Autophagy Plays a Critical Role in the Degradation of Active RHOA, the Control of Cell Cytokinesis, and Genomic Stability

Cells are faced with the tasks of dividing, of keeping the genome intact, and even of dying when appropriate. At their heart, these cell fates are dictated by tumor suppressor mechanisms. One such mechanism is autophagy, which is commonly mutated or downregulated in human cancers (1). Indeed, the essential autophagy gene BECN1 is deleted or mutated in 40% to 75% of breast, ovarian, colon, and prostate cancers. Consistently, the notion that autophagy suppresses tumor development came from the demonstration that allelic loss of Becn1 predisposes mice to lymphomas, hepatocellular carcinomas, and lung carcinomas (2, 3). Likewise, defects in other autophagy genes (Atg4c, Atg5, Uvrag, Ambra1, and Bif-1/Sh3glb1) render cells or mice tumor prone (4–8).

Physiologically, autophagy ensures in all cell types the turnover of all organelles and most long-lived proteins by a pathway, which begins with the formation of a double-membrane compartment, termed a “phagophore” that sequesters them. The phagophore expands into a completed vesicle, an “autophagosome,” and subsequently, the autophagosome rapidly fuses with a lysosome to become an “autolysosome” where the content is finally degraded. Originally identified as a housekeeping process, emerging data suggest that constitutive autophagy (i.e., under nutrient-rich conditions) might also fight cancer by limiting inflammation (9), facilitating senescence (10), or clearing signaling proteins (11). Likewise, recent studies reveal that both BECN1 and ATG5 function as “guardians” of cellular genome. Epithelial cells with loss of Becn1 or Atg5 display gene amplification and aneuploidy (7, 8). In support, activation of autophagy was shown to reduce genomic instability within hepatocarcinoma cells (12). However, despite the importance of aneuploidy in cancer development (13, 14), the mechanisms underlying how autophagy deficiency compromises genomic stability are still unknown.

To address this issue, we explored the possibility that signaling proteins essential for cell growth might be degraded by autophagy. Whereas significant advances have been made in the discovery of autophagy machinery, less is known about the nature of the autophagy substrates. Therefore, a key feature of our strategy was to inhibit the autophagy pathway at the degradation step, to achieve sequestration and accumulation of substrates within autolysosomal structures (Supplementary Fig. S1A).

To inhibit the maturation of autophagosomes into degradative autolysosomes, renal cells derived from proximal convoluted tubules of wild-type (WT, a3^+/+) or the lysosomal v-ATPase a3/TCIRG1-null mice (a3^−/−) were isolated and immortalized with the pSV3 neo vector. The renal epithelial cell lines that do or do not express a3 were referred to as WT and a3^−/− cells, respectively. As controls, cells were stimulated with an inhibitor of v-ATPase activity, bafilomycin A1 (100 nmol/L, bafA1; Sigma); or a weak base that raises intralysosomal pH, chloroquine (100 μmol/L, CQ; Sigma). Alternatively, the formation of autophagosomes was inhibited at the initiation step by Atg5 or Atg7 short hairpin RNA (shRNA). As a further control, we analyzed the phenotype of Atg5 KO MEFs (provided by N. Mizushima; ref. 15) and ATG5-depleted A549 lung epithelial cells. We also prevented the sequestration of autophagy substrates within the autophagic vesicles by p62 shRNA. For details on cell culture and shRNA sequences, see Supplementary Information.

Primary non–small cell lung cancer (NSCLC; pairs of pathologic and control tissues from the same patient) were obtained from patients in Nice (France) and collected by the Tumor Biobank of Nice Hospital (Nice CHU, agreement 2010–06).

The activity of the autophagy pathway was monitored by 4 hallmarks: (i) the formation of autophagic vesicles; and the degradation of 3 well-established autophagy substrates: (ii) membrane-associated LC3-II, (iii) p62/SQSTM1, and (iv) long-lived proteins.

At 70% confluency, cells were arrested with colchicine (Invitrogen) in metaphase. Chromosomes were stained with Giemsa, and about 150 mitotic figures per cell line were photographed and the number of chromosomes was counted by Metafer M-Search Metaphase Finder and Ikaros softwares (Metasystems).

For monitoring cell progression through mitosis, exponentially growing cells cultured in complete growth medium were imaged every 5 minutes during 18 hours on an inverted microscope (Carl Zeiss) equipped with a CO₂-equilibrated chamber.

The activity of RHOA pathway was monitored by (i) the levels of active GTP-bound RHO (RHOTEKIN RHO-binding domain pull-down and ELISA-based G-LISA assays, Cytoskeleton Inc.), (ii) the recruitment of RHOA to membranes, and (iii) the downstream phosphorylation of myosin regulatory light chain (P-MLC) and reticulation of actin cytoskeleton.

Cells were lysed in radioimmunoprecipitation assay (RIPA) and RHOA was immunoprecipitated with anti-RHOA antibody (clone 26C4; Santa-Cruz Biotechnology) followed by Western blotting with anti-ubiquitin, anti-p62, anti-LC3, and anti-RHOA antibodies.

HEK 293 cells and A549 cells (control, ATG5, or p62 shRNA-transduced cells) were transfected with FuGene^HD (Promega) and plasmids encoding the active (RHOA Q63) or inactive (RHOA N19) RHOA mutants. Twenty hours after transfection, cells were treated with cycloheximide (CHX; Sigma; C-4859; 10–20 μg/mL) to stop de novo protein synthesis for 7 to 57 hours, alone or in combination with proteasomal (MG132, Sigma; 10 μmol/L) and lysosomal (CQ; 100 μmol/L) inhibitors, and the drop in the levels of RHOA mutants was assayed by anti-myc Western blotting (Millipore; P01106; 1:1,000).

Complete and detailed description of all methods used is available as Supplementary Data.

When adequate, results are presented as means ± SD from the indicated number n of separate experiments. Statistical comparisons were done using χ² or Student t tests as appropriate. P < 0.05 was considered significant.

To gain a deeper insight into the role of autophagy, we established cell lines from v-ATPase a3/TCIRG1-null mice (16). PCT (proximal convoluted tubule) cells were chosen as they express the highest level of v-ATPase (17); the a3 subunit is localized in the lysosomal limiting membrane (18). In agreement with the other reported defect of v-ATPase (19, 20), we show that the a3 loss increased autophagy sequestration and simultaneously impaired autophagic degradation, as evidenced by the accumulation of ATG12–ATG5 conjugate, of autolysosomes and of autophagic substrates (long-lived proteins, LC3-II, and p62; Fig. 1A and Supplementary Fig. S1B). In contrast, a3-null cells seemed to have functionally intact proteasomes (Supplementary Fig. S1C). Remarkably, this a3-dependent autophagy defect was characterized by an increase in cell size and a flattened morphology with the accumulation of vesicles and nuclei (Fig. 1B and C), supporting the importance of autophagy in maintaining cell size (21, 22).

By using different approaches, we excluded a role for senescence as a cause of enlarged morphology (Supplementary Fig. S2). Consistently, fluorescence-activated cell-sorting (FACS) analysis revealed that a3^−/− cells were dividing and displayed increased DNA content (3–4 N; Supplementary Fig. S2C). Of interest, no subdiploid or subtetraploid cells were detected, indicating that the a3 loss did not induce cell death. Subsequent karyotypes of a3^−/− cells confirmed a near-triploid karyotype (group average, 131; Fig. 1D). Therefore, despite aneuploidy, the a3^−/− cells continued to proliferate and escaped apoptosis or senescence. All these abnormalities, which are hallmarks of cancer cells, were reminiscent of that reported for Atg5^−/− or Becn1^−/+ cells (8). Collectively, these data support the notion that defects of the entire autophagy pathway [i.e., either at the step of formation (Atg5^−/− or Becn1^−/+) or degradation (herein, V-ATPase a3^−/−) of autophagosomes] could result in aneuploidy.

We hypothesized that the aneuploidy of a3^−/− cells might arise through cytokinesis failure. Multiple mitotic defects leading to aneuploidy were indeed identified in a3^−/− cells, as these cells often showed multipolar spindles and several chromosome segregation defects: improper attachment of chromosomes to spindle microtubules, lagging chromosomes, chromosome bridges connecting daughter cells, and micronuclei. Strikingly, a large proportion of a3^−/− cells were connected via an asymmetric bridge, in contrast to the short intracellular bridge observed in the middle of the two WT daughter cells (Supplementary Fig. S3).

Using real-time imaging, we showed that the WT cells completed cytokinesis in only 15 min (Fig. 2A, Supplementary Movie S1). In contrast, the cytokinesis was incomplete upon v-ATPase inhibition by bafilomycin A1 treatment (Fig. 2B) or a3 loss (Fig. 2C–G, Supplementary Movies S2–S5). About 72% of a3^−/− cells that entered mitosis normally failed abscission (Fig. 2C) and instead remained connected by an intracellular bridge for up to 8 hours before separating (Fig. 2D, Supplementary Movie S2), re-entering mitosis synchronously (still bound to the sister cell, Fig. 2E, Supplementary Movie S3) or collapsing back, forming a single binucleate cell (Fig. 2F and G, Supplementary Movies S4 and S5). Moreover, we did not observe cell–cell fusion, cell engulfment, and endoreplication during live cell imaging (n = 200). Thus, impairment of cytokinesis at the membrane abscission step was the key event responsible for the formation of multinucleate a3^−/− cells.

We then explored which signaling proteins might be degraded by autophagy and could underlie this phenotype. One candidate was the small GTPase RHOA that dictates cell shape and completion of cytokinesis via F-actin reticulation (23). In this regard, a striking hallmark of a3^−/− cells was a dramatic remodeling of actin cytoskeleton with the loss of stress fibers and the formation of actin patches (Fig. 3A and Supplementary Fig. S4A and S4B). The current consensus is that fine-tuning of RHO activity involves the guanine nucleotide exchange factors (GEF) that activate them, the GTPase-activating proteins (GAP) that inactivate them, and the guanine nucleotide dissociation inhibitors (GDIs) that maintain RHO inactive within the cytoplasm (23). Exciting findings have revealed that RHO GTPases, their upstream regulators and downstream targets might also be subjected to irreversible proteasome-dependent degradation (24–32).

Instead of proteasome, however, we determined that active RHOA was constitutively maintained at low levels by autophagy. Indeed, the active RHOA was barely detected at the plasma membrane of a3^−/− cells but instead accumulated intracellularly within autolysosomes positive both for the autophagy marker LC3 and lysosomal marker LAMP1 (Fig. 3B, Supplementary Fig. S4B, inset). Notably, the level of RHOA-GTP was elevated in resting a3^−/− cells, as evidenced by the RHOTEKIN-binding pull-down assay, the recruitment of RHOA to cellular membranes and the downstream phosphorylation of myosin regulatory light chain (P-MLC; Fig. 3C, Supplementary Fig. S4B).

A role for autophagy in controlling RHOA-GTP level was then suggested by the shRNA-mediated inhibition of autophagosome formation: Expression of Atg5 shRNA increased the localization of active RHOA at the plasma membrane of a3^−/− cells, which allowed actin polymerization into filaments (Fig. 3D). Consistently, the Atg5 shRNA-transduced a3^−/− cells displayed a smaller size and tight cohesion within the colony (Fig. 3D). Importantly, the control of RHOA by autophagy was remarkably specific, as the related GTPase RAC (Supplementary Fig. S4E), as well as the RHOA regulator, RHOGDI, were not affected (Fig. 3C). Together, these data highly suggest a working model in which autophagy might sequester and degrade active RHOA. Inhibition of v-ATPase by a3 loss would stabilize RHOA-GTP within autolysosomal structures, protecting it from autophagy degradation, and at the same time, this would preclude reticulation of actin cytoskeleton (Supplementary Fig. S1A).

As proof-of-concept, pharmacologic inhibition of autophagy degradation by bafA1 or CQ treatment similarly increased the levels of membrane-bound RHOA together with the autophagy substrates LC3-II and p62 (Supplementary Fig. S5). This occurred in multiple cell types including fibroblast, kidney, and lung epithelial cells (Supplementary Fig. S5A). In contrast, impairment of proteasome by MG132 failed to alter the levels of the membrane-bound RHOA in every cell line tested (Supplementary Fig. S5A). As expected, all features of a3^−/− cells (i.e., RHOA autolysosomal recruitment, F-actin depolymerization, and increased cell size) were mimicked by bafA1, but not MG132 treatment, further supporting the model that activity of the RHOA pathway can be controlled by the autophagy–lysosome pathway (Supplementary Fig. S5B and S5C). Consistently, the active, but not inactive, RHOA mutants were long-lived proteins selectively degraded by autophagy (Fig. 4). Indeed, upon inhibition of protein synthesis with cycloheximide, the active RHOA (RHOA⁺, Q63) was degraded within 28 hours and stabilized by inhibiting lysosomal degradation (CQ, Fig. 4A), or autophagosome formation (ATG5 shRNA, Fig. 4B and Supplementary Fig. S6A and S6B) but not proteasomal activity (MG132, Fig. 4A). Inversely, the inactive form of RHOA (RHOA⁻, N19) was unstable and as expected stabilized by inhibition of proteasome but not by inhibition of lysosomal activity or of autophagy (Fig. 4A and B), in agreement with previous reports (24–30).

At this stage, it was of interest to address how autophagy may specifically target the active RHOA. One adaptor by which autophagy acquires specificity is p62 (SQSTM1) that targets ubiquitinated substrates to the autophagy machinery (33). Of interest, p62 appears as a prime candidate for regulating RHOA as it is the sole autophagy adaptor involved in cell mitosis (34), cell spreading (35), and tumor growth (36–38). We observed that p62 not only co-immunoprecipitated (Fig. 4C) and co-localized (Fig. 4D) with ubiquitinated RHOA and LC3-II in CQ-treated cells but was also essential in the selective clearance of active RHOA by autophagy (Fig. 4B and D). Upon p62 silencing and CQ treatment, autophagosomes formed normally (as evidenced by LC3-II conversion, Supplementary Fig. S6A), but the autophagic sequestration (Fig. 4D) and degradation (Fig. 4B) of active RHOA were defective. Collectively, these results strongly suggest a new signaling role for p62-dependent autophagy in the control of RHOA pathway.

The multinucleate phenotype of a3^−/− cells raised the question of whether autophagy may control cytokinesis through regulation of RHOA. Autophagy was suggested to clear the midbody ring after cytokinesis completion (39, 40). Likewise, the depletion of several autophagy genes (Atg5, Becn1, Uvrag, Bif-1, And Vps34) was shown to lead to polynucleation, but the role of autophagy in controlling RHOA activation during cytokinesis was not documented (8, 41).

Although several key RHOA activators have been identified (42), little is known about the mechanisms that confine active RHOA at the midbody. Using the a3^−/− cells, we observe that little, if any, active RHOA was at the equatorial furrow and was instead sequestered within autolysosomes, close to the midbody (Fig. 5A), likely as a result of increased sequestration. Inversely, when we blocked autophagosome formation by ATG5 shRNA (Supplementary Fig. S6A), RHOA was highly enriched at the equatorial furrow of cytokinetic A549 lung cancer cells (Fig. 5B, Supplementary Fig. S7A). Notably, the RHO activity zone in ATG5-depleted cells was 3 times as wide and bright as in controls (control shRNA = 3.7 ± 0.8 μm; ATG5 shRNA = 11.4 ± 2.6 μm; mean ± SD; Fig. 5C and D). Outside the cell equator, RHOA activity was also abnormally high at the cell cortex (Fig. 5B, Supplementary Fig. S7). Accordingly, Atg5 knockdown in mouse embryonic fibroblasts (Fig. 5E) and ATG7 or p62 depletion in A549 cells (Supplementary Fig. S6) faithfully recapitulated the same phenotype with regard to the RHOA pathway. Likewise, closer examination indicated that the upstream regulators required for narrowed activation of RHOA such as the kinesin MKLP1, the RHOA GEF ECT2, and spindle microtubules concentrated at the midbody of cytokinetic cells (Supplementary Fig. S8A and S8B; ref. 43). It is also highly likely that the loss of ATG5 would create defects in the generation and the delivery of new membranes to the cleavage furrow, a process that is central to cytokinesis. As shown in Supplementary Fig. S8C, the delivery of endosomes to the midbody appeared not to be affected by ATG5 depletion: the endosomes were delivered to the cleavage furrow and clustered on either side of midbody of ATG5-depleted cells, as observed in control cells. In contrast, ATG5-depleted cells exhibited in addition to RHOA a broader distribution of downstream F-actin that triggers the formation of actomyosin ring (Supplementary Fig. S7B). Our data therefore suggest that the degradation of RHOA by autophagy may function as a key, but hitherto uncharacterized, mechanism concentrating active RHOA at midbody.

Considering the apparent connection between autophagy and RHOA, a key issue is how defects in autophagy might affect cell behavior in a way relevant for cancer progression. Maintaining the appropriate amount of active RHOA at midbody is critical for faithful cytokinesis as it dictates the position, the formation and the contraction of actomyosin ring (42, 44). If our model were correct, one would expect that the inhibition of autophagy sequestration in autophagy-competent A549 tumor cells would be sufficient to disturb cytokinesis and thereby drive the genomic instability required for tumor progression. As shown in Fig. 6, in control A549 cells, the clustering of RHOA in a narrow zone resulted in the formation of a unique and compact ring whose position and size remained constant throughout furrow ingression (Fig. 6A and B, top). As a result, control cells successfully completed cytokinesis (Supplementary Movie S6). Without functional autophagy, the subsequent hyperactivation of RHOA zone would jeopardize the assembly of an efficient contractile ring, as expected from the cytokinesis defects observed for active RHOA mutants (Supplementary Fig. S4C). Indeed, ATG5-depleted A549 cells progressed through mitosis until the furrow started to constrict, then 52% of the cells showed unstable and loose furrowing that suddenly fell apart, reformed, and constricted more slowly than control furrows, delaying cytokinesis completion (Fig. 6A and B, bottom, Supplementary Movie S6). As result of cytokinesis failure, these autophagy compromises (ATG5 shRNA and p62 shRNA) were sufficient to increase the percentage of cells with multiple nuclei (Fig. 6C), and the frequency of chromosomal gains and losses in nearly all of the chromosomes (Fig. 6D), one hallmark of aggressive cancers. Together, these findings highly suggested that autophagy may act, in part, as a master safeguard mechanism of active RHOA localization during cytokinesis for faithful genome inheritance.

The biochemical, cellular, and genomic changes (increased RHOA activity, increased cellular sizes, and aneuploidy) we consistently observed herein following autophagy inhibition (irrespective of particular autophagy defects) are noteworthy because they accompany the onset and progression of cancers. RHOA overexpression has been observed in many aggressive cancers, such as breast, colon, prostate, and lung cancers, but has been attributed largely to increased RHOA transcription (45). Consistent with a role of autophagy in RHOA degradation, we found that RHOA, together with p62 and LC3-II, were overexpressed in late stages of NSCLCs (T; pTNM stage IIIA), compared with normal epithelia (N) and stage I adenocarcinomas (Fig. 7A and B). An impaired autophagic degradation rather than an increased transcription was correlated with RHOA overexpression, as the levels of RHOA expression and activity increased progressively with the accumulation of the autophagy substrates p62 and LC3-II (Fig. 7A), and no significant changes in RHOA mRNA expression were measured (Fig. 7A, right). Of interest, the inactivation of autophagy and the subsequent RHOA upregulation were not early events but instead were late events in the progression of lung cancer, when these cancers acquire a more genomic instability. All tumor cells positive for p62 were also RHOA-positive (Fig. 7B). p62 overexpression, as a readout of an autophagy defect, was associated with a reduced or absent RHOA membrane localization, which instead displayed intracellular localization (T, right). In sharp contrast, normal bronchial epithelial cells that barely expressed p62 showed strong membrane expression of RHOA primarily at their apical ciliated cell surface (N, left).

So far, most studies suggest that signal termination and irreversible progression through cell cycle depend largely on the proteasomal degradation of key regulatory proteins (46). Recently, Gao and colleagues provide the first evidence that autophagy negatively regulates Wnt signaling by degrading Dishevelled; however, this occurs under nutrient starvation (11). Therefore, the ability of autophagy to degrade signaling proteins under basal conditions and thereby to ensure tumor-suppressive functions remains to be established.

We show here that autophagy promotes the degradation of active RHOA. In support of this, (i) RHOA-GTP was elevated in cells deficient in autophagosome clearance, (ii) importantly, RHOA failed to localize to midbody and instead accumulated within autolysosomes, (iii) a role for autophagy in regulating RHOA-GTP was then suggested by the shRNA-mediated inhibition of autophagosome formation: ATG5 shRNA dramatically increased the localization of RHOA-GTP at the equatorial furrow of cytokinetic cells. One related RHO GTPase, RAC, was not targeted by autophagy. As a result, defects of the entire autophagy pathway [i.e., either at the formation (ATG5 or ATG7 shRNA), sequestration (p62 shRNA), or degradation of autophagosomes (v-ATPase a3^−/− cells)] similarly drive the formation of unstable and loose furrowing, disturbing cytokinesis completion and genome inheritance; processes that directly contribute to cancer development.

The regulation of RHOA is unique in that it involves GEF, GAP, and GDI proteins, along with the proteasome (47) and autophagy. Depending on its activation state, we provide the first lines of evidence that RHOA used distinct routes for degradation: while the proteasome degraded the cytosolic and inactive forms, the autophagy pathway specifically degraded the membrane-associated and active pool of RHOA. This is consistent with the recently reported degradation of 2 constitutively active RHO, RHOH and RHOB, within lysosomes (48, 49) and the redistribution of active RHOA to undefined perinuclear localization after treatment of enterocytes with an autophagy inducer, lipopolysaccharide (LPS; ref. 50). We therefore propose that autophagy may act as a tumor suppressor pathway, in part, by turning off RHOA activation (Fig. 7C). In this model, the remarkable dynamics of autophagy together with its integration of extracellular cues might dictate the time and place where a RHOA is active and is able to interact with its downstream substrates. Accordingly, we reported the targeting of autolysosomes at midbody during cytokinesis, the same subcellular and temporal localizations where RHOA should be controlled. Therefore, autophagy might be critical for localized degradation of active RHOA at midbody and thereby proper actin dynamics during accurate completion of cytokinesis and faithful genome inheritance.

The development of a cancer depends on the ability of tumor cells to acquire growth advantages. Whatever the mutations, deletions, and epigenetic silencing of autophagy genes, our findings provide new information on how autophagy defects can drive tumor progression through deregulation of RHOA pathway. As autophagy is commonly downregulated in cancers (1) where RHOA is overexpressed (47), this new paradigm may be a general mechanism for the acquisition of aneuploidy and progression of human cancer cells.

No potential conflicts of interest were disclosed.

Conception and design: A. Belaid, B. Mograbi

Development of methodology: A. Belaid, M. Cerezo, B. Mograbi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Belaid, A. Chargui, F. Pedeutour, S. Giuliano, M. Ilie, I. Rubera, M. Tauc, S. Barale, C. Bertolotto, G.F. Carle, B. Mograbi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Belaid, A. Chargui, B. Mograbi

Writing, review, and/or revision of the manuscript: M. Ilie, V. Vouret-Craviari, D.J. Klionsky, G.F. Carle, B. Mograbi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Corcelle-Termeau, M. Ilie, P. Brest, P. Hofman, B. Mograbi

Study supervision: B. Mograbi

The authors thank Nathalie Rochet, Nathalie Singer, Etienne Boulter, Jean Albrengues, and Emmanuel Lemichez for helpful discussions; Laurence Cailleteau, Thibault Fabas, Annie-Claude Peyron, Valérie Pierrefite-Carle, Isabelle Mothe, Adrien Botta, and Virginie Tanga-Gavric for their technical assistance; and Prof. Mouroux and Prof. Vénissac (Thoracic Surgery Department, Nice, France) and Dr. N. Mizushima (Tokyo, Japan) for providing us with surgical lung specimens and Atg5 KO MEFs, respectively.

This work was supported by grants from “Institut National de la Santé et de la Recherche Médicale”, “Agence de l'Environnement et de la Maîtrise de l'Energie” (A. Belaid and A. Chargui: ADEME n° 0862C0044), Agence régionale santé Provence Alpes Côte d'Azur and Direction régionale de l'Environnement, de l'aménagement et du logement (A. Belaid: plan régional santé environment PRSE PACA n°6.3.3.3 et 6.3.3.4), “Association pour la Recherche contre le Cancer” (ARC Grants no SL220110603478), “Programme Hospitalier De Recherche Clinique” (P. Hofman: PHRC Nice CHU 2003), “Cancéropole PACA” (PROCAN 2007, axe II), “Institut National du Cancer” (P. Hofman: PNES POUMON INCa), and NIH (D.J. Klionsky: GM053396).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.