SUN4 is a spermatid type II inner nuclear membrane protein that forms heteromeric assemblies with SUN3 and interacts with lamin B3.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (for 'linkers of the nucleoskeleton and the cytoskeleton'). Previous studies have demonstrated that the testis-specific SUN domain protein SUN4 (also known as SPAG4) is a vital player in the directed shaping of the spermatid nucleus. However, its molecular properties relating to this crucial function have remained largely unknown, and controversial data for the organization and orientation of SUN4 within the spermatid nuclear envelope have been presented so far. Here, we have re-evaluated this issue in detail and show robust evidence that SUN4 is integral to the inner nuclear membrane, sharing a classical SUN domain protein topology. The C-terminal SUN domain of SUN4 localizes to the perinuclear space, whereas the N-terminus is directed to the nucleoplasm, interacting with the spermiogenesis-specific lamin B3. We found that SUN4 forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Together, our results contribute to a better understanding of the specific function of SUN4 at the spermatid nucleo-cytoplasmic junction and the process of sperm-head formation.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.