Approaches & Tools

Breaking cells apart to get the best out of them: lysosomes

Characterization of components involved in CMA and eMI

We can isolate intact lysosomes and late endosomes/multivesicular bodies from several rodent and human tissues. We are using a global proteomic and lipidomic approaches in the isolated fractions to identify essential components of CMA and eMI. We have also developed a photoswitchable reporter which allows us to identify changes in CMA in intact cells and a split-venus reporter to measure eMI. We are currently using these reporters to perform CRISPRi screenings in order to discover novel effectors and unknown regulators of these selective autophagy pathways.

Learn more about our CMA and eMI reporters.

Read more in our publications.

Consequences of the age-related defect in CMA

We have generated conditional and inducible transgenic mouse models incompetent for CMA in different tissues and have started to investigate possible tissue-dependent differences in the requirements for functional CMA. We are also analyzing the cellular response to CMA blockage and the different compensatory mechanisms elicited. We have found that blockage of CMA leads to important alterations in cellular quality control and cellular metabolism and deficiencies in the response to different stressors. We are now using genetic and chemical approaches to restore CMA.

Impaired autophagy in age related-disorders

We are analyzing changes in autophagy in three main groups of age-related diseases:

neurodegeneration
metabolic disorders
cancer

By combining metabolic assays, cellular fractionation procedures and our in vitro lysosomal transport assays in different animal and cellular models of these diseases, we have found that autophagy malfunctions in conditions such as diabetes, obesity and familial forms of Parkinson’s disease and of taupathies such as Alzheimer’s disease.

KFERQ-finder

Specificity in substrate selection by CMA is obtained through a special recognition mechanism between hsc70 and the KFERQ-like motif present in the amino acid sequence of substrate proteins.

To facilitate further analyses by the scientific community, we have developed a free web-based resource (KFERQ finder) for direct identification of KFERQ-like motifs in any protein sequence.