Eeva-Liisa Eskelinen

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene

Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

Role for Rab7 in maturation of late autophagic vacuoles

The small GTP binding protein Rab7 has a role in the late endocytic pathway and lysosome biogenesis. The role of mammalian Rab7 in autophagy is, however, unknown. We have addressed this by inhibiting Rab7 function with RNA interference and overexpression of dominant negative Rab7. We show here that Rab7 was needed for the formation of preferably perinuclear, large aggregates, where the autophagosome marker LC3 colocalised with Rab7 and late endosomal and lysosomal markers. By electron microscopy we showed that these large aggregates corresponded to autophagic vacuoles surrounding late endosomal or lysosomal vesicles. Our experiments with quantitative electron microscopy showed that Rab7 was not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles. Finally, we showed that the recruitment of Rab7 to autophagic vacuoles was retarded in cells deficient in the lysosomal membrane proteins Lamp1 and Lamp2, which we have recently shown to accumulate late autophagic vacuoles during starvation. In conclusion, our results showed a role for Rab7 in the final maturation of late autophagic vacuoles.

Regulation of starvation- and virus-induced autophagy by the eIF2α kinase signaling pathway

The eIF2α kinases are a family of evolutionarily conserved serine/threonine kinases that regulate stress-induced translational arrest. Here, we demonstrate that the yeast eIF2α kinase, GCN2, the target phosphorylation site of Gcn2p, Ser-51 of eIF2α, and the eIF2α-regulated transcriptional transactivator, GCN4, are essential for another fundamental stress response, starvation-induced autophagy. The mammalian IFN-inducible eIF2α kinase, PKR, rescues starvation-induced autophagy in GCN2-disrupted yeast, and pkr null and Ser-51 nonphosphorylatable mutant eIF2α murine embryonic fibroblasts are defective in autophagy triggered by herpes simplex virus infection. Furthermore, PKR and eIF2α Ser-51-dependent autophagy is antagonized by the herpes simplex virus neurovirulence protein, ICP34.5. Thus, autophagy is a novel evolutionarily conserved function of the eIF2α kinase pathway that is targeted by viral virulence gene products.

At the acidic edge: emerging functions for lysosomal membrane proteins

It has recently become clear that lysosomes have more complex functions than simply being the end-point on a degradative pathway. Similarly, it is now emerging that there are interesting functions for the limiting membranes around these organelles and their associated proteins. Although it has been known for several decades that the lysosomal membrane contains several highly N-glycosylated proteins, including the lysosome-associated membrane proteins LAMP-1 and LAMP-2 and lysosomal integral membrane protein-2/lysosomal membrane glycoprotein-85 (LIMP-2/LGP85), specific functions of these proteins have only recently begun to be recognized. Although the normal functions of LAMP-1 can be substituted by the structurally related LAMP-2, LAMP-2 itself has more specific tasks. Knockout of LAMP-2 in mice has revealed roles for LAMP-2 in lysosomal enzyme targeting, autophagy and lysosomal biogenesis. LAMP-2 deficiency in humans leads to Danon disease, a fatal cardiomyopathy and myopathy. Furthermore, there is evidence that LAMP-2 functions in chaperone-mediated autophagy. LIMP-2/LGP85 also seems to have specific functions in maintaining endosomal transport and lysosomal biogenesis. The pivotal function of lysosomal membrane proteins is also highlighted by the recent identification of disease-causing mutations in cystine and sialic acid transporter proteins, leading to nephropathic cystinosis and Salla disease.

Maturation of Autophagic Vacuoles in Mammalian Cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions, because a block in autophagic maturation causes a disease.

Autophagy: A lysosomal degradation pathway with a central role in health and disease

Autophagy delivers cytoplasmic material and organelles to lysosomes for degradation. The formation of autophagosomes is controlled by a specific set of autophagy genes called atg genes. The magnitude of autophagosome formation is tightly regulated by intracellular and extracellular amino acid concentrations and ATP levels via signaling pathways that include the nutrient sensing kinase TOR. Autophagy functions as a stress response that is upregulated by starvation, oxidative stress, or other harmful conditions. Remarkably, autophagy has been shown to possess important housekeeping and quality control functions that contribute to health and longevity. Autophagy plays a role in innate and adaptive immunity, programmed cell death, as well as prevention of cancer, neurodegeneration and aging. In addition, impaired autophagic degradation contributes to the pathogenesis of several human diseases including lysosomal storage disorders and muscle diseases.

3D tomography reveals connections between the phagophore and endoplasmic reticulum

Autophagosomes have been reported to form in the vicinity of the endoplasmic reticulum (ER). In many cases, the phagophore membrane is observed between two cisternae of rough ER, but it is not known whether these two membranes are directly connected. To investigate the relationship of the phagophore membrane and the ER, we used electron microscopic tomography of serum and amino acid starved normal rat kidney cells. The cells were fixed in glutaraldehyde and reduced osmium tetroxide and embedded in Epon. Dual axis tilt image series were acquired from two successive 250-nm sections. To analyze the three-dimensional (3D) morphology of phagophores and the associated rough ER, 3D tomograms were used to model the ER and phagophore membranes. The tomographic reconstructions revealed connections between the phagophore/autophagosome membrane and the closely located ER cisternae, especially with the ER located inside the autophagosome. The connections were typically formed by narrow extensions from the phagophore/autophagosome to the ER. This finding has potential implications on the origin of autophagosome membranes, and on the mechanism of phagophore membrane extension. In addition, we observed lipid droplets in very close contact with the phagophores/autophagosomes.

The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death

Autophagic cell death is morphologically characterized by an accumulation of autophagic vacuoles. Here, we show that inactivation of LAMP2 by RNA interference or by homologous recombination leads to autophagic vacuolization in nutrient-depleted cells. Cells that lack LAMP2 expression showed an enhanced accumulation of vacuoles carrying the marker LC3, yet a decreased colocalization of LC3 and lysosomes, suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. While a fraction of mitochondria from starved LAMP2-expressing cells colocalized with lysosomal markers, within autophagolysosomes, no such colocalization was found on removal of LAMP2 from the experimental system. Of note, LAMP1 depletion had no such effects and did not aggravate the phenotype induced by LAMP2-specific small interfering RNA. Serum and amino acid-starved LAMP2-negative cells exhibited an accumulation of autophagic vacuoles and then succumbed to cell death with hallmarks of apoptosis such as loss of the mitochondrial transmembrane potential, caspase activation and chromatin condensation. While caspase inhibition retarded cell death, it had no protective effect on mitochondria. Stabilization of mitochondria by overexpression of Bcl-2 or the mitochondrion-targeted cytomegalovirus protein vMIA, however, blocked all signs of apoptosis. Neither caspase inhibition nor mitochondrial stabilization antagonized autophagic vacuolization in LAMP2-deficient cells. Altogether, these data indicate that accumulation of autophagic vacuoles can precede apoptotic cell death. These findings argue against the clear-cut distinction between type 1 (apoptotic) and type 2 (autophagic) cell death.

LAMP proteins are required for fusion of lysosomes with phagosomes

Lysosome‐associated membrane proteins 1 and 2 (LAMP‐1 and LAMP‐2) are delivered to phagosomes during the maturation process. We used cells from LAMP‐deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP‐1‐ or LAMP‐2‐deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp‐1 and lamp‐2 genes yields an embryonic‐lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcγIIA receptors. Phagosomes formed by FcγIIA‐transfected MEFs obtained from LAMP‐1‐ or LAMP‐2‐ deficient mice acquired lysosomal markers. Remarkably, although FcγIIA‐transfected MEFs from double‐deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP‐1 and LAMP‐2 double‐deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3‐phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time‐lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP‐1 and LAMP‐2 had reduced ability to move toward the microtubule‐organizing center, likely precluding their interaction with each other.

μ1A‐adaptin‐deficient mice: lethality, loss of AP‐1 binding and rerouting of mannose 6‐phosphate receptors

The heterotetrameric AP‐1 complex is involved in the formation of clathrin‐coated vesicles at the trans‐Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse μ1A‐adaptin gene causes embryonic lethality at day 13.5. In cells deficient in μ1A‐adaptin the remaining AP‐1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of μ1A‐adaptin‐deficient embryos that show γ‐adaptin binding to membranes, indicating the formation of an epithelial specific AP‐1B complex and demonstrating the absence of additional μ1A homologs. Mannose 6‐phosphate receptors are cargo molecules that exit the TGN via AP‐1–clathrin‐coated vesicles. The steady‐state distribution of the mannose 6‐phosphate receptors MPR46 and MPR300 in μ1A‐deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP‐1 is required for retrograde endosome to TGN transport of the receptor.