA-treated barley protoplasts. These data suggest that the aquaporin activity of TIP is involved in the deterrent effect of TIP on PSV coalescence. TIP3-GFP barley transgenic seeds showed prolonged expression of the TIP3;1 transcript. Moreover, PSV fusion progressed at a much slower rate compared to wild type. Additionally, the degradation of storage proteins was not as efficient, suggesting that a metamorphic transition of PSVs to lytic organelles is closely correlated with the disappearance of HvTIPs and the PSV fusion process.

Autophagic degradation of the endoplasmic reticulum.

Autophagy is an intracellular degradation system that is present in most eukaryotes. In the process of autophagy, double membrane vesicles called autophagosomes sequester a wide variety of cellular constituents and deliver them to lytic organelles: lysosomes in mammals and vacuoles in yeast and plants. Although autophagy used to be considered a non-selective process in its target sequestration into autophagosomes, recent studies have revealed that autophagosomes can also selectively sequester certain proteins and organelles that have become unnecessary or harmful for the cell. We recently discovered that the endoplasmic reticulum (ER) is degraded by autophagy in a selective manner in the budding yeast Saccharomyces cerevisiae, and identified “receptor proteins” that play pivotal roles in this “ER-phagy” pathway. Moreover, several ER-phagy receptors in mammalian cells have also been reported. This report provides an overview of our current knowledge on ER-phagy and discuss their mechanisms, physiological roles, and possible links to human diseases.

How To Identify Autophagy Modulators.

Autophagy is the ubiquitous process in eukaryotes leading to the degradation of intracellular components, during which a portion of the cytoplasm, including organelles, is sequestered by nascent autophagic membranes and delivered into the lytic organelles for degradation ([Mizushima and Komatsu,

Three Distinct Types of Microautophagy Based on Membrane Dynamics and Molecular Machineries.

Microautophagy is originally defined as lysosomal (vacuolar) membrane dynamics to directly enwrap and transport cytosolic components into the lumen of the lytic organelle. Molecular details of microautophagy had remained unknown until genetic studies in yeast identified a set of proteins required for the process. Subsequent studies with other experimental model organisms resulted in a series of discoveries that accompanied an expansion of the definition of microautophagy to also encompass endosomal membrane dynamics. These findings, however, still impose puzzling, non-integrated images as to the molecular mechanism of microautophagy. By reviewing recent studies on microautophagy in various experimental systems, we propose the classification of microautophagy into three types, as the basis for developing a comprehensive view of the process.

Fluorescence Imaging of Autophagy-Mediated ER-to-Vacuole Trafficking in Plants.

Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism in eukaryotic cells that delivers unneeded cellular components for degradation in the lytic organelle. In plants, as in other eukaryotes, autophagy begins in the formation of cup-shaped double membranes that engulf cytosolic material. The double membrane closes to form autophagosomes that are then transported to the vacuole for degradation. Autophagy can function as a bulk nonselective process or as a selective process targeting specific proteins, protein aggregates, organelles, or other cellular components for degradation. The endoplasmic reticulum (ER) is linked to autophagy-related processes in multiple ways. The ER was suggested as a possible site for the nucleation of autophagosomes, and as a source for autophagosomal membranes. Furthermore, autophagy has an important role in ER homeostasis, and the ER is a target for a selective type of autophagy, ER-phagy, in response to ER stress. However, the detailed molecular mechanisms, especially in plants, are only now starting to be revealed.In this chapter, we describe the use of confocal imaging to follow the delivery of fluorescently tagged ER-associated proteins to the vacuole. We also describe the utilization of fluorescent protein fusions to look at the co-localization of a protein of interest with the autophagosome marker protein ATG8, a core autophagy machinery protein that is essential for selective autophagy processes.

Involvement of vacuolar processing enzyme SlVPE5 in post-transcriptional process of invertase in sucrose accumulation in tomato

Enhancing the flavor of fruits plays a fundamental role in improving fruit quality, and volatile compositions as well as acid and sugar accumulation are significant factors that have an impact on the acceptability of sensory responses by human beings. Vacuoles in plants not only function as cell compartments that store amino acids, sugars and other metabolites but also act as lytic organelles where vacuolar proteins are post-translationally processed into mature forms or degraded by the action of vacuolar processing enzyme (VPE). We have previously characterized VPE genes (SlVPE1-5) during fruit development in tomato and discovered that the VPE enzyme activity negatively interfered with sugar accumulation in mature fruits. Comparative proteomic analysis demonstrated that acid invertase was one of the molecular targets of SlVPE5, which is involved in the hydrolysis of sucrose. This study also showed that decreased VPE enzyme activity due to suppression of SlVPE5 by RNAi strategy (RNAi-SlVPE5) accompanied with decreased enzyme activity of acid invertase. Further, we identified the enzyme activity of acid invertase was not well correlated with mRNA levels in the RNAi-SlVPE5 line. These results suggest that SlVPE5 regulates post-transcriptional processing through de novo synthesis of the acid invertase protein to suppress enzyme activity, thereby eventually ensuring sucrose hydrolysis.

Lack of association of the autophagy-related gene polymorphism ATG16L1 rs2241880 in RA predisposition

Rheumatoid arthritis (RA) is a chronic inflammatory disease, in which pathogenesis many genetic, environmental and hormonal factors have been implicated. Up to date, the known genetic factors account for the 60 % of the genetic basis of the disease. The molecular pathway of autophagy and the related genes have not yet been studied for their role in RA liability. Autophagy has proved to be important in the maintenance of cellular homeostasis and energetic balance, in cellular and tissue remodeling and in cellular defense against extracellular insults and pathogens [1]. In addition, recently, it was reported the role of autophagy and related proteins in human immune responses and inflammatory diseases’ pathogenesis [2, 3]. Therefore, mutations and polymorphisms of autophagic genes could possibly be related to autoimmune diseases’ predisposition such as RA. Specifically, during autophagy, large portions of the cytoplasm as big as whole organelles (e.g., mitochondria) are captured by isolation membranes (phagophores) and sequestered into autophagosomes for degradation within the specialized lytic organelles termed autolysosomes [1]. The genes (Atg) involved in this pathway have been identified in species from yeast to humans, and their variants could be related to diseases’ pathogenesis. Polymorphism rs2241880 in exon 8 of ATG16L1 (autophagy-related 16-like 1) gene results in the amino acid substitution Thr300Ala. This polymorphism has mainly been studied for its association with autoimmune diseases’ susceptibility, and it has been revealed to play a role in Crohn’s disease, ulcerative colitis and palmoplantar pustulosis, while altered expression profiles of ATG16L1 have been described in psoriatic arthritis [4–7]. The aim of the present research protocol was to study the association of ATG16L1 polymorphism rs2241880 with RA liability. One hundred and thirty-four unrelated RA patients, who satisfied the American College of Rheumatology criteria, were enrolled in the study. Additionally, 147 ethnicmatching healthy volunteers with no personal or family history of chronic autoimmune, metabolic or infectious diseases were included in the study [8]. All subjects gave informed written consent in accordance with Helsinki Declaration of 1975/83 and the local ethics committee granted approval. Genomic DNA was extracted from peripheral blood lymphocytes according to the standard salt extraction procedure. ATG16L1 polymorphism rs2241880 was amplified using the following primer pair: rs2241880F: 50-TCA AGC GTG GTA GGG TTC GGG G-30, rs2241880R: 50-CTT CCT TCC CAG TCC CCC AGG-30. Polymerase chain reaction– restriction fragment length polymorphism (PCR–RFLP) assay was conducted using the restriction endonuclease SfaN I. Double digestion of each PCR product was performed so as to confirm the results of the RFLP assay, and no discrepancies between the two digestions were revealed. The SPSS statistical package was used to test differences in polymorphisms’ distribution between RA patients and controls (Pearson’s chi-square). Furthermore, the odds ratio (OR) with a confidence interval (CI) of 95 % was calculated. A difference at p B 0.05 was considered as statistically significant. A. Chatzikyriakidou Laboratory of General Biology and Genetics, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

Alternative pathways for MHC class I presentation: a new function for autophagy.

The classical view that endogenous antigens are processed by the proteasome and loaded on MHC class I molecules in the endoplasmic reticulum, while exogenous antigens taken up by endocytosis or phagocytosis are degraded and loaded on MHC class II in lysosome-derived organelles, has evolved along with the improvement of our understanding of the cell biology of antigen-presenting cells. In recent years, evidence for alternative presentation pathways has emerged. Exogenous antigens can be processed by the proteasome and loaded on MHC class I through a pathway called cross-presentation. Moreover, endogenous antigens can be targeted to lytic organelles for presentation on MHC class II through autophagy, a highly conserved cellular process of self-eating. Recent evidence indicates that the vacuolar degradation of endogenous antigens is also beneficial for presentation on MHC class I molecules. This review focuses on how various forms of autophagy participate to presentation of these antigens on MHC class I.

Trafficking and Subcellular Localization of Multiwalled Carbon Nanotubes in Plant Cells

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

Cytochemistry of proteolytic activity and pH status of vacuoles in Medicago truncatula root nodules

The vacuole development in root nodules of Medicago truncatula was analyzed by light and electron microscopy. Histochemistry of protease activity in root nodules was studied using fluorogenic substrates for proteolytic enzymes, 7-amino-4-methylcoumarin, CBZ-L-phenylalanyl-L-arginine amide, hydrochloride (AMC), and rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide) dihydrochloride (RPA). Furthermore, the topology of acidification of the central vacuoles in infected and noninfected cells in root nodules of Medicago truncatula was analyzed with the fluorescent pH-sensitive acidotropic dye Neutral Red. It was shown that vacuoles were acidic, lytic organelles in noninfected cells and young infected cells of the nodule where they displayed protease activity. Mature vacuoles of infected cells had high pH and did not show any substantial protease activity.

Mechanisms of pathogen entry through the endosomal compartments

Several pathogens - bacteria, viruses and parasites - must enter mammalian cells for survival, replication and immune-system evasion. These pathogens generally make use of existing cellular pathways that are designed for nutrient uptake, receptor downregulation and signalling. Because most of these pathways end in lysosomes, an organelle that is capable of killing microorganisms, pathogens have developed remarkable means to avoid interactions with this lytic organelle.

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.

Barley Aleurone Cells Contain Two Types of Vacuoles: Characterization of Lytic Organelles by Use of Fluorescent Probes

Barley Aleurone Cells Contain Two Types of Vacuoles: Characterization of Lytic Organelles by Use of Fluorescent Probes

Effect of methanol and gamma irradiation on enzymatic activity of apple pollen

In order to characterize devitalized pollen (which can be used as a “mentor” in mixed pollinations), certain hydrolase activities were spectrophotometrically tested in normal and in methanol or gamma irradiation-treated pollen of Malus domestica Borkh. cultivar ‘Golden Delicious’, and in normal pollen of the cultivar ‘Starkrimson’. Enzymatic activities of methanol-treated pollen were somewhat decreased, except for acid phosphatase which was enhanced over the control. This increase could be due to a higher hydrolysis rate by the alcohol, and/or to enzyme activation after damage of the lytic organelle membranes. Amylase, cellulase, ribonuclease and particularly acid phosphatase activities were stimulated in irradiated pollen. It was hypothesized that there was an enzyme leakage from the lytic compartment following pollen irradiation. On the basis of these data on enzymatic activities, it can be concluded that comparable effects were induced by either methanol or irradiation treatment of apple pollen.