Browsing by Author "Honkanen, Riina"

Autophagy is a major cellular catabolic pathway which is responsible for the degradation of protein aggregates and damaged organelles, as well as the replenishment of the cellular energy levels during starvation by degrading dispensable cytoplasmic components. In autophagy, cytoplasmic material is sequestered in double-membrane vesicles termed autophagosomes, which ultimately fuse with the endosomal and lysosomal compartment to form organelles called autolysosomes, in which the secluded cellular constituents are digested. The degradation products are transported back to the cytoplasm, and the cell can use them for biosynthetic reactions or energy production. Rab GTPases are key regulators of intracellular membrane trafficking, functioning in multiple processes including autophagy. They undergo a so-called Rab GTPase cycle, where they mediate downstream signalling according to the bound guanine nucleotide. Rab GTPases are generally small, 20-25 kDa of size, and are structurally conserved throughout phylogeny with the exception of the hypervariable C-termini. However, Rab24 GTPase contains unique amino acids that confer special characteristics not found among other Rab GTPases, such as low intrinsic GTPase activity. Rab24 GTPase has been implicated in the late stages of autophagy, where it has been suggested to function in autolysosomal clearance. The role of Rab24 GTPase in autophagy was further studied in this thesis using Rab24 siRNA and control siRNA transfected HeLa cells with stable expression of mRFP-GFP-LC3, labelled with a mixture of LAMP1 and LAMP2 antibodies. LAMP1 and LAMP2 are lysosomal membrane proteins. The tandem fluorescent-tagged mRFP-GFP-LC3 construct localises to autophagic vacuoles and fluoresces both mRFP and GFP under non-acidic conditions. However, the maturation of the autophagic vacuoles with the simultaneous decrease in the pH abolishes the GFP fluorescence, while mRFP is more acid resistant and continues to fluoresce. The formation of autolysosomes was followed by indirect immunofluorescence labelling with LAMP1/2 antibodies in HeLa mRFP-GFP-LC3 cells. The cells were imaged with a confocal microscope and colocalization of the three colours was analysed in three dimensions with Imaris software. The volumes of GFP and mRFP-positive vesicles (autophagosomes) as well as mRFP and LAMP1/2-positive vacuoles (autolysosomes) between control and Rab24 silenced cells were similar in full culture medium and up to 4 h of serum and amino acid starvation. However, the volume of autolysosomes as well as of the LAMP1/2-labelled compartments was substantially higher in Rab24 depleted cells compared to control cells after 6h of starvation. Taken together, these findings indicate that Rab24 is dispensable for autophagosome formation and maturation, and that Rab24 may be involved in autolysosomal clearance upon prolonged serum and amino acid starvation.