Snapshots from NAS Conference grant receivers
Reports from Lab exchange grantees
One of the aims of the Nordic Autophagy Society (NAS) is to promote excellent research in the autophagy field. To achieve this goal, the NAS offer https://nordicautophagy.org/lab-exchange-grants/, giving the opportunity for a research stay in another group in a period from 5 to 90 days. These research exchanges allow the application of methodologies which are not available in the NAS member’s laboratory, and eventually they will facilitate valuable collaborations between research groups.
Apsana Lamsal (Lab Exchange Grantee)
Does arginine starvation induce selective or bulk autophagy?
Host: Dr. Nikolai Engedal (Oslo University Hospital)
Duration of visit: 4 weeks (Nov 22nd to Dec 17th, 2021)
Cancer cells can alter the Tumor Microenvironment by several mechanisms. They can have altered metabolism and recruit immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs) or neutrophil-like cells, which can secrete arginase-1 (ARG1). We had observed a close link between ARG1 expression and reduced arginine in breast cancer tumors. We therefore hypothesized that local arginine depletion induces autophagy in the tumor. We observed arginine starvation was a strong signal for induction of selective autophagy receptors in breast cancer cells. So, we wanted to know if arginine starvation induces selective or bulk autophagy. To understand this, a lab visit to Dr. Nikolai Engedal’s group was arranged for measuring cytosolic cargo sequestration using the Lactate Dehydrogenase Sequestration (LDH) Assay. This assay allows quantitative measurement of autophagic sequestration of the endogenous cytosolic cargo protein lactate dehydrogenase. The assay was used with success during my stay and already provided some interesting results. In collaboration with Dr Engedal’s lab, we also made mKeima-based reporter cell lines. This further allows us to understand if arginine starvation induces only bulk autophagy, or also selective autophagy of mitochondria. From the very first day to the last, I had a pleasant lab visit with a very good lab environment and the visit was a great success. We will maintain an active collaboration with Dr. Nikolai Engedal. I thank NAS for providing me this opportunity.
Florentina Negoita (Lab Exchange Grantee)
The role of SIK2 in autophagy and its link to human adipose tissue dysfunction in the development of type 2 diabetes
Host: Pr. Eeva-Liisa Eskelinen (University of Turku, Finland)
Duration of visit: 2 weeks (September 30 – October 11, 2019)
The aim of this project is to characterize the molecular mechanisms underlying the regulation of autophagy by SIK2 in adipocytes. Transmission electron microscopy (TEM) is currently considered to be the gold standard test for the evaluation of autophagy, but identification of autophagic structures is challenging, without proper expertise, and might lead to false conclusions. Thus, in order to learn how to correctly identify and differentiate autophagic structures from other cellular structures and how to quantify the TEM images, a research visit was organized in the lab of Pr Eeva-Liisa Eskelinen, a leading scientist on TEM imaging of autophagic structures. Adipocytes treated with a SIK inhibitor were imaged and images were then analyzed. We found a marked change in the number and volume of autolysosomes, in line with our previous data. With the protocol for TEM sample processing obtained through this collaboration, we are now planning to further explore our findings by establishing the TEM method in our own laboratory, and applying it to 3T3-L1 adipocytes treated with SIK2 siRNA. We will maintain an active collaboration with Pr Eeva-Liisa Eskelinen. In conclusion, the research visit was very successful. I received excellent training in TEM analysis of autophagic structures, and we also generated novel, interesting data, which has opened up a new direction of study in the project.
Florentina Negoita (2nd from the left) with the group of Prof Eeva-Liisa Eskelinen (1st from the left)
Pauline Verlhac (Lab Exchange Grantee)
WIPIng out Flu: Role of WIPI2 in restricting Influenza A virus
Host: Prof. Jörn Dengjel (University of Fribourg, Switzerland)
Duration of visit: 7 days (June 16th-23rd 2019)
The ultimate goal of the project is to decipher how WIPI2 controls viral replication, using influenza A virus (IAV) as a relevant clinical model. Therefore, we plan to use an unbiased and broad approach to identify potential WIPI2 binding partners involved in the control of IAV infection. To do so, we have used stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) with proximity-dependent biotin labelling by fusing WIPI2 with APEX2 in order to identify interacting proteins in both infected and non-infected cells. To determine which WIPI2 neighbours are regulated by IAV infection, we combined APEX2 with SILAC-based MS. Neighbours identified thanks to this Lab Exchange will now be tested for their impact on IAV replication by siRNA interference and their interaction with WIPI2 confirmed by IP. The work was realised with the help of Devanarayanan Siva Sankar, who taught the experimental part, and Professor Jörn Dengjel for the statistical data analysis. The exchange allowed me to implement a new and innovative technique into our laboratory as well as to strengthen the collaboration between our two laboratories.
From left to right: Devanarayanan Siva Sankar, Dr Pauline Verlhac and Prof Jörn Dengjel
Yanyah Aman (Lab Exchange Grantee)
Is mitophagy protective against tau aggregation in Alzheimer’s disease?
Host: Dr. William A. McEwan (University of Cambridge, United Kingdom)
Duration of visit: One month (May 6th to June 7th 2019)
Mitochondrial quality is tightly regulated by mitophagy, in which damaged mitochondria are degraded and recycled. However, the underlying mechanism remains elusive. We wished to investigate how mitophagy inhibits p-tau using a novel in-vitro system. For this purpose, an overseas visit to Dr McEwan’s lab was arranged in order to acquire and apply high-content quantitative assays for measuring cytosolic tau aggregation in situ. This assay not only allows to assess the impact of mitophagy inducers on the development of p-tau aggregates; but also provide a platform to evaluate whether mitophagy can aid in degradation of already established tau aggregates, which further enhances the translational relevance. The assay was used with success during my stay and already provided some interesting results. In collaboration with Dr McEwan’s lab, we will further explore on the underlying molecular mechanisms that contribute to p-tau pathogenesis with the perspective of the mitophagy and its role using this exciting seeded aggregation of tau model system.
The McEwan group visited by Dr. Yahyah Aman in May-June 2019