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 E. A; RAIMUNDA D; BISBAL M; UNSAIN N

San Luis

Congreso; XXXVIII Congreso Reunión Anual - Sociedad Argentina de Investigación en Neurociencia. San Luis, Argentina; 2023

SAN - Sociedad Argentina de Investigación en Neurociencia

Axons and dendrites possess a particular arrangement of their cortical skeleton, referred to as the Membrane-associated actin/spectrin Periodic Skeleton (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 tetramer “spacers”, making the MPS only visible using super-resolution microscopy approaches. Most studies have described the MPS in cell culture and the dynamics of the spectrin “spacers” within each period have not been investigated in detail. Our project will shed light into these aspects in the nervous system 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 a transgenic fly, in which the endogenous β-spectrin gene can be recombined in a cell-type and time-specific manner to include C-terminus tags that can then be detected by nanobodies. Thus, a specific neuronal population will recombine to include a C-terminus “ALFA-tag” and that subpopulation can then suffer a second recombination to replace “ALFA-tag” by “BC2-tag”. We are going to show advances in the molecular cloning steps towards CRISPR/Cas9-mediated editing as well as the production of the nanobodies for the detection of the tags. This transgenic fly will allow the examination of dynamical properties of the MPS in nerve tissue.