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

BRUNO G; BISBAL M; GAZAL N. G.; GOROSTIZA E. A; RAMOS F.O.; BRUNO VIGNOLO A.; UNSAIN N.

Congreso; XXXVI Congreso Reunión Anual ? Sociedad Argentina de Investigación en Neurociencia. Modalidad Virtual; 2021

It has been recently discovered that axons and dendrites possess a particular arrangement of their corticalskeleton, referred to as the membrane-associated actin/spectrin periodic skeleton or MPS. The MPS is aperiodic protein structure consisting of actin "rings" located transversely to the axon and separated every 190nm 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 canonly be described using super-resolution (SR) microscopy approaches, since its special features lies below thediffraction limit of light. All published approaches describe static pictures of the MPS in cultured neurons. Onthe contrary, we anticipate that studying MPS dynamics in tissue (in situ) will provide more relevant evidenceon its possible functions. In order to do this, we present a plan for producing a Drosophila melanogaster line inwhich the endogenous single beta-spectrin gene will incorporate successive peptide tags in the C-terminus, in acell- and time-specific manner. This animal model will allow us to study the dynamics of beta-spectrin within theMPS in situ. To do this, we will perform CRISP/Cas9-mediated gene editing of the beta-spectrin gene, so that itincorporates different terminal codons after recombination by Flippase and Cre, which can be controlled intime and space by existing fly lines.