2d single particle tracking and super-resolution imaging in living cells
The main aim of this experiment has been to use the single particle tracking (SPT) approach to tackle the molecular basis of timely human pathologies, in particular Alzheimer’s disease. Most of the current research on the molecular mechanisms of Alzheimer’s disease is based on averaged results obtained using bulk methods. In this case, many important details can be missed and only the most prominent features are eventually taken into account. This consideration may explain at least part of the discrepancies arising from the several models that have been proposed during the last years. Within this context, single particle tracking experiments carried out during the last five years have provided a better understanding of the pathogenesis of Alzheirmer’s disease by monitoring the dynamics of single cytotoxic oligomeric species and their interaction with the components of the plasma membrane. Overall, the experimental strategy for 2D surface SPT is based on labelling immunochemically the target species with antibodies coupled to small (10-30 nm) and extremely photostable fluorescent probes called ‘quantum dots’ (QDs). Real time recordings of single molecules moving on the plasma membrane of living cells are carried out using a high sensitivity camera. The accurate localization of the fluorescent particle is obtained by fitting its point spread function with a Gaussian function. The analysis of the trajectories allows to calculate the mean square displacement and the diffusion coefficient of each particle. These parameters are used to fit the data to several models of diffusion, and distinguish between different types of motions (random walk, confined motion, directed motion, etc.). In particular, by combining the use of conformation-specific antibodies and SPT techniques, we have investigated the mobility of individual cytotoxic Aβ1-42 oligomers on the plasma membrane of living cells. Distinct structural types of Aβ1-42 oligomers were labeled with two different conformation-specific antibodies. While both types of oligomers showed a heterogeneous dynamic behavior, their overall mobility was found to be significantly different. Conversely, we discovered that other amyloid oligomers sharing a similar conformation but composed of different peptides (amylin and prion Sup35NM) display dynamic behaviors comparable to those found for Aβ1-42 oligomers. This study provides evidence for a link between the quaternary structure and the membrane mobility of proteins, revealing that structurally analogous supramolecular assemblies diffuse similarly in cells. Furthermore, we have showed that amyloid oligomers and aggregates formed by Aβ1-42 and amylin (a peptide associated with the development of type II diabetes) interact with GM1 and decrease dramatically its lateral diffusion on the plasma membrane of living neuroblastoma cells. The confinement of GM1, a constituent of membrane rafts involved in neuroprotection, at the level of both types of amyloid aggregates can interfere with cell signaling pathways and contribute to the loss of neuroprotection. In agreement with these results, also amyloid aggregates formed by the prion protein Sup35 were found to reduce the mobility of GM1.
Research & Technical staff: