Session: 7

PS7-08 | Generation of a Drosophila melanogaster line for studying of the structural dynamics of the actin/spectrin membrane-associated periodic skeleton in situ

Fabian Orlando Ramos

INIMEC-CONICET-Universidad Nacional de Córdoba

It has been recently discovered that axons and dendrites possess a particular arrangement of their cortical skeleton, referred to as the membrane-associated actin/spectrin periodic skeleton or MPS. The MPS is a periodic protein structure consisting of actin “rings” located transversely to the axon and separated every 190 nm by α/β-spectrin tetramers “spacers”. It is speculated that a better understanding of the MPS organization, distribution and dynamics could shed light into understanding the biology of axons and dendrites. The MPS can only be described using super-resolution (SR) microscopy approaches, since its special features lies below the diffraction limit of light. All published approaches describe static pictures of the MPS in cultured neurons. On the contrary, we anticipate that studying MPS dynamics in tissue (in situ) will provide more relevant evidence on its possible functions. In order to do this, we present a plan for producing a Drosophila melanogaster line in which the endogenous single beta-spectrin gene will incorporate successive peptide tags in the C-terminus, in a cell- and time-specific manner. This animal model will allow us to study the dynamics of beta-spectrin within the MPS in situ. To do this, we will perform CRISP/Cas9-mediated gene editing of the beta-spectrin gene, so that it incorporates different terminal codons after recombination by Flippase and Cre, which can be controlled in time and space by existing fly lines.