Endosomal maturation controls trafficking and degradation of macromolecules in eukaryotic cells and represents an important regulatory point in the endocytic pathway. Progression through this phase is primarily recognized through the change of endocytic membrane proteins, particularly the transition from a Rab5 to Rab7 positive compartment. This specific detachment/attachment of Rab5 and Rab7 respectively, seems to regulate this checkpoint in endo-lysosmal pathway via a cascade of complex interactions, emphasizing mutual feedback mechanisms. The Rab proteins have over the last two decades been connected to several important roles in various vesicular trafficking pathways in the cell. While the process of maturation is a complex event, involving many different effector proteins, there is a growing understanding of what roles the different proteins inhabit and the dynamic interplay between them.
In this project we set out to establish a method for investigating if Rab7 has a regulatory role on the coat dynamics of Rab5. We made use of three different inducible cellular systems that have been known to generate enlarged endocytic compartments and transiently transfected these cells with Rab5-wt-mCherry, Rab7-wt-EGFP, Rab7-Q67L-EGFP or Rab7-T22N-EGFP. From this enlarged endocytic structures co-transfected with Rab5-wt-mCherry and Rab7 alternates we were able to accurately measure the kinetics of Rab5-wt-mCherry. The binding dynamics of Rab5-wt-mCherry were measured prior to and during maturation. Moreover, Rab5-mCherry coat detachment during maturation was quantified by calculating the maturation half time (M½) and the maturation end point (Mend) as a function of Rab5-wt-mCherry (see materials and methods). Prior to maturation we measured the on/off-cycling of Rab5-wt-mCherry by FRAP experiments on stable early endosomes and calculated the T½ recovery of Rab5-wt-mCherry. In cells expressing either Rab7-wt-EGFP or the mutants, any perturbations in the Rab5-wt-mCherry coat kinetics prior to and during maturation may imply a regulatory role for Rab7 in Rab5 coat kinetics.
Our results indicate that Rab7 is likely to play an important role in controlling the kinetics of Rab5. The different Rab7 mutants that were transiently transfected into the various systems led to some interesting results in Rab5 coat dynamics, both during and prior to maturation.