A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

Abstract

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.

The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

“…, 1998; Kiseleva et al. , 2004, 2007). Crucially, our IEM studies indicate that Mlps not only form the basket at the NPC, but are also arranged horizontally along the plane of the NE and are found in areas bridging neighboring nuclear pores (also see Strambio-de-Castillia et al.…”

Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus

“…Replication factories were isolated using the spheroplast methodology [39,40]. Specifically, Acanthamoeba polyphaga were cultivated and infected by Mimivirus.…”

Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex

“…Electron microscopic studies revealed that the NPC is a cylindrical structure consisting of a central core with an 8-fold rotational symmetry across a nucleocytoplasmic axis and a 2-fold symmetry in the plane of the nuclear envelope (5)(6)(7)(8). This symmetric core is linked to the asymmetric cytoplasmic filaments and a nuclear basket structure (9).…”