Showing papers by "Eeva-Liisa Eskelinen published in 2019"

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs

Abstract: Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

Correlative Light and Electron Microscopy of Autophagosomes.

Abstract: Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases.

Abstract: Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.

A Computer-Vision-Guided Robot Arm for Automatically Placing Grids in Pioloform Film Preparation

Abstract: Preparing pioloform/formvar support films on transmission electron microscopy (TEM) grids is a routine laboratory procedure in practically all electron microscopy units. In current practice, these grids are manually placed on the support film one by one using special tweezers, a process requiring a steady hand. The work is often ergonomically awkward to continue for a longer period of time. In this article, we describe a low-cost, computer vision-guided robot arm that automatically places the grids on the film. The success rate of the prototype robot is 90%, which is comparable to an experienced laboratory technician.

Driving next-generation autophagy researchers towards translation (DRIVE), an international PhD training program on autophagy.

Abstract: The European autophagy consortium Driving next-generation autophagy researchers towards translation (DRIVE) held its kick-off meeting in Groningen on the 14th and 15th of June 2018. This Marie Sklo...

Abstract 4153: Therapeutic targeting autophagy to sensitize cancer immunotherapy in various cancer types

Abstract: Although cancer immunotherapy has revolutionized cancer treatment, patient response to immunotherapy remains varied. Despite progress, the mechanisms limiting cancer immunotherapy are not yet fully understood. A low number of tumor infiltrating T cells (cold tumor) is one of the limiting factors for cancer immunotherapy. Agents that enhance immunotherapy by shifting cold tumors to hot tumors will greatly benefit cancer immunotherapy. In prostate cancer, the majority of tumors are known to be cold and hence, cancer immunotherapy is not the ideal treatment option. Here, we use a prostate cancer model as an example to demonstrate that modulating the tumor microenvironment through altering autophagy will change the tumor cytokine secretion profile, which in turn attracts immune lymphocytes into the tumor microenvironment. In tandem, we have identified a candidate compound known as ESK981 for such a purpose. Method A small molecule library was used for screening autophagy activity and cytokine secretion. Various types of human cancer cell lines (prostate, renal, bladder, breast etc.) and multiple syngeneic mouse lines were examined for autophagy activity as well as an in vitro response to interferon stimulation with or without ESK981. A syngeneic mouse prostate cancer was used for the in vivo examination of autophagy as well as the anti-tumor effect by ESK981 monotherapy and/or in combination with anti-PD-1 therapy. Conclusion We have discovered a robust, novel autophagy-modulating small molecule, named ESK981, for the treatment of various cancer types as a monotherapy. In addition, we have demonstrated that autophagy has an essential role in the anti-tumor effect of immunotherapy, especially for anti-PD-1 in syngeneic prostate cancer model. Therefore, the use of a small molecule such as ESK981 to target autophagy can enhance immunological infiltration induced by cancer immunotherapy, such as immune checkpoint blockade, for non-immunogenic tumors. Conflict of Interest Statement A.M.C. is a co-founder and serves on the Scientific Advisory Board of Esanik Therapeutics, Inc. which owns to the rights to the clinical development of ESK981. Esanik Therapeutics, Inc. did not fund or approve the conduct of this study. Funding This project is supported by Prostate Cancer Foundation. Citation Format: Yuanyuan Qiao, Jae E. Choi, Josh N. Vo, Jean C. Tien, Lisha Wang, Lanbo Xiao, Stephanie A. Simko, Andrew D. Delekta, Nathan B. Hodge, Parth Desai, Kristin Juckette, Alice Xu, Fengyun Su, Rui Wang, Xuhong Cao, Xiaoju Wang, Xiaoming Wang, Javed Siddiqui, Zhen Wang, Amelie Bernard, Ester Fernandez-Salas, Nora M. Navone, Ke Ding, Eeva-Liisa Eskelinen, Elisabeth I. Heath, Daniel J. Klionsky, Weiping Zou, Arul M. Chinnaiyan. Therapeutic targeting autophagy to sensitize cancer immunotherapy in various cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4153.

Autophagy, Inflammation, and Metabolism (AIM) Center in its second year

Abstract: The NIH-funded center for autophagy research named Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center is now completing its second year as a working center with a mission to promote autophagy research locally, nationally, and internationally. The center has thus far supported a cadre of 6 junior faculty (mentored PIs; mPIs) at a near-R01 level of funding. Two mPIs have graduated by obtaining their independent R01 funding and 3 of the remaining 4 have won significant funding from NIH in the form of R21 and R56 awards. The first year and a half of setting up the center has been punctuated by completion of renovations and acquisition and upgrades for equipment supporting autophagy, inflammation and metabolism studies. The scientific cores usage, and the growth of new studies is promoted through pilot grants and several types of enablement initiatives. The intent to cultivate AIM as a scholarly hub for autophagy and related studies is manifested in its Vibrant Campus Initiative, and the Tuesday AIM Seminar series, as well as by hosting a major scientific event, the 2019 AIM symposium, with nearly one third of the faculty from the International Council of Affiliate Members being present and leading sessions, giving talks, and conducting workshop activities. These and other events are often videostreamed for a worldwide scientific audience, and information about events at AIM and elsewhere are disseminated on Twitter and can be followed on the AIM web site. AIM intends to invigorate research on overlapping areas between autophagy, inflammation and metabolism with a number of new initiatives to promote metabolomic research. With the turnover of mPIs as they obtain their independent funding, new junior faculty are recruited and appointed as mPIs. All these activities are in keeping with AIM's intention to enable the next generation of autophagy researchers and help anchor, disseminate, and convey the depth and excitement of the autophagy field.