A method for single molecule localization microscopy of tissues reveals non-random distribution of nuclear pores in Drosophila

Abstract

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles

“…This enabled us to perform DNA-PAINT super-resolution microscopy of the large flight muscles without dissecting individual myofibrils. Such large specimen have rarely been investigated with DNA-PAINT (Cheng et al, 2021; Lelek et al 2021). This shows that DNA-PAINT can be readily applied to super- resolve structures in large tissues if mounting and labelling protocols are optimised.…”