Beyond lamins other structural components of the nucleoskeleton

Abstract

The nucleus is bordered by a double bilayer nuclear envelope, communicates with the cytoplasm via embedded nuclear pore complexes, and is structurally supported by an underlying nucleoskeleton. The nucleoskeleton includes nuclear intermediate filaments formed by lamin proteins, which provide major structural and mechanical support to the nucleus. However, other structural proteins also contribute to the function of the nucleoskeleton and help connect it to the cytoskeleton. This chapter reviews nucleoskeletal components beyond lamins and summarizes specific methods and strategies useful for analyzing nuclear structural proteins including actin, spectrin, titin, linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, and nuclear spindle matrix proteins. These components can localize to highly specific functional subdomains at the nuclear envelope or nuclear interior and can interact either stably or dynamically with a variety of partners. These components confer upon the nucleoskeleton a functional diversity and mechanical resilience that appears to rival the cytoskeleton. To facilitate the exploration of this understudied area of biology, we summarize methods useful for localizing, solubilizing, and immunoprecipitating nuclear structural proteins, and a state-of-the-art method to measure a newly-recognized mechanical property of nucleus.

Figure 1. Actin-based pore linked filaments in the interior of Xenopus oocyte nuclei

(A) Nuclear content fixed and visualized by feSEM after “peeling” away the nuclear envelope. Intranuclear filaments associated with spherical bodies are seen in the figure. (B) The Immunogold feSEM identify the nuclear actin in filamentous network (pseudo-colored yellow). Adapted from (Kiseleva et al. 2004)

Figure 2

Indirect immunofluorescence microscopy indicate that cells expressing GFP-NLS (nuclear localization signal) fused titin fragment M-is7 had a high frequency of aberrantly-shaped nuclei and nuclear envelope herniations (arrowheads) that contained GFP-is7, but not chromatin or lamin B. Negative control use cells transfected with GFP-NLS fused to pyruvate kinase. Bar: 2μm. Adapted from (Zastrow et al. 2006)

Figure 3. Altered membrane permeablization allows visualization of INM versus ONM proteins

Digitonin permeabilizes the plasma membrane but not the nuclear membrane whereas Triton X-100 permeabilizes all membranes. During immunocytochemistry this allows for altered antibody accessibility to different regions of the nucleus. INM proteins cannot be detected with digitonin permeabilization (A and A′), but can be detected with Triton X-100 treatment (B and B′). ONM protein can be detected both with digitonin (C and C′) and Triton X-100 (D and D′) treatment. This has been shown functionally with the nucleoskeleton protein lamin A/C which is inside the nucleus, and the ONM protein Nesprin 4 (A′ through D′ adapted from (Roux et al. 2009)).

Figure 4. Micropipette aspiration (MPA) of nuclei and cells to show subtle nuclear structural and mechanical changes

(A) Schematic of aspirated cell showing nuclear deformation into the pipette of ΔA (crosshatched) under applied pressure ΔP. (B) Aspiration of cells into the micropipette under constant pressure shows nuclear deformation into the pipette. (C) When pressure released to zero, the wide type (WT) cell recoiled back gradually, while cell with loss of αII spectrin (KD αII-sp) failed to recoil.