Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is gradually exchanged with Rab7a on the same endosome. Recent publications show that Rab5 detachment is sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a rapid diffusion-like exchange between the cytosol and endosomal membrane, and the following second phase is slower where Rab5 converges into a specific domain that specifically detaches as a Rab5 indigenous endosome. Both Rab5 and Rab7 have to be dephosphorylated to be localized to the endosomal membranes. Tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that have been shown to regulate the localization of Rab7 to endosomes. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183 that could regulate the recruitment of Rab7 on to late endosomes and may also be involved in the Rab5 endosomal binding dynamics.
Aim of Study:
Recent discoveries indicate a new pathway for maintenance of the endosomal homeostasis and cell growth. However, the molecular mechanisms of this particular regulation is not yet deciphered.
In this master project the candidate will focus on the molecular mechanisms of endosomal maturation. More precisely, try to locate the shared activator/deactivator of Rab5 detachment and Rab7 attachment through phosphorylation/dephosphorylation.
Methods:
In this project we will approach this predicament through different high-end microscopy techniques as Fluorescence Recovery After Photobleaching (FRAP), photoactivation 2, 3 and 4D live cell super-resolution imaging and high-content screening microscopy.
- Cloning techniques for construction of the different GFP/RFP/mCherry fusion proteins localized to the endosomal organelles.
- RNA interference, tetracycline transcriptional activator
- PCR, Western Blot
- Cell culturing techniques for producing stably transfected cell-lines
- Transient transfections of fusion proteins and RNA interference (RNAi)
- Quick change mutations and primer design
- Advanced image processing and analysis
Progress:
- First part of this project will be to learn basic cell work and performing transient transfections of fluorescently fused RabGTPases.
- The master student will be introduced to a number of high-end microscopes, Olympus SoRa, Andor Dragonfly and Zeiss Fast Airyscan, all three microscopes are super resolution microscopes and Total Internal Reflection Microscopy (TIRF). –
- The candidate will become an expert user in the different microscopy techniques
- Second step of this project we utilize an already published method to inhibit the activity of PTEN, an Inducible PTEN gene silencing by a tetracycline transcriptional activator-regulated short hairpin RNA.
- We will analyze of the coat kinetics through FRAP imaging/analysis on the endosomal endosomes after inhibition of the RabGTPases and PTEN.
- Additionally, we will track and identify endosomal interacting vesicles, involved in the maturation process.
- Endosomal tracking will be done in 2, 3, and 4 dimensions utilizing sophisticated software as Imaris (Bitplane) and ImageJ.
- Biochemical analysis of receptor phosphorylation and protein concentration in treated cells
Our goal:
One step closer to understand the endosomal maturation process regulated by the Rab5 and Rab7 switch. This can prove to be important understanding of the future role of RabGTPases as a therapeutic target for cancer treatment.
Main advisor Professor Oddmund Bakke
Daily advisor: Frode Skjeldal and Linda Haugen – at NorMIC-Oslo, IBV?s advanced imaging platform.