Microautophagy in mammalian cells: revisiting a 40-year-old conundrum

The term microautophagy was first used in 1966 by de Duve and Wattiaux and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen ("multivesicular lysosomes"). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.     

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.     

Shedding light on mammalian microautophagy

ESCRT complexes are implicated in mediating membrane protein degradation, whereas hsc70 mediates cytosolic protein degradation via chaperone-mediated autophagy. In this issue of Developmental Cell, Sahu et?al. (2011) describe in mammalian cells the involvement of ESCRT complexes and hsc70 in the degradation of cytosolic proteins in a process resembling microautophagy.     

Microautophagy in the visceral endoderm is essential for mouse early development

During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.     

Piecemeal microautophagy of the nucleus requires the core macroautophagy genes

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.     

Chasing the elusive mammalian microautophagy

Different mechanisms for delivery of intracellular components (proteins and organelles) to lysosomes and late endosomes for degradation co-exist in almost all cells and set the basis for distinct autophagic pathways. Cargo can be sequestered inside double-membrane vesicles (or autophagosomes) and reach the lysosomal compartment upon fusion of these vesicles to lysosomes through macroautophagy. In a different type of autophagy, known as chaperone-mediated autophagy (CMA), single individual soluble proteins can be targeted one by one to the lysosomal membrane and translocated into the lumen for degradation. Direct sequestration of proteins and organelles by invaginations at the lysosomal membrane that pinch off into the lumen has also been proposed. This process, known as microautophagy, remains poorly understood in mammalian cells. In our recent work, we demonstrate the occurrence of both "in bulk" and "selective" internalization of cytosolic components in late endosomes and identify some of the molecular players of this process that we have named endosomalmicroautophagy (e-MI) due to its resemblance to microautophagy.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).     

Exploiting cell death pathways by an E. coli cytotoxin: autophagy as a double-edged sword for the host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when "feeding" materials are exhausted, do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.     

Ultrastructure of autophagy in plant cells

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.     

Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.     

The unobtrusive majority: mononucleated bone resorbing cells in teleost fish and mammals

The canonical view of a mammalian (usually shown as human) bone resorbing cell is that of a giant macrophage‐like cell (osteoclast) that dissolves bone minerals and digests bone matrix proteins. The cells’ presence and activity is easily recognised based on three distinct morphological features: (i) multinuclearity, (ii) a multiply folded apical cell membrane (ruffled border), and (iii) deep lacunae (Howship’s lacunae) that the cells eroded into the bone surface. Mononucleated osteoclasts without these features are considered to be inactive precursors. We challenge the view that bone resorbing cells must be multinucleated giant cells, based on our comparative studies on the teleost skeleton, on what is currently known – but often disregarded – about mononucleated mammalian osteoclasts, and on what is know about osteocytic osteolysis.     

The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy

We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). In cells of a constructed ATG25 deletion strain (atg25) peroxisomes are constitutively degraded by nonselective microautophagy, a process that in WT H. polymorpha is only observed at nitrogen limitation conditions. This suggests that nonselective microautophagy is deregulated in H. polymorpha atg25 cells.     

Exploiting Cell Death Pathways by an E. coli Cytotoxin: Autophagy as a Double-Edged Sword for the Host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when “feeding” materials are exhausted, they do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.

Addendum to:

Is the Rac GTPase-Activating Toxin CNF1 a Smart Hijacker of Host Cell Fate?

W. Malorni and C. Fiorentini

FASEB J 2006; 20:606-9     

ESCRTing endoplasmic reticulum to microautophagic degradation

ABSTRACT

Changing conditions necessitate cellular adaptation, which frequently entails adjustment of organelle size and shape. The endoplasmic reticulum (ER) is an organelle of exceptional morphological plasticity. In budding yeast, ER stress triggers the de novo formation of ER subdomains called ER whorls. These whorls are selectively degraded by a poorly defined type of microautophagy. We recently showed that ESCRT proteins are essential for microautophagic uptake of ER whorls into lysosomes, likely by mediating the final scission of the lysosomal membrane. Furthermore, ER-selective microautophagy acts in parallel with ER-selective macroautophagy. The molecular machineries for these two types of autophagy are distinct and their contributions to ER turnover vary according to conditions, suggesting that they serve different functions. Our study provides evidence for a direct role of ESCRTs in microautophagy and extends our understanding of how autophagy promotes organelle homeostasis.     

Physiopathologic dynamics of vesicle traffic in astrocytes

The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.     

转录因子EB和自噬在神经退行性疾病发病机制中的作用

自噬广泛存在于真核细胞中,与机体生理和病理过程的发生发展密切联系.自噬主要参与长寿蛋白质的降解,以清除受损或多余的蛋白质和细胞器,是细胞自我降解的过程之一.自噬通常被分为三类:大自噬、分子伴侣介导的自噬和小自噬.自噬溶酶体途径(ALP)功能障碍导致蛋白质聚集,从而产生异常蛋白质和无效细胞器的积累,这些特征是阿尔茨海默病(Alzheimer disease,AD)、帕金森病(Parkinson disease,PD)和亨廷顿病等神经退行性疾病(Huntington disease,HD)的标志.自噬的过程受一系列复杂的信号分子的调控,其中一个主要调节因子是转录因子EB(TFEB),是转录因子MiT家族的成员之一.研究表明,TFEB可通过积极调节自噬体形成和自噬体-溶酶体融合参与自噬,此外它还通过溶酶体胞吐作用提高细胞内的清除作用.因此作为自噬溶酶体生物发生的主要调节因子,TFEB已被广泛证明激活后可以从病理方面改善这些疾病.我们回顾分析ALP和TFEB的调节及其对神经退行性疾病的影响,同时展望ALP和TFEB在疾病病理中的复杂作用及其治疗意义.     

RBM4: a multifunctional RNA-binding protein

RBM4, also known as Lark, was described initially as having a role in circadian rhythm control in Drosophila. In the last 5 years data have emerged from studies of mammalian cells. It is now clear that RBM4 is an RNA-binding protein involved in diverse cellular processes that include alternative splicing of pre-mRNA, translation, and RNA silencing. Its structure, similar to other RNA-binding proteins, contains two RNA recognition motifs and a CCHC-type zinc finger. Here we review current information about the function of RBM4 and its localization within the cell. We then speculate about its possible relationship to disease.     

Heparan sulfate proteoglycans in the nervous system: their diverse roles in neurogenesis, axon guidance, and synaptogenesis

Development of the mammalian nervous system involves generation of neurons from neural stem cells, migration of generated neurons toward genetically determined locations, extension of axons and dendrites, and establishment of neuronal connectivity. Recent progresses revealed diverse role of heparan sulfate proteoglycans in these processes. This article reviews our current knowledge about the functional roles of heparan sulfate proteoglycans in three critical events in mammalian neural development, namely neurogenesis, axon guidance, and synapse development.