The Eskelinen group aims to understand membrane dynamics during autophagosome biogenesis.
Autophagy is a lysosomal degradation pathway for cytoplasmic material and organelles that maintains cellular homeostasis during nutrient deprivation and stress. Autophagy is an important survival mechanism during short-term starvation: by degrading non-essential components, cells acquire nutrients for energy production and vital biosynthetic reactions. Autophagy also contributes to the clearance of damaged organelles and aggregate-prone proteins, protection against metabolic stress and DNA damage, and even to longevity of the organism. Defective autophagy has been connected to many human diseases including cancer, myopathies, metabolic diseases, and neurodegenerative diseases.
In macroautophagy, autophagosomes are formed in a subcompartment of the endoplasmic reticulum. A flat membrane cistern called the phagophore emerges first, which then elongates and forms a double-membrane bound autophagosome. Autophagosomes acquire hydrolytic capacity by fusing with endosomes and lysosomes. The origin of phagophore and autophagosome membranes is one of the longest lasting open questions in the field. We address this question using three-dimensional electron microscopy, live-cell imaging and correlative light-electron microscopy.
Highlighted publications of Eskelinen group are listed here.
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Autophagosomes have a close relationship with the endoplasmic reticulum (ER). Using electron tomography, we showed that phagophores are lined by rough ER on both sides and have tubular membrane connections with ER. We also found that the same phagophore can have contacts with several organelles simultaneously, including ER, ER exit sites, mitochondria, Golgi complex, and endosomes. Our findings suggest that these organelles may have a role in delivering material for the growing phagophore.
Biazik J, Ylä-Anttila P, Vihinen H, Jokitalo E and Eskelinen EL: Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 2015, 11:439-451. DOI:10.1080/15548627.2015.1017178
Biazik J, Vihinen H, Jokitalo E, Eskelinen EL: Untrastructural characterization of phagophores using electron tomography on cryoimmobilized and freeze substituted samples. Methods Enzymol (in press)
Ylä-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL: 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 2009, 5:1180-1185.
In order to further clarify the origin of phagophore membranes, we aim to visualize phagophore precursors at high resolution in three dimensions. We are currently using live-cell imaging and correlative light-electron microscopy to trace nascent phagophores. Another appoach is to use various knockout cell lines in which phagophore biogenesis is halted at an early stage. To preserve close to native fine structure, we use high-pressure freezing and freeze substitution, which also effectively preserves the cytoskeletal filaments.
Biazik J, Vihinen H, Anwar T, Jokitalo E, Eskelinen E-L: The versatile electron microscope: an ultrastructural overview of autophagy. Methods 2015, 75:44-53.
The beclin 1 gene is monoallelically deleted in 40-75% of human sporadic breast, ovarian, and prostate cancers. Heterozygous disruption of beclin 1 gene in mice results in increased cellular proliferation and reduced autophagy. Thus beclin 1 is a haplo-insufficient tumor-suppressor gene.
Beclin 1 is part of the PI3-kinase complex that is needed for autophagosome biogenesis. The PI3-kinase produces PI3P, which recruits downstream autophagy proteins to the forming phagophore. We aim to elucidate the role of Beclin 1 in autophagosome biogenesis by using constructs in which Beclin 1 is artificially targeted to different organelles.
Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen E-L, Mizushima N, Ohsumi Y, Cattoretti G, Levine B: Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003; 112:1809-1820.
RAB24 is a small GTPase that has unusual features, such as unusual amino acids in the GTP-binding region and preferential occurrence in the GTP-bound form. RAB24 has been know to localize to autophagic vacuoles and it has been suggested to regulate autophagy.
We showed that RAB24 is required for the clearance of autolysosomes in cells under basal conditions; autolysosomes accumulate when RAB24 is silenced. RAB24 localizes to the limiting membranes of autophagosomes, and this localization depends on prenylation and GTP-binding. RAB24 also enhances the clearance of an aggregate-prone autophagy substrate. However, when the cells are starved, macroautophagy is independent of RAB24.
Ylä-Anttila P, Mikkonen E, Happonen KE, Holland P, Ueno T, Simonsen A, Eskelinen EL: RAB24 facilitates clearance of autophagic compartments during basal conditions. Autophagy 2015, 11:1833-48.
LAMP2, or lysosome associated membrane protein 2, is a transmembrane protein of late endosomes and lysosomes. Mutations in lamp2 gene cause Danon disease that leads to fatal cardiomyopathy, myopathy and varying degree of mental retardation. LAMP2 protein is needed for efficient maturation of autophagosomes into degradative autolysosomes, and deficiency of LAMP2 leads to accumulation of autophagic vacuoles in several tissues including the heart and skeletal muscle.
We have shown that LAMP2 plays a role in intracellular cholesterol transport. If LAMP2 is missing, cholesterol accumulates in late endosomes and lysosomes, and intracellular cholesterol esterification and storage in lipid droplest are drastically reduced. We have performed structure-function analyses of the LAMP2 molecule to elucidate its role in cholesterol transport, and further monitored the lipid species that accumulate in LAMP2-deficient cells.
Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M, Blanz J, Domanskyy M, Breiden B, Brodesser S, Landgrebe J, Sandhoff K, Ikonen E, Saftig P, Eskelinen E-L: Role for LAMP-2 in endosomal cholesterol transport. J Cell Mol Med 2011, 15:280-295