Study of MPS in transgenic Drosophila melanogaster line using nanobodies for his detection

RAMOS FO; SOSA MJ; BRUNO VIGNOLO A; GAZAL NG; BRUNO G; MIHELJ P; GOROSTIZA A; RAIMUNDA D; BISBAL M; UNSAIN N

Santa Cruz

Congreso; IUBMB EMBO Workshop Emerging Concepts of the Neuronal Cytoskeleton. Santa Cruz, Chile; 2023

EMBO

Axons and dendrites possess a particular arrangement of their cortical skeleton, referred to as theMembrane-associated actin/spectrin Periodic Skeleton (MPS). The MPS is a periodic protein structureconsisting of actin “rings” located transversely to the axon and separated every 190 nm by α/β-spectrintetramer “spacers”, making the MPS only visible using super-resolution microscopy approaches. Moststudies have described the MPS in cell culture and the dynamics of the spectrin “spacers” within eachperiod have not been investigated in detail. Our project will shed light into these aspects in the nervoussystem of Drosophila melanogaster. Since β-spectrin is expressed in all fly cells, it is necessary to “tag”β-spectrin in a cell specific manner. For this, we are using CRISPR/Cas9-mediated editing to produce atransgenic fly, in which the endogenous β-spectrin gene can be recombined in a cell-type andtime-specific manner to include C-terminus tags that can then be detected by nanobodies. Thus, aspecific neuronal population will recombine to include a C-terminus “ALFA-tag” and that subpopulationcan then suffer a second recombination to replace “ALFA-tag” by “BC2-tag”. We are going to showadvances in the molecular cloning steps towards CRISPR/Cas9-mediated editing as well as theproduction of the nanobodies for the detection of the tags. This transgenic fly will allow the examinationof dynamical properties of the MPS in nerve tissue.