text:
A Model for p38MAPK-Induced Astrocyte Senescence

Experimental evidence indicates that aging leads to accumulation of senescent cells in tissues and they develop a secretory phenotype (also known as SASP, for senescence-associated secretory phenotype) that can contribute to chronic inflammation and diseases. Recent results have showed that markers of senescence in astrocytes from aged brains are increased in brains with Alzheimer’s disease. These studies strongly involved the stress kinase p38MAPK in the regulation of the secretory phenotype of astrocytes, yet the molecular mechanisms underlying the onset of senescence and SASP activation remain unclear. In this work, we propose a discrete logical model for astrocyte senescence determined by the level of DNA damage (reparable or irreparable DNA strand breaks) where the kinase p38MAPK plays a central role in the regulation of senescence and SASP. The model produces four alternative stable states: proliferation, transient cycle arrest, apoptosis and senescence (and SASP) computed from its inputs representing DNA damages. Perturbations of the model were performed through gene gain or loss of functions and compared with results concerning cultures of normal and mutant astrocytes showing agreement in most cases. Moreover, the model allows some predictions that remain to be tested experimentally.

Introduction
Cellular senescence is an anti-tumor program that is triggered by different insults like telomere shortening, oxidative stress and oncogene activation [1][2][3]. Experimental evidences support that senescent cells accumulate in aging mammal tissues and have an altered phenotype, called SASP (senescence-associated secretory phenotype), that apparently contributes to several aging diseases including Alzheimer's disease (AD) [3][4][5][6]. SASP contributes to 'inflamm-aging' (the development of a systemic proinflammatory status with normal aging) which involves an increase of blood plasma levels of inflammatory cytokines like interleukin 6 (IL-6) [7]. AD is an example of inflammaging disease, other cases are atherosclerosis, osteoporosis and diabetes [7]. In the case of AD, astrocyte senescence is claimed to be an important contributor to the development of the pathology [5]. Astrocytes are the most numerous cell type in the human brain and are involved in many essential physiological functions that keep the brain homeostasis, among them the clearance of the Amyloid-β peptide that accumulates in brains with AD [5]. Astrocytes are sensitive to oxidative stress (caused by reactive oxygen species or ROS) which increases with aging and causes DNA damage [8]. The question of whether astrocyte senescence contributes to age-related dementia was recently addressed by Bhat and coworkers who proposed that it is an age-related risk factor for AD [9]. The authors observed in vitro that under oxidative stress, astrocytes of brains from patients with AD expressed more senescence and SASP markers than brains from healthy, aged individuals. The chief markers observed include secretion of β-galactosidase, expression of cyclin-dependent kinase inhibitor 2A (p16 INK4a ) and senescence-associated heterochromatin foci [5,10]. The authors verified that astrocytes exposed to Amyloid-β peptides triggered a senescence response and produced high quantities of interleukin 6 (IL-6), a mediator of chronic inflammation that is increased in the central nervous system of AD individuals [5]. In addition, Bath et al. observed a strong expression correlation between IL-6 and the mitogen activated protein kinase 14 (p38MAPK) that is an important regulator of cell cycle checkpoints [11,12]. IL-6 in pre-senescent and senescent astrocytes could be abolished by drug inhibition of p38MAPK [9].
These experimental results suggest that astrocyte senescence is strongly connected to p38MAPK activation. However, the exact molecular mechanisms that drive astrocytes into senescence remain obscure [5]. p38MAPK can induce checkpoint arrest and its overexpression induces senescence in fibroblasts which are cells that share functional similarities with astrocytes [5,13]. Based on a previous, specific model of senescence onset at G1/S checkpoint [12], in this work we propose that p38MAPK induction can explain astrocyte senescence and SASP and we propose an extended logical model of the process integrating checkpoints G1/S and G2/M [14] as both have similar mechanisms of checkpoint activation by p38MAPK upon DNA damage [11,15]. The model corroborates several experimental findings and make some predictions.
In what follows we describe the organization of the paper. The logical modeling method is described in the next section. Then after an overview of general molecular mechanisms of checkpoint and cell fate decisions, our model is defined and studied in the Results section. The Discussion section summarizes the implications of this work and indicates future work.

Methods
Logical models were used to study cell cycle control [16] and cell fate decisions [17], for a review see [14]. A logical model [18][19][20] is defined by a directed regulatory graph where discrete variables are associated with the nodes and logical rules determine the evolution of these variables. Nodes in this type of graph symbolize molecular components as genes and/or proteins, biological processes (for example, a pathway) or phenomenological events (e.g. apoptosis, senescence etc.). Edges represent activatory or inhibitory effects and variables denote activity levels with two or more states (multi-valued). In most cases the variables are Boolean (0 or 1), but multi-valued variables can represent different influences of a node affecting its targets.
The evolution of the level of each component is defined by a logical rule subjected to the regulators of this component. Input components are not regulated and symbolize extrinsic constant conditions. The dynamics of logical models can be characterized in terms of state transition graphs, where the states are nodes comprising the level of each component in the model and the edges, connecting the nodes, represent state transitions resulting from the logical rules that change the levels of the model components. End nodes in state transition graphs correspond to attractors that can be a stable state (which has no successor state) or a cycle.
The logical framework allows the consideration of diverse molecular processes associated with different time scales in a unique model as it happens with transcriptional regulation and protein phosphorylation [16]. In addition, the logical method permits analysis of perturbations consisting in retaining a variable to its lowest levels, known as loss of function experiment (LoF), or to its positive levels, known as gain of function experiment (GoF).
This framework is implemented in the tool GINsim (http://ginsim.org), which permits different types of analysis of logical models including the determination of stable states [14].

Results
Cell fate decisions between apoptosis or senescence upon DNA damage occur at cell cycle checkpoints [21]. In what follows, we give an overview of the molecular processes responsible for the induction of cell cycle checkpoints as a result of DNA damage. These responses constitute the focus of the logical regulatory model of Fig 1. Then, we describe our proposal for the mechanisms involved in the regulation of astrocyte senescence and SASP upon checkpoint induction. In a previous work, we introduced a model for the role of p38MAPK on the onset of senescence limited to the G1/S checkpoint [12]. Here, we enlarge this model including the mechanisms activation of the checkpoint G2/M to build a unified framework of checkpoint activation in which p38MAPK regulates the senescence fate [11]. Checkpoint regulation and apoptosis (Fig 1) DNA damage activates checkpoints arresting cell cycle progression for a transient arrest for DNA repair or, if the damage is irreparable, a decision is taken between apoptosis or senescence [21,22]. Arrest of the cell cycle can be triggered at G1/S and G2/M checkpoints which have similar molecular mechanisms, in particular, the inhibition of cell division cycle 25 protein family (CDC25A/B/C) required for cell cycle, occurs at both checkpoints.
DNA double-strand breaks activate the kinase ataxia telangiectasia mutated (ATM), either DNA single-strand breaks (SSB) or DSB activate Rad3-related (ATR). Phosphorylations downstream ATM and ATR lead to activation of p53 [22,23]. The cascade phosphorylations triggered by ATM and ATR is shown in Fig 1 [15,21]. The kinase checkpoint kinase 2 (CHEK2) is phosphorylated by ATM while the kinase checkpoint kinase 1 (CHEK1) is phosphorylated by ATR. CHEK2 and CHEK1 start the arrest upregulating Wee1 G2 checkpoint kinase (Wee1) and inactivating CDC25A/B/C required for both checkpoints to activate protein complexes involving cyclins and cyclin-dependent kinases (CDKs) that determine cell cycle progress [15,21]. These complexes are cyclin-dependent kinase 4, 6 and cyclin D (Cdk4/6-Cyclin-D) complex, cyclin-dependent kinase 2 and cyclin E (Cdk2/Cyclin-E) complex for checkpoint G1/ S, and cyclin-dependent kinase 1 and cyclin B (Cdk1/Cyclin B) complex (which is inhibited by Wee1) for checkpoint G2/M [21]. In addition, phosphorylated p53 mediates the maintenance of arrest through the activation of cyclin-dependent kinase inhibitor 1A (p21), which also inhibits Cdk4/6-Cyclin-D [24,25]. In the case of checkpoint G1/S, the inhibition of these complexes prevents the phosphorylation of retinoblastoma 1 protein (pRB) and the release of E2F transcription factors that induce the expression of genes required for the cell to enter the S phase [21,26]. In the case of reparable damage, the complexes are reactivated driving the cell to the next phase of the cycle.
The choice between cycle arrest and apoptosis occurs through a threshold mechanism dependent on the activation level of p53 that, when exceeded, triggers apoptosis [28]. Owing to this, in our model, apoptosis is activated only when p53 reaches its highest level which is a strong simplification. p14ARF (the alternate reading frame product) and cyclin-dependent kinase inhibitor 2A (p16 INK4a ) contribute to cell cycle regulation and senescence [6,27], deletion of the locus (CDKN2A) that produces these two proteins enhances astrocyte proliferation [29].
Astrocyte senescence, p38MAPK and SASP (Fig 1) Experimental results strongly suggest that astrocyte senescence in AD is entangled with the activation of the kinase p38MAPK [9] which, when overexpressed, induces senescence in fibroblasts [5,13,30]. The p38 MAPK family of proteins in which p38α has a prominent role is activated in a ATM/ATR dependent manner by cellular stresses induced, for example, by ROS [8], and it also seems to regulate the secretion of IL-6 in senescent astrocytes [5,9]. IL-6 plays a central role in SASP and inflammaging diseases [3,7].
DNA damage can induce a checkpoint arrest through p38MAPK upon joint mechanisms like: upregulation of p16 INK4a and p14ARF, inhibition of the protein family Cdc25A/B/C and phosphorylation of p53 which, additionally, can lead to apoptosis [11,15,31,32].
Senescence requires the activation of p53-p21 and p16 INK4a -pRB pathways in different cell types. p16 INK4a contributes along with p53 to block proliferation as it inhibits cyclin-dependent kinases [6,33,34]. The molecular mechanisms of regulation of p16 INK4a (and p14ARF) are not completely understood, but p38MAPK affects the expression of CDKN2A locus [35,36].

Logical model for astrocyte fate
Based on the biological facts mentioned above, we define a logical model for astrocyte cell cycle checkpoint regulation and fate. The main hypothesis underlying the model is as follows: In astrocytes senescence activation by p38MAPK upon DNA damage utilizes similar mechanisms for checkpoints G1/S and G2/M. Tables 1 and 2 include a brief description of the model nodes and of the logical rules governing the states of the nodes, respectively. The logical rules were built based on our interpretation of the biological information, the process also involves several manual rounds of consistency analysis between model predictions and experimental knowledge. The interactions among the nodes in Fig 1 are reported in the literature and detailed bibliographic information can be found in S2 Dataset. Only direct interactions are considered.
The input nodes of the network, SSB and DSB, can assume three values denoting DNA damage levels: absence of damage = 0, reparable damage = 1 and irreparable damage = 2. SSB and DSB values define ATR and ATM levels, respectively. ATM and ATR activate CHEK2, CHEK1, p38MAPK, Wee1 and p53. DSB can activate CHEK1 through ATM. p53 and p38MAPK are multi-valued and have 3 and 4 levels, respectively, they strongly affect fate decisions. Reparable damage induces p53 to its middle level (p53 = 1) which is involved in several fates. When p53 reaches its highest value 2, apoptosis is triggered but it only occurs for highest DNA damage, i.e. DSB = SSB = 2 [28]. p38MAPK activation has a stronger influence from ATM than ATR and is controlled in the following way: to reach its first positive level (1) it requires activation of ATR, or ATM but not at its highest level [11]. p38MAPK reaches its level (2) when ATR is not at its maximum level but ATM is. p38MAPK reaches its highest level (3) only when ATM and ATR are both at their maximum levels.
Absence of DNA damage implies the activation of nodes CdkCyclin (representing the protein complexes that promote cell cycle for both checkpoints G1/S and G2/M) and 'proliferation'. The 'cycle_arrest' node represents an arrest for repair and it is inhibited by CdkCyclin and E2F. The p16 INK4a -pRB and p53-p21 pathways in astrocytes seem to have redundant function in promoting inhibition of proteins involved in cell cycle progression [37]. Hence, we defined the activation of node 'senescence' to require the activation of both, p21 and p16 INK4a , inactivation of Cdc25ABC and p53 < 2. Nevertheless, if Cdc25ABC is active, senescence can be activated if p16 INK4a = 2. SASP requires activation of p38MAPK and senescence [6,9]. Cdc25ABC has 3 levels and can be inactivated only in presence of CHEK1, CHEK2 and p38MAPK [32,38].  In what follows we analyze the model predictions in terms of stable states for the wild-type situation and some selected in silico mutations.

Model results: wild type case
This model presents deterministic behavior since each combination of the levels of the input nodes DSB and SSB (nine in total) leads to a unique stable state (see Fig 2) characterized by the activation or deactivation of the nodes representing fates. The synopsis of the results for the wild type case is that no DNA damage (input: DSB = SSB = 0) leads to proliferation, as expected, and the main active components are those involved in cell cycle progression. The highest level of irreparable damage DSB = SSB = 2 yields apoptosis, irreparable DSBs lead to senescence (and SASP) and reparable DSBs result in transient cycle arrest indifferent to the value of SSBs. 'cycle_arrest' requires the inhibition of CdkCyclins and it accompanies 'senescence' and 'apoptosis' which are selected according to the level of DNA damage. DSB = 2 and SSB<2 yield 'senescence' which is compatible with the fact that irreparable SSBs seem not able to induce senescence [6,23]. Lastly, 'apoptosis' is activated by p53 = 2 when SSB = DSB = 2.

Comparison of in silico mutations with experiments
The model can be perturbed through in silico mutations corresponding to loss of function or gain of function experiments. These model perturbations affect the stable states with respect to the wild type case and changes can be related to the predominant growth trend observed in cultures of astrocyte cells undergone LoF or GoF experiments involving one or more genes.
Comparisons of LoF and GoF experiments with model perturbations can be classified in two cases according to DNA damage: (i) absence or (ii) presence of damage that activates checkpoints. DNA damage is assumed to happen in stress situations as seems to be the case of astrocytes in aged brains or in AD and also induced in cultures by ROS, ionizing radiation and other methods.
Only a few experiments with astrocytes were found in the literature and they use primarily mice cells. Given to this lack of information, we report also some interesting perturbations using human or mouse fibroblasts [5,12,13]. Although they will not necessarily yield correct results for astrocytes, they can be considered as model predictions and remain to be verified. Table 3 presents comparisons between model outcomes and LoF and GoF experiments for mutant astrocytes. The full set of stable states of each perturbation is listed in S1 Dataset. In what follows we comment the comparisons in Table 3.
Pharmacological inhibition of p38MAPK in pre-senescent and senescent human astrocytes prevents SASP [9], while the effect of its GoF is yet unknown. However, in human and mouse fibroblasts p38MAPK GoF induces senescence [30]. According to our model p38MAPK LoF abrogates apoptosis, senescence and SASP, while its GoF, with p38MAPK maintained between states 1 and 2, abrogates proliferation and enhances senescence and SASP compatible with fibroblasts experiments. If p38MAPK is maintained at its maximum state 3, then an apoptotic phenotype prevails.
CDKN2A-null mice astrocytes have high proliferation and are used as a model for glioblastoma development [29]. Using the model, this perturbation corresponds to p16INK4a and p14ARF LoF which yields abrogation of senescence and apoptosis which we interpret as compatible with the observed increase of proliferation in experiments. Given to the importance of p16INK4a in senescence, we studied its perturbations. The model predicts enhanced senescence for GoF of p16INK4a maintained at its state 2 and loss of senescence for its LoF, compatible with observations in other cell types [4].
p53-null mice astrocytes present increased proliferation in culture with small sensitivity to ionizing radiation [39]. The simulation of p53 LoF in absence of DNA damage yields proliferation, in presence of DNA damage it abrogates senescence and apoptosis which should contribute to increased proliferation observed in experiments. GoF of p53 maintained at its state 1 enhances senescence.
ATM-null mice astrocytes show a slight decrease in proliferation in presence or absence of ionizing radiation [39,40]. ATM LoF in the model implies increased proliferation in absence or presence of DNA damage contrasting with experiments. Actually, this is a surprising experimental behavior, in other cell types ATM LoF contributes to senescence suppression [41], however for astrocytes this perturbation seems to have a different effect probably because ATM has some other additional important function in astrocytes that we ignore and that is not contemplated by the present model.
Deletion of CDKN2A and simultaneous overexpression of CDK4 in mice astrocytes generates high proliferating immortal cells and is also studied as a model for glioblastoma development [42]. We simulated a related perturbation with the model by combining the LoF of both p16INK4a and p14ARF with the GoF of CdkCyclin (last line in Table 3). The result is a single output: proliferation, which strongly agrees with the model. Double mutant ATR;p53-null mice astrocytes show increased proliferation in relation to wild type cultures [43]. For the simulation, the LoF of both ATR and p53 yields proliferation in absence of damage and abrogates senescence and apoptosis compatible with an increase in proliferation.
In what follows we refer to model predictions based on experiments with human or mice fibroblasts, some results are similar to those obtained with our previous model [12]. p21 ectopic expression decreases proliferation and induces senescence in human and mouse fibroblasts [44,45]. For the model, p21 GoF abrogates proliferation in absence of damage agreeing with experiments and its LoF predicts abrogation of senescence.
CDC25ABC LoF and GoF respectively induce or prevent checkpoint arrest in mice fibroblasts [46]. For CDC25ABC LoF, the model enhances senescence and for GoF abrogates senescence agreeing partially with experiments [46].
Human fibroblasts do not proliferate without E2F which agrees with the model that indicates decrease of proliferation with E2F LoF [47]. E2F GoF in human fibroblasts induces apoptosis, however the model only shows this result in presence of DNA damage [47].
pRB-null mice fibroblasts present increased apoptosis [48,49], a phenotype recovered by our model only for the highest damage case. For pRB GoF the model predicts decrease of proliferation in absence of DNA damage.

Conclusion
Recent experiments suggest that astrocyte senescence (and SASP) is an important component of Alzheimer´s disease [5,9,10]. Motivated by these experiments, in this paper we presented an original model for astrocyte cell fate where p38MAPK plays a central role in the explanation of senescence and SASP induction due to DNA damage [5]. The in silico perturbations of the model are consistent with the available experimental data. The model predictions remain to be tested experimentally and one, in particular, the confirmation that p38MAPK GoF in astrocytes induces senescence, if confirmed, would be a strong support for the model [30].


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

Background: The Vps4 ATPase powers the endosomal sorting complexes required for transport (ESCRT) pathway. Results: Peptide binding to hexameric Vps4 is promoted by nucleotides that can mimic ADP, ATP, and the transition state. Conclusion: ESCRT-III substrates bind Vps4 MIT domains and then bind the central pore of an asymmetric, nucleotide-bound Vps4 hexamer. Significance: Mechanistic understanding of Vps4-substrate interactions is advanced by this work. The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.
Eukaryotic Vps4 enzymes comprise the N-terminal MIT domain, an ϳ40-residue linker, a two-domain AAA ATPase cassette, a "␤-domain" that is inserted within the small domain of the ATPase cassette, and a C-terminal helix that binds against the large ATPase domain. Vps4 functions as a higherorder oligomer. Although the subunit stoichiometry and structure have been controversial (33)(34)(35)(36), we have shown that the active Vps4 enzyme is a hexamer (37), like all other well characterized Type I AAA ATPases (38,39). Our working structural model for the Vps4 hexamer is based on superposition of the known crystal structure of the Vps4 ATPase cassette (33, 37, 40 -43) onto the structure of the p97 D1 hexamer (44) and is supported by mutational analysis of proposed hexameric interface residues (37,40,41). This model places the Vps4 ␤-domains on the periphery, where they can bind the VSL domain of the Vta1p/LIP5 cofactor that promotes Vps4 assembly and stimulates ATPase activity (32,40,(45)(46)(47)(48)(49). The Vps4 hexamer has a central pore that is lined by two conserved loops: pore loop 1 and pore loop 2. Vps4 pore loop 1 displays an aromatic hydrophobic dipeptide that is conserved across AAA ATPases that have polypeptide substrates (40,41), and pore loop 2 contains a series of charged residues that are conserved and functionally important in the related ATPase spastin (50,51). Mutations in either Vps4 pore loop inhibit HIV budding (40,41), although these mutations may also destabilize the Vps4 hexamer to some extent.
Hanson and colleagues (52) have reported that overexpressed human ESCRT-III subunits CHMP2A and CHMP1B that lack their terminal MIM elements nevertheless co-sediment with human VPS4B(E235Q) from cell lysate. This interaction requires ESCRT-III helix 5 and surrounding loops, and this same region also contributes to stimulating VPS4A ATP hydrolysis when it is present within C-terminal fragments of ESCRT-III proteins (53). These observations suggest that the ESCRT-III helix 5 region may bind preferentially to the pore of the Vps4 hexamer. To investigate this idea further, we characterized the Vps4-ESCRT-III interaction using purified recombinant Saccharomyces cerevisiae proteins Vps4p and Vps2p (CHMP2 homolog). We found that the AAA ATPase cassette of a Vps4p construct that lacked the MIT domain formed a stable hexamer with the flexible nucleotide mimics ADP⅐AlF x or ADP⅐BeF x , and that this hexameric complex bound a single Vps2p helix 5 peptide in an interaction that was mediated by the Vps4p pore loop residues. Vps2p helix 5 binding was autoinhibited by the MIT domains, and this inhibition was alleviated by association of the MIT domain with an MIM1 or an MIM2 sequence. These observations, together with previous studies (21,(52)(53)(54), support a model in which ESCRT-III complexes are disassembled by pulling ESCRT-III helix 5 into the central pore of asymmetric Vps4p hexamers in a manner that is initially primed and activated by binding of MIT domains to ESCRT-III MIM sequences.

Experimental Procedures
Proteins and Peptides-Vps4p and the VSL domain of Vta1p (Vta1p VSL , residues 280 -330) were expressed and purified as described (35,37,40,41). Bacterial expression vectors are listed in Table 1. Mutations were generated by QuikChange mutagenesis and verified by DNA sequencing. Peptides were synthesized on a Prelude peptide synthesizer (Protein Technologies, Inc.) using standard Fmoc chemistry (55). Following cleavage from resin, the peptides were precipitated with icecold ether, washed thoroughly with ether, dissolved in water/ acetonitrile, and lyophilized for long-term storage. 5(6)-Carboxyfluorescein (Acros Organics) was used to fluorescently label the peptides.
For N-terminally labeled peptides, the fluorescein group was coupled to the N-terminal ␣-amine by standard coupling conditions. All other amines within the peptide were t-butyloxycarbonyl-protected. For C-terminally labeled peptides, an orthogonal lysine deprotection method was used. At the first position of the peptide (C terminus), Dde-Lys(Fmoc)-OH (AAPPTec) was coupled to the resin. Fmoc removal was performed with 20% piperidine, followed by fluorescein coupling to the ⑀-amine on the lysine side chain. The Dde group (on the lysine ␣-amine) was then removed with 3% hydrazine in N,Ndimethylformamide, and the remaining peptide was synthesized using standard protocols. Unlabeled N termini were blocked with an acetyl group, and unlabeled C termini were amides. Peptide quality was verified by reversed phase C18 HPLC. For experiments described below, peptide concentrations were determined by infrared spectroscopy using a Direct Detect spectrometer (EMS Millipore). The identities of all peptides and recombinant proteins were verified by mass spectrometry.
Analytical Size-exclusion Chromatography-A Superdex 200 column was equilibrated in running buffer (10 mM MgCl 2 , 100 mM NaCl, 20 mM HEPES, pH 7.0, and when appropriate, 3 mM nucleotide). To prepare ADP metal fluoride stock solutions, 1 M NaF was added dropwise to a 10 mM solution of ADP to a final concentration of 62.5 mM with stirring, and then 1 M AlCl 3 or BeCl 2 was slowly added to a final concentration of 12.5 mM. Vps4p proteins (200 or 50 M in subunit concentration, corre- sponding to 33 or 8.3 M hexamer, respectively) were preincubated with a 2-fold molar (subunit) excess of Vta1p VSL in running buffer at 4°C for at least 2 h. The column was calibrated using protein standards (Bio-Rad).
Fluorescence Anisotropy Binding Assays-To generate the data shown in Figs. 1B, 3A, 4B, and 6A, a dilution series of Vps4p-Vta1p VSL complex (Vta1p VSL was in 2-fold excess over Vps4p subunits, total volume 60 l) in binding buffer (10 mM MgCl 2 , 100 mM NaCl, 20 mM HEPES, pH 7.0, and where indicated, 3 mM nucleotide) was incubated with fluorescein-labeled peptides (1 nM) at 25°C for at least 3 h before measuring parallel and perpendicular fluorescence intensity using excitation/ emission wavelengths of 485/535 nm on a Tecan Infinite 200 microplate reader. Anisotropy values were calculated and plotted against the Vps4p hexamer concentration. Dissociation constants (K D values) were estimated by fitting the data to the where FA is the normalized fluorescence anisotropy and corresponds to "fraction bound," using GraphPad Prism 5 (GraphPad Software, Inc.). Error bars show the S.D. from three independent experiments and are shown for every data point in the figures.
To generate the data shown in Fig. 2, fluorescence anisotropy was measured during titration of Vps4p ⌬MIT -Vta1p VSL hexamer into 40 M peptide C in the binding buffer (above). Fraction bound was plotted versus the [Vps4p ⌬MIT hexamer]/[peptide] ratio. At these high peptide concentrations (17-fold over K D ), the stoichiometry of peptide binding to the Vps4p ⌬MIT hexamer can be estimated by the ratio at which saturation binding was reached. Data are shown for three independent experiments performed with protein from independent preparations.
GST Pulldown Assays-An N-terminal GST fusion of Vps2p residues 165-201 was used for pulldown assays. The fusion protein was expressed from a pET151-D-Topo vector with an N-terminal tobacco etch virus protease-cleavable His 6 tag and purified using the procedure described above for Vps4p proteins. Purified Vps4p at the indicated subunit concentration, Vta1p VSL (2-fold excess over Vps4p), and GST fusion proteins (1 M) were mixed in wash buffer (10 mM MgCl 2 , 100 mM NaCl, 20 mM HEPES, pH 7.0, with or without 3 mM ADP⅐AlF x ) and incubated for 3 h at 4°C. 20 l of glutathione agarose (Amersham Biosciences) was added to 1 ml of protein samples and incubated for 1 h at 4°C. Unbound proteins were removed by washing with wash buffer (four washes with 1 ml of buffer). The glutathione resin was subsequently resuspended in SDS-PAGE loading buffer and incubated for 5 min at 95°C, and bound proteins were visualized by SDS-PAGE.
ATPase Assay-To generate the data shown in Fig. 5A, Vps4p ⌬MIT (final concentration 0.3 M subunits, corresponding to 50 nM hexamer) was mixed with Vta1p VSL (2-fold excess over Vps4p subunits) and 2 mM ATP in reaction buffer (100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, and 20 mM HEPES, pH 7.5), together with the indicated concentrations of Vps2p peptide C. After 10 min, 2 volumes of malachite green solution (14 mM ammonium molybdate, 1.3 M HCl, 1.5 mM malachite green) were added, followed by 21% citric acid (same volume as the malachite green solution). Absorbance at 650 nm was mea-sured using a POLARstar OPTIMA (BMG Labtech) plate reader, and phosphate concentrations were calculated using a sodium phosphate standard curve. To generate the data shown in Fig. 4C, Vps4p ⌬MIT and its pore loop 2 mutants (final concentration 0.5 M subunits) were mixed with Vta1p VSL (2-fold excess over Vps4p subunits), and their ATPase activity was measured as described above.

Results
Vps2p Peptides Bind the ATPase Cassette of an Asymmetric Vps4p Hexamer-Quantitative fluorescence anisotropy assays were performed to test whether peptides that spanned the C-terminal end of Vps2p helix 5 (Fig. 1A) bound a pure recombinant protein complex comprising a Vps4p construct that lacked the N-terminal MIT domain (Vps4p ⌬MIT ; residues 81-437) and the Vta1p VSL domain (Vta1p VSL , residues 280 -330). Tight peptide binding (K D ϭ 2-10 M) was observed in the presence of ADP⅐AlF x or ADP⅐BeF x , but not in the presence of ATP, ADP, AMPPNP, or ATP␥S (Fig. 1B). Binding specificity was validated by the observation that a control peptide corresponding to the downstream Vps2p MIM1 motif (residues 218 -232) bound 15-fold less tightly than Vps2p helix 5 peptides A and C, and by the demonstration that different versions of peptide A that were fluorescently labeled at the N or the C terminus bound equivalently (within 2-fold, data not shown).
Peptide binding affinities to the Vps4p ⌬MIT -Vta1p VSL complex in the presence of ADP⅐AlF x were calculated assuming that Vps4p ⌬MIT is present as a hexamer, and that one Vps4p hexamer binds one peptide (see below). Peptides A (residues 165-201; K D ϭ 2.5 Ϯ 0.5 M) and C (residues 165-184; K D ϭ 2.4 Ϯ 0.5 M) bound modestly more tightly than peptide B (residues 144 -184; K D ϭ 6.1 Ϯ 2.4 M) (Fig. 1B), indicating that the minimal peptide C construct contains the primary binding site, and suggesting that the patch of highly acidic residues within the N-terminal extension of peptide B may weaken the binding slightly.
The unique ability of ADP⅐AlF x and ADP⅐BeF x to support binding likely reflects the ability of these nucleotide analogs to mimic multiple states, including ATP, ADP, and transition state-bound nucleotide complexes (56 -59), and suggests that different subunits within each Vps4p hexamer may need to bind different nucleotide states and adopt different conformations to generate a stable, asymmetric substrate-binding site. Consistent with this idea, hexameric Vps4p ⌬MIT -Vta1p VSL complexes were more stable in the presence of ADP⅐AlF x or ADP⅐BeF x than in the presence of ATP, ADP, or ATP␥S, or in the absence of nucleotide, as shown by size-exclusion chromatography (note the relative mobilities of the different complexes at 50 M Vps4p ⌬MIT subunit concentrations, corresponding to 8.3 M hexamer concentrations, Fig. 1C). Thus, Vps4p ⌬MIT is hexameric in the concentration range used for the fluorescence anisotropy binding assay in the presence of ADP⅐AlF x or ADP⅐BeF x , and the fluorescence anisotropy signal can be used as a readout to quantify peptide binding, especially since substrate binding further stabilizes hexamer formation (see below).
The Vps4p Hexamer Binds a Single Peptide-To verify the stoichiometry of binding, we performed a fluorescence anisot-ropy binding experiment in which Vps4p ⌬MIT -Vta1p VSL hexamers in the presence of ADP⅐AlF x were titrated into a solution of fluorescently labeled peptide C at high concentration (ϳ20fold above the K D ). Peptide binding saturated when the stoichiometry was close to one peptide per Vps4p hexamer (Fig. 2), supporting the idea that each asymmetric Vps4p hexamer contains a single binding site for ESCRT-III helix 5.
Pore Loop Residues Bind Directly to Vps2p-derived Peptides-To determine whether residues of the Vps4p hexamer pore contribute to binding, we tested binding to pore loop mutants that have been previously shown to inhibit HIV budding when equivalent mutations are present in human Vps4 homologs (40,41). These correspond to W206A (pore loop 1), R241A (pore loop 2), and R251A (adjacent to pore loop 2) of S. cerevisiae Vps4p (40,41). None of these mutants bound peptides with appreciable affinity (Fig. 3A). However, gel filtration experiments, performed at 100 M Vps4p ⌬MIT subunit concentration (17 M hexamer) for a more sensitive readout of hexamer dissociation, indicated that the hexamer stability of these mutants was reduced (Fig. 3B).
To identify pore loop mutations that do not destabilize the Vps4p hexamer, we substituted 10 different pore loop residues individually with alanines and assayed oligomerization by analytical size-exclusion chromatography at a Vps4p subunit concentration of 50 M (8.3 M hexamer). At this concentration, the wild-type Vps4p ⌬MIT protein elutes as a stable hexamer in  2. One Vps4p ⌬MIT hexamer binds one Vps2p helix 5 peptide. Fluorescence anisotropy was measured during titration of 40 M (ϳ20-fold over K D ) peptide C with the Vps4p ⌬MIT -Vta1p VSL hexamer. Saturation of the signal occurred when the stoichiometry of the complex was close to 1:1. Data from three independent experiments are shown here, and the indicated stoichiometry represents the ratio of Vps4p ⌬MIT -Vta1p VSL hexamer to peptide C at saturation when all data sets were fit globally. MAY (Figs. 1C and 4A). Three of the Vps4p substitution mutants (T240A, E243A, and E247A) displayed essentially unchanged oligomerization, even at low protein concentrations (Fig. 4A). These pore loop alanine substitution mutants were therefore assayed for peptide binding. Remarkably, introducing E243A or E247A point mutations increased the affinity of peptide C binding (by 3-and 4-fold, respectively, Fig. 4B). Moreover, the double mutant, Vps4p ⌬MIT (E243A, E247A), bound peptides 4-fold (peptide C) or 10-fold (peptide B, not shown) more tightly than wild-type Vps4p (Fig. 4B, lower  panel). Because different alanine pore loop substitutions can either increase or decrease peptide binding affinity (Fig. 4B) without substantially changing Vps4p hexamerization, we con-

Vps4-Substrate Interactions
clude that substrates bind the pore loops directly. This conclusion is reinforced by the observation that the double mutation, which gives the tightest binding, actually reduces hexamerization slightly (Fig. 4A, lower panel), thereby emphasizing that the mutations uncouple reduction of binding from reduction of hexamerization. Similar to the effect of removing acidic pore loop residues, we also find that introducing a positive charge (T240K) enhances peptide binding (Fig. 4B, top panel) without affecting oligomerization (Fig. 4A, top panel), whereas the T240F mutation has no effect.
We also tested the effects of the pore loop 2 glutamate mutations on the basal ATPase activity of Vps4p ⌬MIT . As shown in Fig. 4C, both the E243A and the E247A mutations reduced ATPase activity, but the double mutation did not further reduce ATPase activity (p value between E243A and the double mutant is 0.17). Although hexamerization was not impaired when assayed by gel filtration, a possible explanation is that reduced ATPase activity reflects reduced hexamerization at the concentration used to assay ATP hydrolysis (0.3 M versus 50 M subunit concentrations), whereas the increased peptide binding reflects improved interactions between the pore loop residues and the substrate. Alternatively, the reduced activity of these mutants may reflect allosteric coupling of the enzymatic active site to the central peptide-binding site (and altered coupling in the mutants).
Peptide Binding Stimulates ATPase Activity and Stabilizes Hexamerization-A previous study showed that VPS4A ATPase activity is stimulated by C-terminal ESCRT-III constructs that span the MIM motif and helix 5 (53). Having mapped relatively high affinity binding to the helix 5 region of Vps2p, we tested whether this pore-binding element could also stimulate Vps4p ⌬MIT ATPase activity. As shown in Fig. 5A, the addition of peptide C stimulated the Vps4p ⌬MIT ATPase activity by 2.5-fold at the highest concentration tested. To test the possibility that this effect resulted from increased stabilization of the Vps4p hexamer in the presence of peptide C, we characterized the oligomerization of Vps4p ⌬MIT in the presence of Vta1p VSL and ADP⅐AlF x and in the presence or absence of peptide C. Analytical gel filtration was performed at (low) Vps4p concentrations where the hexameric complex was appreciably dissociated (10 M Vps4p ⌬MIT subunits, 1.7 M hexamer). Under these conditions, Vps4p ⌬MIT eluted as a hexamer in the presence but not the absence of peptide C (Fig. 5B). Thus, peptide binding stabilizes the Vps4p hexamer. We note that ATPase activity was assayed at a concentration where inactive monomers and dimers of Vps4p ⌬MIT likely predominated over active hexamers, and the observed peptide stimulation of ATPase activity is therefore likely due, at least in part, to substrate-mediated stabilization of the Vps4p ⌬MIT hexamer.
The MIT Domain Inhibits Substrate Binding in the Absence of an MIM Interaction-To determine whether the MIT domains influence substrate binding to the Vps4p pore, we compared binding of the A, B, and C peptides to various Vps4p constructs (Fig. 6B) and found that full-length Vps4p binds each of these peptides with ϳ10-fold lower affinity than does Vps4p ⌬MIT (Fig. 6, A and C). To determine whether this inhibitory effect of the MIT domain could be alleviated by MIM binding, we designed a Vps4p variant (MIM1-Vps4p) that included an N-terminal extension of the Vps2p MIM1 motif (residues 218 -232) covalently linked to the MIT domain through a (GGGGS) 3 linker that was long enough to allow an intramolecular MIM1-MIT interaction (Fig. 6B). The equivalent construct bearing the MIM1 L225D mutation (MIM1(L225D)-Vps4p) was used as a negative control because this mutation disrupts the interaction between MIM1 and MIT (26). The MIM1-Vps4p fusion and Vps4p ⌬MIT proteins bound peptides with similar affinities, whereas the binding affinity of MIM1(L225D)-Vps4p was similar to that of full-length Vps4p (Fig. 6, A and C). Additional validation was provided by a GST affinity co-purification pulldown assay using Vps2p helix 5 residues fused to GST. This construct bound both Vps4p ⌬MIT and MIM1-Vps4p, whereas binding to both Vps4p and MIM1(L225D)-Vps4p was greatly diminished (Fig. 7). These data indicate that MIM1 binding alleviates inhibition of substrate pore binding by the MIT domains. This effect does not appear to reflect MIT domain inhibition of Vps4p oligomerization because MIM1-Vps4p and MIM1(L225D)-Vps4p displayed equivalent migration on gel filtration chromatography in the presence of ADP⅐AlF x (Fig. 6D). Essentially identical results were found for binding of peptide C to MIM2-Vps4p constructs (Vfa1p residues 183-203) (Fig. 6C) that were designed in light of the recently reported Vfa1p MIM2-MIT complex structure (60).

Discussion
ESCRT-mediated membrane fission requires remodeling and depolymerization of ESCRT-III filaments by the Vps4 AAA ATPase, which is recruited, at least in part, by binding of its N-terminal MIT domain to C-terminal ESCRT-III MIM elements. Earlier studies indicated that an additional interaction occurs for human VPS4A between the ATPase cassette and sequences from the helix 5 region of ESCRT-III subunits, that deletion of the MIT domain and linker sequence enhances ATPase activity in human VPS4A, and that mutation of pore loop residues could either increase or reduce ESCRT-III-induced ATPase activity (52,53). We have verified and extended these findings by performing quantitative binding studies using purified reagents, delineating effects due to peptide binding versus those due to stabilization or destabilization of the active hexamer, and demonstrating effects of MIM1 and MIM2 interactions on substrate engagement. The earlier studies on VPS4A were prompted in part by the relatively low ATPase activity of human VPS4 proteins (53), which suggested that fundamental differences in Vps4 regulation might exist between species but might be explained by the different assay conditions such as the presence/absence of Vta1/LIP5. In addition to providing new insights, our work using the S. cerevisiae Vps2p and Vps4p proteins therefore indicates that key principles of Vps4 regulation are likely to be conserved across species.
Our fluorescence anisotropy studies demonstrated that a 20-residue peptide from the helix 5 region of Vps2p/ESCRT-III binds the hexameric Vps4p AAA ATPase cassette with an affinity similar to that of the primary contact between the isolated Vps4p MIT domain and the Vps2p/ESCRT-III MIM (albeit without enhancing avidity effects). This interaction seems unlikely to have absolute sequence specificity, and we anticipate that many protein sequences could similarly engage the Vps4 ATPase cassette, consistent with the reports that overlapping fragments of multiple ESCRT-III family members are able to co-sediment with VPS4B(E235Q) (52) and stimulate ATPase activity of human VPS4A (53) or yeast Vps4p (61,62). We also demonstrated that binding occurs with a stoichiometry of one peptide per hexamer. This further validates the conclusion that Vps4 is active as a hexamer (37) and supports the model that substrates bind in the central Vps4 pore. We also found that peptide binding stimulates ATPase activity, at least in part by stabilizing the hexamer.
Although residues of the pore loops have previously been implicated in Vps4 function (40,41), it was formally possible that these mutations inhibited activity by reducing the stability of the active Vps4 oligomer. We have now identified point mutants in Vps4p pore loop 2 that increased binding without decreasing hexamerization, thereby solidifying the idea that substrates bind in the central pore of the hexamer. We were

Vps4-Substrate Interactions
unable to perform equivalent experiments with pore loop 1 mutants, however, because although we identified many pore loop 1 mutations that diminished binding, they also diminished hexamerization, at least to some degree.
An important conclusion from our work is that Vps4p functions as an asymmetric hexamer that translocates substrate into the central pore. This follows from our observations that a single peptide binds per hexamer, which necessarily requires an asymmetric interface, and is dependent upon the presence of ADP⅐AlF x or ADP⅐BeF x , which allow the ATPase to adopt multiple nucleotide-bound states. This is consistent with models of other ring-like ATPases, including F 1 ATPase (63), E1 helicase (64), Rho helicase (59), the 26S proteasome (65,66), the bacterial enhancer-binding protein NtrC1 (58), and N-ethylmaleimide-sensitive factor (NSF) (67), which all display asymmetry of the subunits that results in a spiral staircase arrangement of the pore loops. An attractive model is that Vps4 translocates substrate, at least partially, through the central pore, driven by changes in pore loop conformations that propagate around the hexamer ring in concert with the ATP hydrolysis cycle.
Peptide binding was not observed with ADP or ATP, which presumably gave a mixture of ATP and ADP because the binding assay was performed at a temperature that allows for hydrolysis, and nor was binding observed in the presence of nonhydrolyzable analogs of ATP. Presumably, ADP metal fluorides enable the formation of asymmetric, peptide-bound complexes but do not drive translocation, whereas binding under conditions of active ATP turnover is transient and therefore not captured by our assay. The transcriptional activator PspF, a related AAAϩ ATPase, also requires ADP⅐AlF x to form a complex with 54 that can be detected in vitro (68).
Our observations that the pore loop mutants E243A and E247A bind more tightly (especially for peptide B) indicate that the negatively charged pore residues Glu-243 and Glu-247 diminish binding of peptides rich in acidic residues (such as peptide B). Thus, the pore is not optimized to bind all peptides equally or with maximal binding affinity. Our findings are somewhat discordant with conclusions from pioneering work from the Hanson laboratory, which provided motivation for this study but also concluded that acidic residues preferentially bind (52) and stimulate ATPase activity (53) of human VPS4A. Regardless, the overall impression is that there is a pore-selective-binding element located within helix 5 of Vps2p, but also that different amino acid sequences can bind the Vps4p pore with appreciable affinity, which is consistent with the idea that once an ESCRT-III subunit is engaged, it can be processively translocated through the hexamer pore. In principle, this action could translocate the ESCRT-III substrates completely, although the extent of translocation remains to be determined experimentally.
In contrast to human VPS4A (53), deletion of the MIT domain does not seem to increase the basal rate of ATP hydrolysis for yeast Vps4p (36,61,62). However, our observation that the MIT domain inhibits peptide binding to the hexamer pore in the absence of an MIT-MIM interaction suggests a two-step model of Vps4-mediated ESCRT-III lattice disassembly (Fig. 8). We propose that the MIM-MIT interaction serves to recruit Vps4 to ESCRT-III polymers and to unmask or activate the binding site at the hexamer pore, which possesses an inherent ability to bind and translocate a wide variety of sequences starting at an internal loop, such as has been described for the proteasome (69,70). An attractive feature of this model is that it provides a mechanism for avoiding Vps4 pore engagement of inappropriate substrates. The mechanism of MIT inhibition is unclear at this time, and might include direct occlusion or indirect stabilization of an inhibited pore conformation. It is also unclear whether all of the MIT domains in a Vps4 hexamer need to be engaged by MIMs to allow binding at the pore and stimulate ATPase activity, although it is attractive to speculate that multiple MIT-MIM interactions are required because that would provide an effective mechanism for restricting activity to ESCRT-III polymers. This model provides a foundation for future structural and mechanistic studies to understand in more detail how Vps4 functions in membrane fission and the dissociation of ESCRT-III filaments.


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Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages

Here we report a novel regulatory mechanism for autophagy-mediated degradation of Mycobacterium tuberculosis (Mtb) and specific strategy exploited by the virulent Mtb to evade it. We show while both avirulent (H37Ra) and virulent (H37Rv) mycobacteria could readily localize to autophagosomes, their maturation into autolysosomes (flux) was significantly inhibited by the latter strain. The inhibition of autophagy flux by the virulent strain was highly selective, as it did not perturb the basal autophagy flux in the macrophages. Selective inhibition of flux of Mtb-containing autophagosomes required virulence regulators PhoP and ESAT-6. We show that the maturation of Mtb-containing autophagosomes into autolysosomes required recruitment of the late endosome marker RAB7, forming the intermediate compartment amphisomes. Virulent Mtb selectively evaded their targeting to the amphisomes. Thus we report a crosstalk between autophagy and phagosome maturation pathway and highlight the adaptability of Mtb, manifested by selective regulation of autophagy flux.

Autophagy involves sequestration of cellular cargo in a double-membrane structure called autophagosomes. Autophagy induction is conventionally monitored as MAP1LC3B (LC3) puncta formation through microscopy. It emerges now, autophagy induction does not always lead to degradation of the cargo. Degradation typically involves fusion of autophagosomes with the lysosomes thereby forming autolysosomes and subsequent action of lysosomal hydrolases. However during the degradation process, LC3 on the inner membrane of autophagosomes too gets degraded due to the activity of lysosomal hydrolase 12 . Therefore when active autophagic degradation is taking place, LC3 would be constantly degraded leaving only the outer membrane associated LC3 intact and thereby making it difficult to accurately assess autophagy induction. The degradation of LC3 however can be blocked by vacuolar ATPase inhibitors like bafilomycin A1 or lysosomal protease inhibitors like E64-d and pepstatin A 12 . Strangely several studies in the past, which showed an important role for autophagy in case of Mtb infections, did not monitor autophagy flux 9,13,14 . In some studies however, in case of Mtb infections autophagy flux as a means of bacterial degradation were monitored [15][16][17] . One such report suggests strain dependent variations in autophagy induction which also correlated with the autophagic degradation 17 . Yet another study identified the immunity regulator molecule TBK1 as key regulator of autophagy maturation 15 .
In the present study we aimed to investigate the autophagy flux in THP-1 macrophages upon Mtb infection in detail using laboratory strains H37Ra (avirulent) and H37Rv (virulent). Interestingly both the virulent and avirulent strains localized to the autophagosomes. Later in the infection, the flux of Mtb-containing autophagosomes was specifically inhibited by the virulent Mtb. We then show that the inhibition of autophagy flux by H37Rv was the result of its ability to block recruitment of RAB7 on Mtb-containing autophagosomes. Therefore through this study we report a RAB7-dependent autophagy pathway of mycobacterial killing in macrophages.

Results
Virulent Mycobacterium tuberculosis selectively inhibits autophagy flux. The laboratory strains H37Rv and H37Ra of Mtb, consistent with previous reports, showed distinct survival ability within the macrophages (Fig. S1A) [18][19][20] . To check how autophagy gets regulated under the two conditions, we monitored targeting of H37Ra or H37Rv to LC3 positive autophagosomes by immuno-staining followed by confocal microscopy. THP-1 macrophages were infected with H37Ra or H37Rv at 1:10 MOI following the protocol described previously 10 . We monitored autophagosome targeting of the two strains at 6, 12, 24, 48 and 72 hours post-infection. Throughout the course of infection, both H37Ra and H37Rv efficiently co-localized with LC3 containing compartments (Fig. 1A). The antibody used to stain endogenous LC3 was highly specific and did not show any cross-reactivity with the Mtb (Fig. S1B and S1C). We calculated the M1 Coefficient with respect to Mtb (herein after referred to as co-localization coefficient, see methods) to measure co-localization of both the strains to autophagosomes. In parallel we also had groups where samples were treated with vacuolar ATPase inhibitor Bafilomycin A1 (BafA1, 100 nM) for three hours before the end of each of the time points. Treatment with BafA1 inhibits LC3 degradation upon maturation by blocking the acidification of autolysosomes. We observed a significant increase in the co-localization coefficient of H37Ra with LC3 in BafA1 treated samples relative to the untreated ones from 24 hours post infection (Fig. 1B). Importantly no such increase was observed in case of H37Rv across all time points (Fig. 1C).
We also tested these results in mouse bone marrow derived primary macrophages (BMDMs). Consistent with the results above, BafA1 treatment in BMDMs led to an increase in the co-localization coefficient of H37Ra and LC3, while there was no effect of BafA1 treatment in the case of H37Rv (Fig. 1D,E). Interestingly, Western blot analysis of LC3 in the whole cell lysates from H37Ra or H37Rv infected as well as uninfected THP-1 macrophages showed increased levels of lipidated LC3 (LC3II, Fig. 1F) upon BafA1 treatment, implying maturation of autophagosomes at certain basal state in these cells. We also noted both in THP-1 macrophages and in BMDMs, presence of autophagosomes that did not contain either H37Ra or H37Rv (arrows in Fig. 1A,D).
Differential flux of H37Ra and H37Rv containing autophagosomes was also confirmed by monitoring co-localization of Mtb with autolysosomes ( Fig. 1G,H). Since BafA1 treatment diminishes LysoTracker staining of lysosomes, we used lysosomal protease inhibitors E64-d and pepstatin A to monitor H37Ra or H37Rv in LC3:LysoTracker double positive compartments representing autolysosomes. We observed a significantly higher percentage of H37Ra in autolysosomes, as compared to H37Rv, from 24 hour post infection (Fig. 1H). We also noted, in both the cases, presence of LC3:LysoTracker double positive compartments that did not contain any Mtb (Fig. 1G, arrows).

Mycobacterium tuberculosis PhoP and ESAT-6 help H37Rv inhibit autophagy flux. The strain
H37Ra differs from H37Rv mainly due to a point mutation in the master regulator gene PhoP 21 and expression of wild-type (WT) PhoP (from H37Rv) in H37Ra is shown to revert the virulent phenotype 21,22 . We asked whether PhoP, a known Mtb virulence regulator, could help H37Rv modulate host xenophagy flux. We monitored co-localization of WT-PhoP complemented H37Ra strain (H37Ra:PhoP) with LC3 in THP-1 macrophages. PhoP complementation of H37Ra was confirmed by real-time analysis of several genes known to be regulated by PhoP (Fig. S2). The co-localization coefficient of H37Ra:PhoP The Mtb PhoP is a global transcriptional factor involved in regulating the expression of RD1 region. H37Ra shows much diminished secretion of ESAT-6, the major antigen on RD1 locus and a well-known mycobacterial virulence effector 21,23 . To test if PhoP mediated block in the autophagy flux was effected by ESAT-6, we used ESAT-6 deleted mutant of H37Rv (∆ESAT-6). BafA1 treatment led to significant increase in the co-localization of ∆ESAT-6 strain with LC3 (Fig. 2C). To note here is that the block in autophagy flux observed in case of H37Rv was released with the deletion of ESAT-6. As expected, ∆ESAT-6 strain showed compromised survival in the THP-1 macrophages compared to the wild type H37Rv (Fig. 2D). Moreover treatment with 3-MA, an autophagy inhibitor, was able to rescue ∆ESAT-6 from killing (Fig. 2D).
H37Rv inhibits amphisome formation. We next wanted to understand the cellular events in the host that gets perturbed by virulent Mtb in order to inhibit autophagy flux. Maturation of autophagosomes involves direct fusion with the lysosome to form auto-lysosome or they can first recruit RAB7 to form amphisomes which eventually fuse with the lysosomes 2,24 . It is well known that H37Rv inhibits RAB7 recruitment to escape lysosomal targeting and killing 8 . Therefore, we tested whether RAB7 recruitment on autophagosomes (amphisomes) could also represent a mechanism of H37Rv to evade lysosomal fusion. Therefore, we monitored co-localization of H37Ra and H37Rv to LC3-RAB7 double positive compartment (hereby defined as amphisomes) at 48 hours post-infection.
As expected we observed significantly higher co-localization of H37Ra with either LC3 or RAB7 as compared to H37Rv (Fig. 3A,B). Consistent with that, the percentage of H37Ra was significantly higher in amphisome (LC3:RAB7 double positive) as compared to H37Rv at 48 hours post infection (Fig. 3C). However, LC3-RAB7 co-localization, representing overall amphisome population in the cell, was similar for both the strains. These results clearly suggest that RAB7 recruitment to autophagosomes is significantly higher in H37Ra as compared to H37Rv. Therefore, we next tested whether the inhibition of autophagic flux by virulent Mtb is associated with RAB7 recruitment. Panel (B) shows relative survival percentage in THP-1 macrophages with respect to H37Rv, H37Ra or H37Ra:PhoP at 48 hours post-infection (mean ± SD, *p-value < 0.05). M1 co-localization coefficient of ∆ESAT-6 mutant of H37Rv with LC3 compartment in BafA1 treated and untreated condition is shown in 2C (mean ± SEM, **p < 0.001). In 2D, relative survival was estimated in H37Rv and ∆ESAT-6 infected THP-1 macrophages, 48 hours post-infection. The ∆ESAT-6 strain infected cells were treated with 3-MA (5 mM, 12 hours) to observe their rescue upon autophagy inhibition (mean ± SD, *p-value < 0.05, **p-value < 0.001).
Scientific RepoRts | 5:16320 | DOI: 10.1038/srep16320 Exclusion of RAB7 from Mtb-containing autophagosome allows H37Rv to escape lysosome targeting. In order to elucidate the role of RAB7 in modulating flux of Mtb-containing autophagosomes, specific siRNA was used to knockdown RAB7 in infected THP-1 macrophages. Specific knockdown of RAB7 was verified by Western blots (Fig S3A). As shown in Fig. 4A, RAB7 knockdown inhibited the flux of H37Ra-containing autophagosomes. There was no effect on flux of H37Rv-containing compartments (Fig. 4A). Intriguingly RAB7 siRNA also led to inhibition of general autophagy flux in THP-1 macrophages (Fig. S3B and S3C). As expected, co-localization of H37Ra to lysosomes was significantly inhibited in the RAB7 knockdown macrophages (Fig. 4B). Moreover RAB7 depletion also resulted in improved intracellular survival of H37Ra (Fig. 4C).

Discussions
Autophagy as a defence mechanism in case of Mtb infection has been shown by several studies in the past 9,11,25 . Given the fundamental role of autophagy in cells as homeostatic mechanism under various stress conditions, how Mtb could specifically manipulate it, remains poorly understood. There are studies on strain specific regulation of autophagy, suggesting that the virulent strains of Mtb induce autophagy to a lesser extent in contrast to the non-pathogenic, attenuated strains 17 . Some other reports however suggest while the wild-type strains could induce autophagy, the attenuated ones could not 14 . Here we exclusively focused on the regulation of maturation of Mtb-containing autophagosomes and observed a distinct pattern of selectivity between the virulent and avirulent strains of Mtb in regulating autophagy flux.
We show here that both virulent and avirulent strains of Mtb can readily localize to the autophagosomes. Our experiments with BafA1 revealed a higher co-localization of H37Ra with autophagosomes relative to the untreated set post 24 hours of infection. Since BafA1 inhibits LC3 degradation in the autolysosomes, we believe the increase in co-localization was due to the enhanced signals from the accumulated, undegraded LC3 molecules in the autolysosomes. This result implies flux of H37Ra-containing autophagosomes that were maturing into autolysosomes. On the other hand, we did not observe any increase in the co-localization of H37Rv with autophagosomes upon BafA1 treatment throughout the course of infection, thereby suggesting a block in the flux of H37Rv-containing autophagosomes. This differential flux of Mtb-containing autophagosomes was corroborated by higher localization of H37Ra with the autolysosomes as compared to H37Rv. We found a crucial role of the virulence factors PhoP and ESAT-6 in effecting this maturation block of Mtb-containing autophagosomes. Similar role of ESAT-6 in the regulation of autophagy flux in infected dendritic cells has been shown earlier 16 .
Curiously, we observed several autophagosomes devoid of bacteria in the infected cells. Moreover, several of those autophagosomes were at maturation stage co-localizing with the acidified lysosomes. This observation indicated that the autophagosomes, which did not contain Mtb, could mature without being influenced by infection. The LC3 immunoblots provided additional evidence supporting the selective nature of maturation block brought by the virulent strain. This observation opens up an interesting possibility that non-Mtb-containing autophagosomes and Mtb-containing autophagosomes (xenophagosomes) could have distinct maturation dynamics within the same cell. A blanket inhibition of autophagy, under many circumstances, would lead to induction of apoptosis, which may be detrimental to Mtb survival 19 . By specifically blocking the maturation of compartments where H37Rv resides, it avoids being killed by lysosomal mechanism. At the same time by allowing cellular autophagy, it ensures extended survival of the host 26,27 . However, this interesting possibility deserves more detailed investigation.
Our results on the role of RAB7 in autophagosome maturation provide novel insights into functioning of this small GTPase. Exclusion of RAB7 on phagosomes by virulent Mtb has been classically reported as the mechanism for avoiding lysosomal degradation 28 . It seems phagosome maturation goes hand-in-hand with autophagosome maturation and that at least Mtb-containing autophagosomes do not mature into autolysosomes without recruiting RAB7 to form an intermediate amphisome. How the resident virulent Mtb could block autophagosome fusion with late endosomes remains unanswered. However in case of phagosome maturation, it was shown that the failure to recruit RAB7 to H37Rv phagosome was contingent on the lipid product phosphatidylinositol 3-phosphate (PtdIns[3]P) and subsequent binding of RAB5 effector EEA1 29 . However it remains unclear if that holds true even in the case of autophagosomes. Interestingly a number of studies propose, in other systems, role of RAB7 in the maturation phase of autophagy [30][31][32] . The importance of maturation step of autophagy in case of Mtb infections was also supported by the study where TBK1, an innate immunity regulator was shown to regulate autophagy maturation to eliminate BCG infections 15 . Intriguingly, upon RAB7 depletion in THP-1 macrophages, even the general autophagy flux gets perturbed. That raises another question on how the selectivity in autophagosome maturation could be ensured by resident virulent Mtb if RAB7 is essential for autophagosome maturation in general. One plausible explanation for this may be selective modulation of RAB5 effector function by H37Rv leading to altered autophagosome. Thus even though late endosomes could efficiently fuse with other autophagosomes, H37Rv-containing autophagosomes evade this process. Such mechanisms are studied in case of phagosome maturation 29 , whether similar mechanism could be functional in case of autophagy needs further investigations. In this study we could not distinguish autophagy from LC3 associated phagocytosis (LAP). Involvement of LAP in Mtb infections remains debatable despite some reports emerging on it, and needs further investigations 33,34 . The crosstalk between the autophagy pathway and the phagosome maturation pathway has been schematically summarized in Fig. 5. In conclusion we report here a mechanism whereby virulent Mtb strain could selectively block maturation of the resident autophagosomes. We show that the virulent Mtb could achieve this by avoiding RAB7 recruitment to form the amphisomes. The crucial role of amphisome compartment in the regulation of intracellular Mtb survival has added new dimensions to our understanding of cell autonomous defense mechanisms in case of Mtb infections. Understanding the regulation of autophagy maturation at molecular level could help identify novel and perhaps better host directed drug targets against tuberculosis.

Materials and Methods
Ethics statement. All  Bacterial suspension preparation and Infection. Bacterial whole cell suspension was dispersed by aspiration five times each with a 23-and then a 26-gauge needle, followed by an additional dispersion by aspiration 3 times through a 30-gauge needle. The dispersed bacteria were allowed to stand for 5 min to allow the clumps to settle down. The upper half of the suspension was then used for the experiments. Quantitation of bacteria was done by taking absorbance at 600 nm wavelength (0.6 O.D. corresponds to ~100 × 10 6 bacteria). Complete media containing the required number of bacteria was added to the cultured cells at an MOI of 10, followed by a short spin of the culture plates at 700 rpm for 5 minutes. After 4 hours of infection, the cells were washed twice with warm RPMI and treated with Amikacin (200 μ g/ ml, HiMedia laboratories) to remove any extracellular bacteria. After two hours of treatment, the cells were washed and maintained in complete media. For microscopy experiments, the desired number of bacteria were stained with PKH67 (green)(Sigma-Aldrich Co. MINI67-1KT), lipophilic fluorescent dye, as per the manufacturer's protocol. The stained bacteria were passed thrice through a 26-gauge needle and were then used for infection.

Animals and isolation of BMDMs.
Bone marrow derived macrophages (BMDMs) were isolated from femurs of BALB/C mice (4-6 weeks old, female). BMDMs were obtained by culturing the marrow cells in the presence of macrophage-colony stimulating factor (M-CSF, eBioscience, 14-8983-80) for 7 days. Fully differentiated macrophages were harvested and seeded for infection with H37Ra or H37Rv. The infection protocol was same as described above for THP-1 macrophages.
Construction of PhoP over-expressing Mtb H37Ra strain. For expressing H37Rv PhoP in H37Ra, gene encoding H37Rv PhoP (Rv 0757) was PCR amplified and cloned into E.coli-Mycobacterial shuttle vector, pSD5hsp 35 . The pSD5-phoP construct was then electroporated into Mtb H37Ra and transformants were selected on 7H11 medium containing kanamycin (25 ug/ml). The expression of phoP was under strong hsp65 gene promoter from M. leprae. siRNA treatments. We followed established protocols for siRNA treatments of differentiated THP-1 cells as described earlier 10 . RAB7 siRNA were procured from Dharmacon (M-010388-00-0005). Addition of RAB7 siRNA was done after amikacin wash and treatment was done for 48 hours.
Confocal Microscopy. One hour prior to the termination of the experiment, complete media containing LysoTracker dye at a concentration of 200 nM was added to the cells for staining of acidified lysosomes. Following the required treatments, the cells were fixed with 4% paraformaldehyde (Sigma) for 20 minutes, followed by 3 washes with 1X PBS. The cells were permeabilized using 0.2% (w/v) TritonX-100 in 1X PBS for 20 minutes. Blocking was performed using 3%( w/v) BSA and 0.5%Tween20 in 1X PBS for one hour. This was followed by primary and secondary antibody staining for duration of one hour each. The primary antibodies used in the study are LC3 (Novus, NB100-2220, Cell Signaling Technology, 2775S), RAB7 (Santa Cruz Biotechnology, SC-6563). Commercially available Alexa dye conjugated secondary antibodies (Molecular Probes, A31556 and A11057) were used for immunostaining. Antibody dilutions were made in blocking solution as per the supplier's protocol. The coverslips were washed thoroughly with 1X PBS and were mounted onto glass slides with Antifade reagent (Invitrogen Molecular Probes). Images were acquired randomly from each set of stained cells with a Nikon EclipseTi-E laser scanning confocal microscope equipped with a 60X/1.4NA PlanApochromat DIC objective. Images were acquired using the softwares EZ-C1 3.80 and NIS-Elements. Image analyses were performed using the software Imaris version 7.6.4 (BitPlane).
Image Capture and analysis. Stained cells were observed with a Nikon Ti-E microscope equipped with 60X /1.4 NA planapochromat DIC objective lens. Samples were excited at 543 nm with He-Neon laser, 488 nm with an Argon ion laser and in the UV range with a blue diode. Images were acquired sequentially to avoid bleed-through signal, with a scanning mode format of 512 × 512 pixels. The transmission and detector gains were set to achieve best signal to noise ratios and the laser powers were tuned to limit bleaching of fluorescence. The refractive index of the immersion oil used was 1.515 (Nikon Corporation). All settings were rigorously maintained for all experiments. All images were qualitatively assessed using Imaris version 7.6.4 (BitPlane). All the images are in the Tiff RGB 24 format. M1 co-localization coefficient of Mtb and various markers were calculated in the software. The percentage of Mtb in autolysosomes and amphisomes were estimated by calculating the percentage of Mtb voxels in LC3:Lysotracker and LC3:RAB7 colocalized channels. The results represent an average of at least three independent sets of experiments.