Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.Key words: autophagy, tuberculosis, ribosome, ubiquitin, antimicrobial peptidesAutophagy is an evolutionarily conserved cytoplasm-homeostatic process with a multitude of functions supporting, for the most part, cellular viability. During autophagy, cytoplasmic targets ranging from protein aggregates to whole organelles such as mitochondria and intracellular microbes are sequestered into a double-membrane bound organelle called the autophagosome. Autophagosomes mature into autolysosomes through fusion with lysosomes or their transport intermediates, bringing about acidification and acquisition of hydrolases leading to the digestion of the captured substrates. It is generally assumed that autophagy produces terminal degradative products such as free amino acids that are then used by the cell or the body as nutrients at times of starvation. Recently, we have discovered that autophagy generates, by proteolysis of captured cytosolic proteins, a mixture of peptides conferring potential cryptic biological functions, termed “cryptides.” Some of the cryptides with thus far assigned biological functions are the neo-antimicrobial peptides liberated from innocuous cytoplasmic proteins such as the ribosomal protein precursor FAU and ubiquitin.Our study was motivated by the search for factors or ingredients that make autophagic organelles particularly mycobactericidal, as Mycobacterium tuberculosis can survive the environment of the conventional phagolysosome. This was shown in the 1970s by the classical work of Armstrong and D''Arcy Hart at the same time when these authors established the more broadly appreciated and well-ingrained reputation of the tubercle bacillus as inhibiting the conventional phagosome-lysosome fusion. The approach to identifying such hypothetical ingredients was to first examine the steps of the autophagic pathway that are necessary for the mycobactericidal nature of macrophages induced for autophagy by, for example, starvation. We have found that not only are all stages of autophagy (initiation, elongation/closure and maturation) required for full mycobactericidal potency, but that p62, the first autophagic adapter characterized by the Johansen group, and also known as sequestosome 1, is absolutely required for autophagic elimination of M. tuberculosis. Sequestosome 1/p62 recognizes ubiquitinated protein aggregates and possibly ubiquitinated depolarized mitochondria and other targets, and delivers them to nascent autophagosomes; p62 also binds to the mammalian Atg8 paralog LC3 via its LC3-interaction region (LIR), thus conveniently bridging the targets with forming phagophores.At first blush, it may seem that mycobacteria follow the same fate demonstrated for several other bacteria, whereby p62 or another autophagic adapter, NDP52, capture cytosolic microbes and deliver them to autophagosomes. For example, the fraction of Salmonellae that are no longer retained within phagosomes and are free in the cytosol, or Shigella and Listeria that actively escape into the cytosol, are associated with ubiquitinated material or become otherwise recognized by p62 or NDP52, and end up being sequestered into autophagosomes. However, we found no evidence for p62 acting directly to transfer intraphagosomal mycobacteria into autophagic vacuoles. Instead, we observed p62-positive organelles as periodically fusing with mycobacterial phagosomes. At the same time, we found by imaging and biochemical means that proteins recruited by p62 from the cytosol into conventional autophagic organelles are subsequently transferred to model (latex bead phagosomes formed upon feeding 1 µm beads to macrophages) or mycobacterial phagosomes, as they gradually acquire autolysosomal characteristics. Next, we established that p62-captured cytosolic proteins (ribosomal protein rpS30 precursor FAU and ubiquitin) are proteolytically degraded into smaller peptides, and that specific peptides from these complex mixtures show antimycobacterial activity. Thus, the emerging model posits that autophagy captures cytosolic proteins and converts them into neo-antimycobacterial peptides that can then kill M. tuberculosis upon delivery to mycobacteria-containing phagosomes, which in turn gradually acquire autolysosomal properties (Fig. 1).Open in a separate windowFigure 1Elimination of M. tuberculosis by autophagy and p62. Mycobacteria are phagocytosed by macrophages and at least for some time reside within phagosomes. Upon induction of autophagy, p62, as a bifunctional agent interacting with autophagic substrates and with LC3, recruits into autophagosomes pre-antimicrobicidal cytosolic substrates. Autophagosome maturation including acquisition of lysosomal hydrolases leads to the proteolytic cleavage of p62 substrates and their conversion into peptides (cryptides) that can act as antimicrobial peptides.In contrast to the direct mechanism of capturing bacteria employed in some instances described above, in the case of M. tuberculosis, an organism that resides within the phagosomes, the adapter molecule p62 exerts its anti-microbial action through an indirect, but rather sophisticated mechanism. By sequestering into autophagosomes the initially harmless cytosolic components and by proteolytically processing them within maturing autophagosomes, p62 and autophagy liberate antimicrobial peptides from the otherwise innocuous substrates. This amounts to a resourceful utilization by the cell of otherwise spent or to-be-discarded cytoplasmic proteins and gives them an after-function upon completion of their “day jobs” that they performed as whole proteins.Our studies have uncovered a previously unappreciated function for autophagy in generating neo-antimicrobial peptides, and perhaps also opened the prospect for other biological functions potentially engendered by the products of autophagic proteolysis. Given that autophagy has the capacity to capture en masse and subject to digestion large sections of the cytoplasm, most cellular proteins are undergoing, or can undergo, processing into peptides or peptide intermediates within autophagic organelles. We postulate that the antimicrobial peptide production revealed in our studies thus far is only one manifestation of a spectrum of potential biological functions of cryptides generated by autophagy.     

Dual function of CALCOCO2/NDP52 during xenophagy

During xenophagy, pathogens are selectively targeted by autophagy receptors to the autophagy machinery for their subsequent degradation. In infected cells, the autophagy receptor CALCOCO2/NDP52 targets Salmonella Typhimurium to the phagophore membrane by concomitantly interacting with LC3C and binding to ubiquitinated cytosolic bacteria or to LGALS8/GALECTIN 8 adsorbed on damaged vacuoles that contain bacteria. We recently reported that in addition, CALCOCO2 is also necessary for the maturation step of Salmonella Typhimurium-containing autophagosomes. Interestingly, the role of CALCOCO2 in maturation is independent of its role in targeting, as these functions rely on distinct binding domains and protein partners. Indeed, to mediate autophagosome maturation CALCOCO2 binds on the one hand to LC3A, LC3B, or GABARAPL2, and on the other hand to MYO6/MYOSIN VI, whereas the interaction with LC3C is dispensable. Therefore, the autophagy receptor CALCOCO2 plays a dual function during xenophagy first by targeting bacteria to nascent autophagosomes and then by promoting autophagosome maturation in order to destroy bacteria.Xenophagy is the process referring to the selective degradation of intracellular microorganisms by autophagy. Xenophagy is a very potent intrinsic cellular line of defense to fight pathogens and requires first the detection and targeting of microorganisms to growing phagophores prior to autophagosome maturation leading to microbial destruction. The targeting step can be achieved by cytosolic autophagy receptors, which bind on the one hand to the pathogen and on the other hand to LC3, a phagophore membrane-anchored protein. Once entrapped within an autophagosome, bacteria can survive or escape, unless they are rapidly destroyed. Therefore, autophagosome maturation allows the discharge of lysosomal enzymes in autolysosomes, allowing destruction of the bacteria. It is, however, not well known how autophagosomes mature, especially in the context of xenophagy. Recently, the endosomal membrane-bound protein TOM1 and the dynein motor MYO6 have been both shown to be implicated in the transport of endosomes into the vicinity of autophagosomes in order to ensure fusion of autophagosomes with vesicles of the endo/lysosomal pathway. Moreover, the concomitant absence of 3 autophagy receptors, CALCOCO2, TAX1BP1/T6BP, and OPTN/OPTINEURIN, impairs autophagosome biogenesis and maturation. As CALCOCO2 was already shown to have a MYO6 binding domain, we wondered whether CALCOCO2 could also be implicated in autophagosome maturation per se to promote bacterial degradation.We first observed that the binding site of CALCOCO2 to MYO6 was required for cells to control Salmonella Typhimurium intracellular growth. Nevertheless, when the binding of CALCOCO2 to MYO6 was abolished, bacteria were still efficiently targeted to autophagosomes, but yet still able to replicate to levels similar to the one observed in CALCOCO2-depleted cells. Strikingly, in noninfected cells the absence of CALCOCO2 perturbs the autophagy flux, resulting in a strong accumulation of autophagosomes, suggesting a positive role for CALCOCO2 in the autophagosome-lysosome fusion process. Surprisingly, we found that CALCOCO2 binding to LC3C, through its noncanonical LC3 interacting region (CLIR), is not involved in the maturation of autophagosomes. Instead, we identified another motif in the primary sequence of CALCOCO2, which mediates binding to at least LC3A, LC3B, and GABARAPL2 (but not LC3C). We referred to this motif as “LIR-like” as it differs from the canonical LIR motif by the absence of a hydrophobic residue in position X₃. This LIR-like motif was necessary for autophagosome maturation, along with the domain of CALCOCO2 responsible for its binding to MYO6. Eventually, mutation of this LIR-like motif also resulted in an increased Salmonella Typhimurium intracellular proliferation, whereas bacteria were still efficiently targeted within nondegradative autophagosomes. Interestingly, the absence of the autophagy receptor OPTN also led to the accumulation of nondegradative autophagosomes, suggesting that other autophagy receptors could share CALCOCO2 dual functions in xenophagy.Having autophagy receptors ensuring both targeting and degradation of pathogens could be an important evolutionary advantage against infections. Indeed, this mechanism could help to reduce the delay necessary for maturation, thus avoiding adaptation of the pathogen to its new environment (as proposed for Coxiella burnetti, Listeria monocytogenes, and Legionella pneumophila) or its escape from the autophagosome. Conversely, pathogens could avoid autophagy entrapment or autophagic degradation by targeting CALCOCO2 or any other autophagy receptors, which could play similar roles. For instance Chikungunya virus was reported to target CALCOCO2 in human cells leading to increased virus replication. Nevertheless, redundancy among autophagy receptors could also ensure a selective immune advantage against pathogens targeting any one of these receptors.Our results and those from others suggest for now that CALCOCO2 serves as a docking platform for MYO6-bound endosomes, thus facilitating autophagosome maturation (Fig. 1). How this action is coordinated with CALCOCO2 directing pathogens to the phagophore membranes remains unclear. During xenophagy against Salmonella Typhimurium, CALCOCO2 interaction first with LC3C is necessary to further recruit other ATG8 orthologs and ensure the final degradation of bacteria. Since the LIR-like motifs bind several ATG8s, whereas the CLIR motif only mediates binding to LC3C, it is possible that binding of CALCOCO2 to LC3C induces conformational changes and uncovers the LIR-like motif that can be then engaged with other ATG8 orthologs to trigger autophagosome maturation. Moreover, it is still unclear whether the action of CALCOCO2 in autophagosome maturation is coordinated with other partners, such as STX17/SYNTAXIN 17, which is recruited on the external membrane of autophagosomes and regulate fusion with lysosomes. Open in a separate windowFigure 1.Schematic model for the dual role of CALCOCO2 in xenophagy. CALCOCO2 targets bacteria to the phagophore through its LC3C binding site (CLIR motif), and, independently, regulates autophagosome maturation through its LC3A, LC3B, or GABARAPL2 binding site (LIR-like motif) and its MYO6 interacting region.Our findings reveal a new role for the autophagy receptor CALCOCO2 in autophagosome maturation, unravelling another function for CALCOCO2 in cell autonomous defense against pathogens: CALCOCO2 not only targets pathogens to phagophore membranes, but also regulates subsequent maturation of pathogen-containing autophagosomes, thus assuring efficient degradation of autophagy-targeted pathogens.     

Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKC, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.     

Gold for Ubiquitin in Vancouver: FIRST CONFERENCE ON PROTEOMICS OF PROTEIN DEGRADATION AND UBIQUITIN PATHWAYS HELD JUNE 6–8, 2010 IN VANCOUVER,UNIVERSITY OF BRITISH COLUMBIA,ORGANIZED BY LAN HUANG,THIBAULT MAYOR,AND PEIPEI PING*

The rise of proteomics has had tremendous influence on analysis and understanding of the role of post-translational modifications in biological processes. The covalent attachment of small proteins like ubiquitin, SUMO,1 or other ubiquitin-like proteins (Ubls) is one class of post-translational modifications where proteomics has had notable impact. Various proteomics approaches, but in particular mass spectrometry-based analyses, have influenced the field and enabled significant advances over the past few years. The first meeting dedicated to proteomics of protein degradation and ubiquitin pathways showcased these advances and allowed a glimpse at future contributions of proteomics to this field. With its many attractive drug targets, the ubiquitin and proteasome system, as well as other proteolysis pathways, could offer new therapies for various human diseases including cancer and neurodegenerative disorders.The covalent linkage of ubiquitin to other proteins is catalyzed by the E1-E2-E3 cascade of enzymatic reactions whereby the many different E3 ubiquitin ligases provide substrate specificity to the process of protein ubiquitylation (1). Ubiquitylation is best known for targeting proteins for degradation by the proteasome, but other functions for ubiquitylation independent of proteolysis are also known. Likewise, modifications with SUMO or other Ubls generally do not regulate protein degradation but instead control subcellular localization, protein interactions, or change protein conformation and activity (2).The questions addressed by proteomics approaches to ubiquitylation and Ubl modifications are plentiful. They range from very specific, e.g. determination of the modified residue in a substrate protein, to complex, such as protein dynamics in proteome-wide ubiquitin (or Ubl) modification profiles (3). In either case, the rapid technological advancements (particularly in mass spectrometry instrumentation as well as quantitation and separation technologies) have allowed impressive progress, which was evident in the First Conference on Proteomics of Protein Degradation and Ubiquitin Pathways in Vancouver (http://ppdup.org/) (Fig. 1).Open in a separate windowFig. 1.Group picture from First Conference on Proteomics of Protein Degradation and Ubiquitin Pathways held June 6–8, 2010 in Vancouver (http://ppdup.org/).     

Spatiotemporal Dynamics of Phosphorylation in Lipid Second Messenger Signaling

The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events.The alteration of protein or lipid structure by phosphorylation is one of the most effective ways to transduce extracellular signals into cellular actions. Phosphorylation can alter enzyme activity, regulate protein stability, affect protein interactions or localization, or influence other post-translational modifications. A plethora of cellular processes, including cell growth, differentiation, and migration, are tightly regulated by phosphorylation. Cellular homeostasis is achieved by means of a precisely regulated balance between phosphorylation and dephosphorylation, and disruption of this balance results in pathophysiologies. Kinases and phosphatases are antagonizing effector enzymes that respond to second messengers and mediate phosphorylation/dephosphorylation events.Two prominent lipid second messenger pathways are those mediated by diacylglycerol (DAG)¹ and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) (Fig. 1A). These membrane-embedded second messengers recruit effector kinases containing specific membrane-targeting modules to membranes, thus activating them. Specifically, DAG recruits C1-domain-containing proteins, notably protein kinase C (PKC), whereas PIP₃ recruits pleckstrin homology (PH) domain-containing proteins, such as Akt. Specificity and fidelity in signaling are often achieved via the compartmentalization of signaling on protein scaffolds and membrane microdomains, which can control the access of enzymes to particular substrates. In this review, we provide a brief background of the DAG and PIP₃ pathways and their effector kinases, PKC and Akt, respectively. We discuss sensors that have been developed to measure lipid second messenger levels and kinase activity at various subcellular compartments, the role of scaffolds and membrane microdomains in compartmentalizing signaling, and the consequences of dysregulation of second messenger signaling in disease.Open in a separate windowFig. 1.Diagram of diacylglycerol and PIP₃ signaling pathways. A, agonist stimulation activates cell surface receptors such as receptor tyrosine kinases or G-protein-coupled receptors, which active phospholipase C (PLC) or phosphoinositide-3 kinase (PI3K), both of which act on phosphatidylinositol 4,5-bisphosphate (PIP₂). PLC hydrolyzes PIP₂ to produce diacylglycerol (DAG) and inositol triphosphate (IP₃). IP₃ binds to the IP₃ receptor (IP₃R) at the endoplasmic reticulum (ER), releasing Ca²⁺. DAG and Ca²⁺ recruit conventional protein kinase C (PKC) to the plasma membrane (via its C1 and C2 domains, respectively) and activate it. Ca²⁺ release also leads to the production of DAG at the Golgi. Diacylglycerol kinase (DGK) suppresses DAG-mediated signaling by converting DAG to phosphatidic acid (PA). Alternatively, PI3K phosphorylates PIP₂ into phosphatidylinositol 3,4,5-bisphosphate (PIP₃), which recruits Akt to the plasma membrane via its pleckstrin homology (PH) domain, where it gets activated. PTEN terminates PIP₃ signaling by converting it to PIP₂. B, enzymes involved in DAG and PIP₂ production and removal. PIP₂ is converted to PIP₃ by PI3K, whereas PTEN opposes this reaction by dephosphorylating PIP₃. PIP₂ can also be converted to DAG and IP₃ by PLC. DAG can then be removed by DGKs as it gets converted to PA. Phosphatidic acid phosphatases (PAPs) and sphingomyelin synthases (SMSs) convert PA back to DAG.

Lipid Second Messengers

Lipid second messengers are signaling molecules produced in response to extracellular stimuli. Targeting enzymes and their substrates to the same membrane constrains them to a space of reduced dimensionality, thus increasing the apparent concentration of the signaling complex and the likelihood and amplitude of signaling (1).

DAG Pathway

Upon activation by agonists, receptor tyrosine kinases and G-protein-coupled receptors can activate phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) found at the plasma membrane into the second messengers DAG and inositol 1,4,5-trisphosphate (IP₃) (2). DAG recruits proteins that contain C1 domains, small globular DAG-binding domains, to membranes (3), whereas IP₃ freely diffuses inside the cell and binds to the IP₃ receptor at the endoplasmic reticulum. This releases another second messenger, Ca²⁺, which induces further production of DAG at the Golgi (4). Two main classes of proteins that bind DAG are the protein kinases PKC and PKD and the Rac-GAPs chimerins; the affinity of their C1 domains for DAG can vary greatly and is discussed below in the context of PKC. DAG can also be generated from phosphatidic acid (PA) by phosphatidic acid phosphatases or sphingomyelin synthases. The removal of DAG, and thus termination of its signaling, is achieved by diacylglycerol kinases (DGKs) that convert it into PA (Fig. 1B).

PIP₃ Pathway

The second messenger PIP₃ is generated upon the stimulation of receptor tyrosine kinases or G-protein-coupled receptors that activate phosphoinositide 3-kinase (PI3K) to phosphorylate PIP₂ (Fig. 1A). PIP₃ binds certain PH domains, recruiting enzymes such as Akt to the membrane (5). PIP₃ signaling is terminated by the tumor suppressor phosphatase and tensin homologue (PTEN), a lipid phosphatase that converts PIP₃ to PIP₂ (Fig. 1B).

Genetically Encoded Second Messenger Sensors

Second messenger levels can be measured in live cells, in real time, using genetically encoded sensors (6–11). The first generation of sensors comprised a fluorescent protein fused to a domain that specifically binds a second messenger (i.e. C1 or PH domain). Recruitment of the tagged domain to membranes containing the second messenger is monitored and used as a proxy for its levels (6, 10, 11). Recently, reporters containing two fluorescent proteins that undergo changes in fluorescence resonance energy transfer (FRET) as they change their distance or relative orientation upon binding of a second messenger have been developed (7–9). These reporters provide more quantitative data, as they rely on ratiometric measurements of two fluorophores as opposed to the translocation of a single fluorophore to membranes, thus minimizing artifacts resulting from cell movements, photobleaching, or variable cell thickness (12). Targeting of these second messenger sensors to particular subcellular compartments provides information on the kinetics and location of second messenger production.

Measuring DAG

One of the first DAG reporters comprised the C1 domain of PKCγ fused to a green fluorescent protein, and its agonist-evoked translocation to membranes served as a readout for DAG production (6). Reporters employing ratiometric measurements were later developed and contain a C1 domain and a FRET pair, such as the DAG reporters DAGR (Fig. 2A), which uses intermolecular FRET (7), and Daglas (Fig. 2B), which uses intramolecular FRET (8). These reporters can be targeted to specific subcellular compartments using a membrane localization sequence, allowing the detection of changes in DAG levels at various intracellular membranes. Such membrane-specific reports have revealed that the Golgi and endoplasmic reticulum have relatively high basal levels of DAG, whereas the plasma membrane lacks measurable basal DAG (9). DAG at the plasma membrane is produced following acute agonist stimulation, and this signaling is swiftly terminated (Fig. 3) as DGK converts DAG to PA (8, 13). In contrast, stimulated DAG levels at the Golgi are relatively sustained, whereas those at mitochondria are not detectably altered by ATP stimulation.Open in a separate windowFig. 2.Genetically encoded reporters for measuring DAG and PIP₃ levels and PKC and Akt activities. A, the diacylglycerol (DAG) reporter, DAGR, comprises a CFP–YFP FRET pair flanking a DAG-binding C1 domain. Translocation of the reporter to membranes upon elevation of DAG leads to intermolecular FRET. B, the DAG reporter Daglas and PIP₃ sensor Fllip are anchored to the membrane via a membrane localization sequence (MLS) and are composed of a FRET pair flanking a lipid-binding domain (LBD) (C1 domain for Daglas and PH domain for Fllip) and a glycine–glycine (GG) hinge. Engagement of the LBD to the membrane induces a conformational change in the reporter, increasing FRET efficiency. The FRET signal is reversed by enzymes that remove the second messengers (i.e. DGK and PTEN). C, Upward and Downward DAG are based on PKCδ and contain a circularly permuted GFP between the pseudosubstrate (PS) and the C1 domain. Binding of the C1 domain to DAG increases (for Upward DAG) or decreases (for Downward DAG) the fluorescence intensity by altering the conformation of the GFP β-barrel. D, InPAkt is a PIP₃ reporter comprising the PH domain of Akt, a pseudoligand that binds the PH domain with a lower affinity than PIP₃, and a FRET pair. Upon PIP₃ production, the PH domain releases the pseudoligand and binds PIP₃, increasing FRET. E, the PKC reporter, CKAR, and the Akt reporter, BKAR, are made up of an FHA2 domain, a PKC- or Akt-specific substrate peptide, and a FRET pair. When phosphorylated, the substrate sequence binds the FHA2 domain, and this conformational change results in a decrease in FRET. F, the nPKC and atypical PKC reporter KCP-1 is based on the PKC substrate pleckstrin. PKC phosphorylation at three sites between the PH and dishevelled–Egl-10–pleckstrin domains of pleckstrin results in a conformational change that increases FRET between enhanced YFP and GFP2. G, Aktus and AktAR measure Akt activity as phosphorylation of an Akt-specific substrate peptide causes it to bind a phosphopeptide-binding domain (PPBD) (14–3-3η for Aktus and FHA1 for AktAR), increasing FRET from CFP to YFP. H, the bioluminescent Akt reporter (BAR) contains a split luciferase, an FHA2 domain, and an Akt substrate peptide. Phosphorylation by Akt causes the split luciferase to dissociate, decreasing bioluminescence. I, Akind is based on Akt''s kinase and PH domains. Upon recruitment of Akt to PIP₃-enriched membranes and its subsequent phosphorylation, Akt undergoes a conformational change that leads to an increase in FRET between the fluorophores.Open in a separate windowFig. 3.Schematic diagram displaying DAG and PIP₃ levels at various intracellular membranes and the PKC and Akt activity they induce at these locations. UTP stimulates rapid but short-lived production of DAG at the plasma membrane. In contrast, stimulated DAG levels at the Golgi are relatively sustained, leading to more sustained PKC activity at the Golgi than at the plasma membrane. Typically cPKCs are recruited to the plasma membrane because their Ca²⁺-regulated C2 domain selectively recognizes PIP₂, which is enriched at the plasma membrane. In contrast, nPKCs signal primarily at the DAG-enriched Golgi (111). The nucleus shows little UTP-induced PKC activity, whereas the cytosol has the greatest, although transient, UTP-stimulated PKC activity. Upon PDGF stimulation, PIP₃ is more rapidly generated at endomembranes such as the Golgi than at the plasma membrane. PDGF-induced Akt activity rapidly increases at the plasma membrane, whereas in the cytosol the kinetics of activation are slower. The nucleus contains high but delayed Akt activity, despite the lack of PIP₃ production at the nuclear membrane, suggesting that active Akt translocates to the nucleus where there is little phosphatase suppression.More recently, a sensor has been engineered to simultaneously measure two second messengers in the same cell. The Green Upward or Downward DAG (Fig. 2C) was paired with a Ca²⁺ sensor (not shown) for the concomitant measurement of DAG and Ca²⁺ (9). Both of these sensors are based on fluorescent proteins that undergo changes in fluorescence intensity upon the binding of second messengers. The DAG sensor (based on PKCδ) and the Ca²⁺ sensor (based on calmodulin and a calmodulin-binding domain (14)) are linked by a peptide that, upon cleavage, produces equal amounts of the two reporters as distinct peptides (15). Considering that DAG and Ca²⁺ are often co-elevated, measuring these second messengers concomitantly gives a more complete view of signal transduction and has the advantage of providing a direct comparison of second messenger production downstream of the activation of various receptors with different agonists. However, because these reporters rely on changes in fluorescence intensity instead of a FRET ratio, care should be taken when using them, as their readout is dependent on the absolute concentration of the sensor, and intensity changes resulting from focus drift or cell movements can occur during imaging.

Measuring PIP₃

Changes in PIP₃ levels can be assessed by monitoring the agonist-dependent relocalization of fluorescently tagged PIP₃-selective PH domains to membranes (10, 12). However, because these methods monitor translocation to all membranes, pools of PIP₃ at specific membranes cannot be readily monitored. Thus, more quantifiable FRET-based PIP₃ sensors that can be targeted to particular subcellular localizations have been developed. For example, an indicator for phosphoinositides based on Akt''s PH domain, InPAkt (Fig. 2D), showed that the plasma membrane contains basal levels of PIP₃ that are maintained through a balance between PI3K and phosphatases, whereas the nucleus has no detectible production of this second messenger (16). Another PIP₃ sensor, Fllip (Fig. 2B), comprising the PH domain of GRP1 and a FRET pair, is anchored to a membrane via a membrane localization sequence (17). This reporter revealed that platelet-derived growth factor (PDGF) stimulates greater production of PIP₃ at the Golgi and endoplasmic reticulum than at the plasma membrane, and that PIP₃ is generated at these endomembranes by endocytosed receptor tyrosine kinases that activate a localized pool of PI3K.

PKC and Akt as Effectors

PKC and Akt, effector kinases of DAG and PIP₃, respectively, phosphorylate myriad downstream targets (reviewed in Refs. 18–20), many of which have been identified through phosphoproteomic screens (21–23).

PKC

The PKC family consists of nine genes that are divided into three categories based on the domain structure of the enzymes they encode, and hence the second messengers they require for activation. Both conventional PKCs (cPKCs) (α, β, γ) and novel PKCs (nPKCs) (δ, ε, θ, η) bind DAG through tandem C1 domains; cPKCs also bind membranes in a Ca²⁺-dependent manner through a C2 domain. Atypical PKCs (ζ and ι/λ) bind neither DAG nor Ca²⁺ and are regulated by protein–protein interactions through a PB1 domain (24). cPKCs and nPKCs are constitutively phosphorylated at three conserved phosphorylation sites termed the activation loop, turn motif, and hydrophobic motif (25). These phosphorylations are necessary for proper PKC folding, and thus for its activation; for PKCα, these modifications occur with a half-time of 5 to 10 min following biosynthesis (26). The activation loop is phosphorylated by the phosphoinositide-dependent kinase PDK-1 (27–29), an event that triggers two tightly coupled phosphorylations at the turn and hydrophobic motifs (25, 30). mTORC2 is required to initiate the phosphorylation cascade, and in cells lacking mTORC2, PKC is not phosphorylated and thus is shunted for degradation (31–33); however, the mechanism of this regulation is unknown. The hydrophobic motif is autophosphorylated by an intramolecular reaction in vitro, but whether this is the mechanism of modification in cells or whether it is the direct target of another kinase such as mTORC2 remains controversial (33, 34). When first translated, PKC is in an open conformation, with the autoinhibitory pseudosubstrate out of the active site (35). Upon phosphorylation, PKC matures into a catalytically competent, but inactive, species that is maintained in an autoinhibited (closed) conformation in which the pseudosubstrate occupies the substrate-binding cavity. Upon intracellular Ca²⁺ release and DAG production, cPKCs are recruited to membranes through their Ca²⁺-sensitive C2 domain. This relocalization reduces the dimensionality in which the C1 domain has to probe for its membrane-embedded ligand, DAG, thus increasing the effectiveness of this search by several orders of magnitude (36). Binding of one of PKC''s C1 domains to DAG provides the energy necessary to expel the pseudosubstrate from the substrate-binding site, allowing the phosphorylation of downstream targets. For some isozymes, such as PKCδ, the second C1 domain (C1B) is the major binder (37). The affinity of C1 domains for DAG is toggled from low to high by a single residue within the DAG binding cavity: a Trp at position 22 confers an affinity for DAG that is 2 orders of magnitude higher than that conferred by a Tyr at that position (38). Consequently, nPKCs, which contain a high-affinity C1B domain, can respond to DAG alone, whereas cPKCs, which have a low-affinity C1B domain, require the elevation of Ca²⁺ concomitantly with DAG production in order to become activated. Reporters such as the Green Upward or Downward DAG (Fig. 2C) paired with a Ca²⁺ sensor (9), described above, would be useful tools for discerning which agonists solely activate cPKCs versus nPKCs.The amplitude of PKC signaling is diligently balanced through its phosphorylation state (controlling its steady-state levels), the presence of its lipid second messenger, DAG (acutely controlling activity), and, for cPKCs, Ca²⁺ levels. Thus PKC activity can be antagonized via direct dephosphorylation by protein phosphatases such as the PH domain leucine-rich repeat protein phosphatase (PHLPP) or by removal of the lipid second messenger through phosphorylation by the lipid kinase DGK (Fig. 1) (39). The peptidyl-prolyl isomerase Pin1 was recently shown to be necessary for cPKC dephosphorylation and degradation following agonist activation, as it isomerizes the phosphorylated turn motif of cPKCs, thus facilitating PKC dephosphorylation (40).

Akt

Akt, also known as PKB because of its homology to PKA and PKC, is a serine/threonine kinase that promotes cell growth and survival (41). The three Akt isozymes (Akt1, Akt2, and Akt3) contain an N-terminal PH domain that mediates PIP₃-dependent membrane recruitment. Akt is maintained in an inactive conformation by an interaction between the PH and kinase domains, and unlike PKC, Akt is directly activated by phosphorylation at its activation loop and hydrophobic motif following agonist-dependent recruitment to membranes (42). Thus, Akt phosphorylation at these sites can be used as a proxy for activity under certain conditions (43). Akt is co-translationally phosphorylated at the turn motif by mTORC2 (contrasting with the post-translational modification of PKC), an event that is not necessary for function but increases the stability of the protein (44). PIP₃ recruits Akt to the plasma membrane via its PH domain, unmasking the kinase domain to permit phosphorylation of the activation loop by PDK-1 (45) and subsequent phosphorylation of the hydrophobic motif. mTORC2 facilitates hydrophobic motif phosphorylation (46, 47), possibly by assisting in unmasking the kinase domain for phosphorylation. Indeed, manipulations that displace the PH domain effectively bypass the requirement for mTORC2 for phosphorylation of the hydrophobic motif (but not the turn motif) (48). Phosphorylated Akt is locked in an active conformation and can disengage from the membrane and relocalize to other intracellular regions, such as the nucleus (49), to phosphorylate diverse substrates (19). Akt signaling is terminated by the hydrolysis of PIP₃ to PIP₂ by PTEN or by direct dephosphorylation of the activation loop by protein phosphatase 2A or of the hydrophobic motif by PHLPP (50–52). PHLPP1 dephosphorylates Akt2 and Akt3, whereas PHLPP2 dephosphorylates Akt1 and Akt3 (52), suggesting that these isozymes are differentially compartmentalized, likely via the unique PDZ (PSD-95, disheveled, and ZO1) ligand of each PHLPP isozyme (53). Interestingly, stoichiometric quantification of Akt phosphorylation at the activation loop and hydrophobic motif sites using LC-MS revealed that in untreated T cells, less than 1% of Akt is phosphorylated at both of these sites, and a low level of Akt phosphorylation is sufficient to contribute to tumorigenesis (54), highlighting the importance of keeping Akt activity low for maintaining cellular homeostasis.

Identifying PKC and Akt Substrates

A number of PKC and Akt substrates have been identified through biochemical methods, and several phosphoproteomic screens have been devised to identify new substrates and novel roles of these kinases. For example, a functional proteomic screen identified enhancer of mRNA decapping 3 as a substrate of Akt that regulates the mRNA decay and translation repression pathways downstream of insulin signaling (21), and another identified a number of chaperone proteins and protein disulfide isomerases as potential Akt substrates in rat mesangial cells, suggesting that Akt might regulate chaperone function (22). An in vivo quantitative phosphoproteomics study employing stable isotope labeling by amino acids in cell culture implicated a highly deregulated kinase network composed of PKC, PAK4, and SRC in squamous cell carcinoma, but not in papilloma (55).Vast efforts combining phosphorylation sites identified in vivo though mass spectrometry, in vitro through protein microarrays, and computationally through prediction algorithms have uncovered more substrates, setting the foundation for the development of human phosphorylation network maps (23, 56). However, despite efforts to enrich for physiologically relevant phosphorylation events by accounting for subcellular localization and scaffolding, these networks still have shortcomings, as the algorithms predict some false positives and miss a large fraction of known phosphorylation sites.

Kinase Activity Reporters

Genetically encoded reporters allow the visualization of the spatiotemporal dynamics of kinase activity in individual cells. These reporters can be targeted to various subcellular localizations and to protein scaffolds to measure localized activity, which can be more physiologically relevant than bulk activity in the cytosol.

PKC

Because cPKCs and nPKCs are constitutively phosphorylated at the C-terminal sites, and because the phosphate at the activation loop does not modulate activity once the C-terminal tail is phosphorylated (25), their activity cannot be measured with phosphorylation-specific antibodies, as is done for most other kinases. However, PKC activity can be monitored using activity reporters such as the C kinase activity reporter (Fig. 2E), which is composed of a CFP–YFP FRET pair flanking a PKC-specific substrate and an FHA2 phosphothreonine-binding domain (7). Upon phosphorylation of this substrate by PKC, the reporter undergoes a conformational change that decreases FRET. As phosphorylation of the reporter is reversible (i.e. phosphatases can dephosphorylate the reporter), it provides a real-time readout of PKC activity.C kinase activity reporter has been targeted to various intracellular locations to enable specific monitoring of PKC activity at these regions. Using these targeted reporters, Gallegos et al. (13) found that the activation of PKC with the agonist UTP leads to rapid and relatively sustained PKC activity at the Golgi, driven by the persistence of DAG at this membrane (Fig. 3). UTP-dependent PKC activity in the cytosol is, however, quickly terminated by phosphatases, and activity in the nucleus is low because of high phosphatase suppression in this compartment. The mitochondria also have little UTP-stimulated activity; however, using a mitochondrially targeted PKCδ-specific activity reporter, Mito-δCKAR, Zheng et al. (57) revealed that PKCδ translocates to, and is active at, the outer membrane of mitochondria upon stimulation with phorbol esters, and that its intrinsic catalytic activity is required for its interaction with the mitochondria.The Schultz lab has developed a reporter for nPKCs and atypical PKCs, KCP-1 (Fig. 2F), that is based on the PKC substrate pleckstrin (58). This reporter does not utilize a phosphopeptide-binding domain; rather, phosphorylation of residues between its PH and dishevelled–Egl-10–pleckstrin domains causes a conformational change in the reporter, resulting in a change in FRET. Thus, interactions between the phosphorylated sites and other endogenous proteins are reduced.

Akt

A number of FRET-based Akt reporters have been developed, most of which measure the phosphorylation of a synthetic substrate of Akt in live cells; these include Aktus, AktAR (Fig. 2G), and BKAR (Fig. 2E) (59–61). Aktus has low sensitivity and requires overexpression of Akt, whereas BKAR and AktAR can sense endogenous Akt activity. Because activated Akt can disengage from the membrane and diffuse to other subcellular locations, targeting these reporters to various subcellular compartments is particularly useful. BKAR targeted to the plasma membrane revealed that phosphatase suppression of Akt is low at this location (60). The phosphatase suppression of Akt activity is greater in the cytosol than it is at the plasma membrane, so Akt is more rapidly inactivated in the cytosol (Fig. 3), most likely by protein phosphatases that dephosphorylate Akt''s substrates as opposed to Akt itself. Conversely, the nucleus has low phosphatase suppression of Akt, so once Akt diffuses to the nucleus its activity is much more sustained (60). AktAR (61) has a greater dynamic range for detecting Akt activity than BKAR and was used to measure Akt activity in plasma membrane microdomains (addressed below).Whereas fluorescent reporters can measure rapid signaling kinetics at subcellular levels, bioluminescent reporters have the advantage of producing their own light and therefore bypass issues with autofluorescence, photobleaching, and tissue damage from the excitation light (62). A bioluminescent Akt reporter, BAR, was engineered using a split luciferase, an Akt specific substrate, and a phosphopeptide-binding domain (Fig. 2H). This reporter has the capability to measure Akt activity in a noninvasive manner in vivo (63). In addition to Akt activity reporters that measure the phosphorylation of a synthetic substrate by Akt, reporters that measure conformational changes of Akt induced by its translocation and phosphorylation (and thus activity state) have also been developed (64–66). The Akt indicator Akind (57), comprising the PH and catalytic domains of Akt and a FRET pair (Fig. 2I), was used to visualize Akt translocation to and activity at lamellipodial protrusions. Conformational changes attendant to the phosphorylation of Akt result in an increase in FRET. The use of another reporter of Akt action, ReAktion (not shown) (58), led to the proposal that activation loop phosphorylation of Akt decreases its membrane binding affinity, thus allowing disengagement from the membrane and relocalization to other cellular compartments. One advantage of these reporters is that both the activity and the translocation of Akt can be measured concurrently. However, the reporters themselves can phosphorylate endogenous substrates of Akt and could theoretically displace Akt from its scaffolds, thus potentially perturbing the system more than the introduction of an exogenous Akt substrate.

Localized Signaling and the Importance of Scaffolding

Protein scaffolds coordinate and allow specificity and fidelity by compartmentalizing kinases and their downstream substrates, as well as the phosphatases that can rapidly terminate the signal (67). Even though lipid second messengers acutely regulate the activity of PKC and Akt, scaffolds can mediate their access to particular substrates.

PKC Scaffolds

Although multiple PKC isozymes respond to the same second messengers, there is some specificity in their function and signaling, mediated in part by their cell-specific pattern of expression, their differential affinities for certain lipids, and by protein scaffolds. PKC is anchored to numerous protein scaffolds through interactions mediated by its regulatory domain, its pseudosubstrate, or, in the case of PKCα and the atypical PKCs, a PDZ ligand. The first PKC scaffolds were identified by Mochly-Rosen and colleagues as receptors for activated C kinase, which are proposed to selectively bind active PKC and enhance its activity toward substrates anchored at that location (68, 69). A kinase anchoring proteins (AKAPs), which were first identified as PKA scaffolds (70), also anchor PKC in proximity to its targets, but in its inactive state, thus enabling rapid downstream signaling upon PKC activation. For example, AKAP-Lbc coordinates PKCη and PKA to phosphorylate PKD and release it from the scaffold (71), whereas AKAP79 functions as a scaffold for PKC, PKA, and protein phosphatase 2B at the postsynaptic density in neurons (72). One critical aspect of scaffolding is that it can alter the pharmacological profile of tethered kinases, which could have clinical implications for drug design. For example, through the use of targeted PKC activity reporters, Scott and colleagues found that PKC bound to the AKAP79 scaffold is refractory to active site inhibitors, but not allosteric ones. Similarly, the processing phosphorylation of cPKCs by PDK-1 is refractory to active site inhibitors of the co-scaffolded PDK-1 (73).PKCα, -ζ, and -ι also bind PDZ-domain-containing scaffolds through their distinct PDZ ligands. In the case of PKCα, scaffolding by its PDZ ligand is required for cerebellar long-term depression (74). One likely PDZ-domain-binding partner involved in this is protein interacting with Cα kinase (PICK1), which interacts specifically with the PDZ ligand of PKCα (75, 76). Interestingly, PICK1 can have opposing effects on PKCα function in neurons, where it can act as either a mediator or an inhibitor of the phosphorylation of downstream targets. PICK1 targets activated PKCα to synapses to phosphorylate the glutamate receptor subunit GluR2, leading to its endocytosis (77), but it can also act as a barrier to phosphorylation of the metabotropic glutamate receptor mGluR7a by PKCα (78). More recently, a family of Discs large homolog scaffolds that interact with the PDZ ligand of PKCα to facilitate cellular migration was also identified (79).

Akt Scaffolds

Akt translocation to different intracellular regions is contingent on which upstream pathway is activated, partially because of the scaffolding proteins that direct its signaling. For example, the stimulation of endothelial cells with insulin leads to the activation of Akt and its translocation to both the Golgi and mitochondria, whereas stimulation with 17β-estrodiol leads to translocation of Akt to the Golgi but not mitochondria (59). Only a few Akt scaffolds have been identified thus far, but it is becoming more apparent that different receptors use different complexes to direct Akt activity (80). For example, Akt kinase-interacting protein 1 was identified as a scaffold for PI3K/PDK-1/Akt that associates with activated EGF receptors (81). This scaffold is necessary for Akt phosphorylation by PDK-1 downstream of EGF signaling. Conversely, Akt scaffolds can also attenuate Akt signaling by scaffolding it in proximity to its phosphatases. β-arrestin 2 scaffolds Akt and its activation loop phosphatase, PP2A, in response to G-protein-coupled receptor stimulation in dopaminergic neurotransmission (82), and β-arrestin 1 scaffolds Akt1 and its hydrophobic motif phosphatase, PHLPP2, downstream of receptor tyrosine kinases (83). Considering that the three Akt isozymes have some overlapping expression, are activated downstream of a multitude of receptors, have numerous cellular functions, and show specificity in their dephosphorylation by PHLPP, an abundance of Akt scaffolds potentially await identification.

Membrane Microdomains

Lipid rafts, which are cholesterol- and sphingolipid-rich microdomains, not only compartmentalize signaling complexes, but also increase signal transduction by aggregating particular signaling complexes in a small area (1).

PKC

DAG and its effector kinase PKC are often enriched in specialized lipid rafts that form membrane invaginations called caveolae (84, 85). For example, activation of the adenosine A1 receptor in cardiomyocytes causes PKCε and PKCδ to translocate to caveolae. PKCα is also enriched within caveolae through interactions with caveolin, which inhibits its activity, or with the serum deprivation response protein (86, 87).

Akt

Akt signaling not only differs at various subcellular localizations, but also varies within microdomains of a particular membrane. The Akt reporter AktAR (Fig. 2G) was preferentially targeted to different plasma membrane microdomains such as lipid rafts (using the N-terminal region of Lyn kinase) and non-raft regions (using the Kras CAAX motif) to analyze the spatiotemporal dynamics of Akt activity (61). These reporters demonstrate that Akt residing in lipid rafts is activated more potently and with faster kinetics than non-raft Akt. PDK-1 is enriched in membrane microdomains, partially accounting for the increased Akt activity in this compartment (88, 89).Membrane microdomains provide sheltered environments in which signaling is protected from immediate termination. For example, PI3K gets activated and produces PIP₃ in lipid rafts where Akt is recruited, enabling Akt to be rapidly and specifically activated by PDK-1 (90). However, PTEN, which terminates PIP₃ signaling, is primarily found in non-raft microdomains, allowing PIP₃ levels to be temporarily maintained in lipid rafts, underscoring the importance of spatially separating kinases from phosphatases. Disturbing this spatial segregation of negative and positive regulators of a pathway can lead to disease states.

Dysregulation of Lipid Signaling in Disease

Dysregulation of the PIP₃ and DAG signaling pathways leads to numerous pathophysiologies, such as inflammation, cardiovascular disease, diabetes, neurodegeneration, and cancer (91). Imbalances in these pathways are caused by mutations, gene amplifications or deletions, chromosomal translocations, or epigenetic changes of genes in these pathways. In general, enzymes that promote signaling such as PKCι, Akt, and the catalytic subunit of PI3K have been identified as oncogenes (92–94), whereas those that terminate signaling—PTEN, PHLPP, and DGK—have been implicated as tumor suppressors (53, 95–100).Although mutations in Akt are uncommon, Akt signaling is often elevated in cancer. Indeed, the PI3K pathway is one of the most frequently up-regulated pathways in cancer (101–103). Mutations that inactivate PTEN or hyperactivate the catalytic subunit of PI3K are very common and induce constitutive Akt signaling. Interestingly, a functional proteomics study using a reverse-phase protein lysate array revealed that in breast cancers, Akt activation loop and hydrophobic motif phosphorylation strongly inversely correlates with PTEN levels, underscoring the importance of PTEN regulation of Akt activity (104).Mutations in receptors upstream of DAG and PIP₃ can also lead to enhanced signaling. For example, certain EGF receptor mutants constitutively associate with the PI3K/PDK-1/Akt scaffold Akt kinase-interacting protein 1, leading to increased Akt signaling in lung cancer (105). The involvement of some of these genes in cancer is further substantiated by their frequent amplification or deletion in tumors. For example, genes encoding PKCι, Akt1, Akt2, and the catalytic subunit of PI3K are amplified in various cancers (92, 94, 106–109), whereas those encoding PTEN and PHLPP are commonly deleted (110).     

PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.PLEKHM1 (pleckstrin homology domain containing, family M [with RUN domain] member 1) is a ubiquitously expressed protein involved in the regulation of osteoclast function and bone resorption. Recently, it was also described in the context of negatively regulating the endocytic pathway but not autophagy. However, in our recent studies, we show that PLEKHM1 positively regulates the terminal stages of both endocytic and autophagy pathways through direct interaction between PLEKHM1, RAB7, the HOPS complex, and mammalian Atg8 proteins (Fig. 1A). In addition, the PLEKHM1-RAB7-HOPS complex is a direct target for the Salmonella (Salmonella enterica Typhimurium) effector protein SifA (Salmonella-induced filament protein A) that together regulate the Salmonella-containing vacuole (Fig. 1B). Open in a separate windowFigure 1.Model of PLEKHM1 function in the endocytic and autophagic pathways. (A) Domain structure of PLEKHM1 and their interactions. RUN (RUNDC3A/RPIP8, UNC-14 and RUSC1/NESCA); PH1 and PH2 (Pleckstrin homology domain 1 and 2); C1/Zinc finger (C1); HOPS (homotypic fusion and protein sorting). (B) Proposed positioning of PLEKHM1 and its associated complexes in the autophagy and endocytic pathway. PLEKHM1 localizes to late endosomes and lysosomes in an RAB7-dependent manner. The interaction between PLEKHM1, RAB7, and HOPS on vesicles positions these vesicles for tethering and fusion with autophagosomes, through direct interaction with MAP1LC3/GABARAP proteins. The autophagosomes may also fuse with late endosomes/MVBs (multivesicular bodies) in a PLEKHM1-RAB7-HOPS-dependent manner to produce amphisomes, prior to fusion with the lysosome. PLEKHM1-RAB7-HOPS can also be subverted by the Salmonella effector SifA, for the proper maintenance of the Salmonella-containing vacuole (SCV) and Sif (Salmonella-induced filament) formation. mAtg8s, MAP1LC3/GABARAP proteins.Using a 2-pronged approach, we identified PLEKHM1 as an interaction partner of RAB7 in its GTP-bound active state, RAB7(GTP), and MAP1LC3/GABARAP proteins. PLEKHM1 interacts directly with all MAP1LC3/GABARAP proteins through a highly conserved LC3-interaction motif (LIR) located between the Pleckstrin homology domain 1 (PH1) and PH2 domains of PLEKHM1 (Fig. 1A). Endogenous PLEKHM1 colocalizes with LAMP1 at the cytosolic-facing membrane, but not the lumenal side, of LC3B-containing amphisomes/autolysosomes, indicating that PLEKHM1 is an autophagy adaptor protein rather than a selective cargo receptor.Using SILAC (stable isotope labeling of cells in culture)-labeled inducible PLEKHM1 cells, we identified the HOPS complex as a significant interaction partner. The hexameric HOPS complex is an essential component of the late endocytic fusion machinery and is required for autolysosome formation. PLEKHM1 interacts directly with the HOPS complex, mediated by the RUN domain of PLEKHM1 and the C terminus of VPS39 (Fig. 1A) Crucially, PLEKHM1 forms an endogenous complex with HOPS. In the context of vesicle fusion, the HOPS complex acts as a tether to anchor and position the vesicles prior to fusion that is driven by SNARE proteins. Multiple SNARE proteins, such as VAMP7, VAMP8, VTI1B, SNAP29, and STX17 have been described to be required for autophagosome-lysosome fusion. Upon autophagy induction, enhanced PLEKHM1 coprecipitation is detected with the HOPS complex and the autophagosome specific SNARE STX17, reinforcing a role for PLEKHM1 in autophagosome-lysosome fusion.Both RAB7 and the HOPS complex are integral components of the endocytic pathway and, as such, we wanted to test the effect of PLEKHM1 loss on EGFR (epidermal growth factor receptor) degradation. We used 2 epithelial cell lines, HeLa and Hke3. In both instances, loss of PLEKHM1 causes a marked decrease in the rate of EGFR degradation and increases retention in early endosomes. This is in stark contrast to previous reports that used A549 cells and showed that a lack of PLEKHM1 accelerates EGFR degradation. Clearly, cell lines and their background mutations will have to be considered for future studies.In addition to the endocytic pathway, RAB7 and the HOPS complex are essential for the autophagosome-to-autolysosome transition. Therefore, we also wanted to explore this facet of PLEKHM1 function. We generated Plekhm1 KO MEFs to analyze the effects of autophagy flux in the absence of PLEKHM1. Plekhm1 KO MEFs show a block in autophagy, with the accumulation of SQSTM1/p62 and LC3B-II and, using tandem-fluorescence-LC3B as a marker, a decrease in autolysosome formation. Taken together, these findings suggest that PLEKHM1 functions at the point of autophagosome-lysosome fusion (Fig. 1B).Finally, we were interested in testing the functional role of PLEKHM1, and in particular the LIR, during selective autophagy of protein aggregates. We treated control and PLEKHM1-depleted cells with puromycin and observed aggregate clearance over time after puromycin removal. Cells lacking PLEKHM1 and those reconstituted with a PLEKHM1-LIR mutant were unable to efficiently remove SQSTM1-ubiquitin-positive aggregates, compared to control or PLEKHM1-wild type reconstituted cells, indicating an important role for the final stages of endosome and autophagosome maturation (Fig. 1B).“No man is an island, entire of itself” seems of particular prudence when considering the intertwined nature of both autophagic and endocytic pathways. Indeed, it is interesting that there are multiple RAB7 effector proteins functioning at the late endocytic step that also contribute to autophagy, including FYCO1, KIAA0226/Rubicon, UVRAG and now PLEKHM1, where only PLEKHM1 and UVRAG have been shown to interact with the HOPS complex. All of which, when mutated or depleted, have effects on both the endocytic and autophagic pathways. Clearly the roles of these proteins in cell-type and tissue-specific settings have to be determined before we fully comprehend the complexities of how the endosomal and autophagic pathways integrate and communicate with each other.     

A new species of Stenoloba Staudinger, 1892 from China (Lepidoptera,Noctuidae, Bryophilinae)

A new species of Stenoloba from the olivacea species group, Stenoloba solaris, sp. n. (Lepidoptera, Noctuidae), is described from Yunnan, China. Illustrations of the male holotype and its genitalia are provided. A diagnostic comparison is made with Stenoloba albistriata Kononenko & Ronkay, 2000, Stenoloba olivacea (Wileman, 1914), and Stenoloba benedeki Ronkay, 2001 (Fig. 4).Open in a separate windowFigures 1–5.Stenoloba spp. adults and biotope. 1 Stenoloba solaris, sp. n., male, holotypus, Yunnan, China (GBG/ZSM) 2 Stenoloba albistriata, male, paratypus, N. Vietnam (ZFMK) 3 Stenoloba olivacea, male, Taiwan (HNHM) 4 Stenoloba benedeki, male, paratypus, N. Vietnam (HNHM) 5 Type locality of Stenoloba solaris, sp. n. China, NW Yunnan, Lijiang/Zhongdian near Tuguancum, 27°29''700"N, 99°53''700"E.     

A photoreceptor going nowhere but the nucleus

Cryptochrome 2 (CRY2) is a blue/UV-A light receptor that regulates light inhibition of cell elongation and photoperiodic promotion of floral initiation in Arabidopsis. We and others have previously shown that CRY2 is a nuclear protein that regulates gene expression to affect plant development. We also showed that CRY2 is phosphorylated in response to blue light and the phosphorylated CRY2 is most likely active and degraded in blue light. Given that protein translation (and probably chromophore attachment) takes place in the cytosol and that a photoreceptor would absorb photon instantaneously, it would be interesting to know where those inter-connected events occur in the cell. Our results showed that freshly synthesized CRY2 photoreceptor is inactive in the cytosol although it may be photon-excited, it is imported into the nucleus where the photoreceptor is phosphorylated, performs its function, becomes ubiquitinated, and eventually gets degraded (Fig. 1).¹ To our knowledge, this is the first example in any organism that a photoreceptor is shown to complete its post-translational life cycle in a single subcellular compartment.Open in a separate windowFigure 1A model depicting the post-translational life cycle of CRY2. Pi, phosphate group; Ubq, ubiquitin.Key words: blue light, cryptochrome, ubiquitination, phosphorylation, Arabidopsis     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

Regulation and Function of Autophagy during Cell Survival and Cell Death

Autophagy is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. Autophagy promotes cell survival by elimination of damaged organelles and proteins aggregates, as well as by facilitating bioenergetic homeostasis. Although autophagy has been considered a cell survival mechanism, recent studies have shown that autophagy can promote cell death. The core mechanisms that control autophagy are conserved between yeast and humans, but animals also possess genes that regulate autophagy that are not present in yeast. These regulatory differences may be explained by the need to control autophagy in a cell context-specific manner in multicellular animals, such as during cell survival and cell death. Autophagy was thought to be a bulk cytoplasmic degradation mechanism, but recent studies have shown that specific cargo is recruited for degradation. This suggests the possibility that either cell survival or death may be regulated by selective autophagic clearance of cytoplasmic material. Here we summarize the mechanisms that regulate autophagy and how they may contribute to cell survival and death.Autophagy (self-eating) is an evolutionarily conserved catabolic process that is used to deliver cytoplasmic materials, including organelles and proteins, to the lysosome for degradation. Three types of autophagy have been described, including macroautophagy, microautophagy, and chaperone-mediated autophagy (Mizushima and Komatsu 2011). Although macroautophagy involves the fusion of the double membrane autophagosome and lysosomes, microautophagy is poorly understood and thought to involve direct uptake of material by the lysosome via a process that appears similar to pinocytosis. By contrast, chaperone-mediated autophagy is a biochemical mechanism to import proteins into the lysosome; it depends on a signature sequence and interaction with protein chaperones. Here we will focus on macroautophagy (hereafter called autophagy) because of our knowledge of this process in cell survival and cell death.Autophagy was likely first observed when electron microscopy was used to observe “dense bodies” containing mitochondria in mouse kidneys (Clark 1957). Five years later, it was reported that rat hepatocytes exposed to glucagon possessed membrane-bound vesicles that were rich in mitochondria and endoplasmic reticulum (Ashford and Porter 1962). Almost simultaneously, it was shown that these membrane-bound vesicles contained lysosomal hydrolases (Novikoff and Essner 1962). In 1965 de Duve coined the term “autophagy” (Klionsky 2008).The delivery of cytoplasmic material to the lysosome by autophagy involves membrane formation and fusion events (Fig. 1). First an isolation membrane, also known as a phagophore, must be initiated from a membrane source known as the phagophore assembly site (PAS). de Duve suggested that the smooth endoplasmic reticulum could be the source of autophagosome membrane (de Duve and Wattiaux 1966), and subsequent studies have supported this possibility (Dunn 1990; Axe et al. 2008). Although controversial, mitochondria and plasma membrane could also supply membranes for the formation of the autophagosomes under different conditions (Hailey et al. 2010; Ravikumar et al. 2010). The elongating isolation membrane surrounds cargo that is ultimately enclosed in the double membrane autophagosome. Once the autophagosome is formed, it fuses with lysosomes (known as the vacuole in yeasts and plants) to form autolysosomes in which the cargo is degraded by lysosomal hydrolases. At this stage lysosomes must reform so that subsequent autophagy may occur (Yu et al. 2010).Open in a separate windowFigure 1.Macroautophagy (autophagy) delivers cytoplasmic cargo to lysosomes for degradation, and involves membrane formation and fusion. The isolation membrane is initiated from a membrane source known as the from the phagophore assembly site (PAS). The isolation membrane surrounds cargo, including organelles and proteins, to form a double membrane autophagosome. Autophagosomes fuse with lysosomes to form autolysosomes in which the cargo is degraded by lysosomal hydrolases.