Automated quantification of protein periodic nanostructures

MASULLO, LUCIANO A.; BARABAS, FEDERICO M.; BORDENAVE, MARTÍN D.; VON BILDERLING, CATALINA; UNSAIN, NICOLÁS; JALIL, SAMI; MARTINEZ, GABY F.; BISBAL, MARIANO; GIUSTI, SEBASTIÁN A.; REFOJO, DAMIÁN; CÁCERES, ALFREDO; STEFANI, FERNANDO D.

Berlin

Simposio; Single Molecule Localization Microscopy Symposium 2018; 2018

European Light Microscopy Initiative (ELMI)

Fluorescencenanoscopy imaging permits the observation of periodic supramolecular proteinstructures in their natural environment, as well as the unveiling of previouslyunknown protein periodic structures. Deciphering the biological functions ofsuch protein nanostructures requires systematic and quantitative analysis oflarge number of images under different experimental conditions and specificstimuli. Here we present a method and an open source software for the automatedquantification of protein periodic structures in super-resolved images [1]. Its performance was characterizedanalyzing simulated and hand-picked images. Then, it was applied to theautomated analysis of hundreds of nanoscopy image and thereby obtaining robustbiological information in two questions. Firstly, the abundance and regularityof the spectrin membrane-associated periodic skeleton (MPS) in hippocampalneurons was quantified as a function of days in vitro (DIV). STED and STORM images of theMPS taken at 2 to 40 DIV were analyzed with the software. The automatedanalysis revealed that both the abundance and the regularity of the MPSincrease over time and reach maximum plateau values after 14 DIV. A detailedanalysis of the distributions of correlation coefficients provides indicationof dynamical assembly and disassembly of the MPS [1].Secondly, westudied the actin/spectrin MPS of dorsal-root ganglions (DRGs) in an invitro model of developmental pruning induced by trophic factorwithdrawal (TFW) [2]. We found that the abundance and organization of the axonalactin/spectrin MPS drop shortly after deprivation, remaining low untilfragmentation. Fragmented axons lack MPS (while maintaining microtubules) andacute pharmacological treatments that stabilize actin filaments prevent MPSloss and protect from axonal fragmentation, suggesting that MPS destruction isrequired for axon fragmentation to proceed.[1] F. M. Barabas, L. A.Masullo et al, Scientific Reports 7(2017)[2] N. Unsain et al, ScientificReports 8 (2018)