The overexpression of nuclear envelope protein Lap2β induces endoplasmic reticulum reorganisation via membrane stacking

Abstract

Some nuclear envelope proteins are localised to both the nuclear envelope and the endoplasmic reticulum; therefore, it seems plausible that even small amounts of these proteins can influence the organisation of the endoplasmic reticulum. A simple method to study the possible effects of nuclear envelope proteins on endoplasmic reticulum organisation is to analyze nuclear envelope protein overexpression. Here, we demonstrate that Lap2β overexpression can induce the formation of cytoplasmic vesicular structures derived from endoplasmic reticulum membranes. Correlative light and electron microscopy demonstrated that these vesicular structures were composed of a series of closely apposed membranes that were frequently arranged in a circular fashion. Although stacked endoplasmic reticulum cisternae were highly ordered, Lap2β could readily diffuse into and out of these structures into the surrounding reticulum. It appears that low-affinity interactions between cytoplasmic domains of Lap2β can reorganise reticular endoplasmic reticulum into stacked cisternae. Although the effect of one protein may be insignificant at low concentrations, the cumulative effect of many non-specialised proteins may be significant.

Keywords: Endoplasmic reticulum; Lap2β; Nuclear envelope; Overexpression.

Fig. 1.. Structural organisation of Lap2β-EGFP-containing complexes in HeLa cells.

(A) Morphological organisation of Lap2β-EGFP-expressing cells. Green, Lap2β-EGFP; blue, DNA. When expressed at low levels, Lap2β-EGFP was localised in the nuclear envelope (arrowheads) and in an ER network (top panels). In cells expressing higher levels of Lap2β-EGFP, bright fluorescent structures (arrows) were observed in the cytoplasm (middle and bottom panels). The brightness of images is the same in both cases due to different exposures used for photographing. (B) Correlative light and electron microscopy of cells expressing high levels of Lap2β-EGFP. Phase contrast and fluorescence microscopic examination (top panels), and the ultrastructural organisation of cropped in the top panel region (bottom panel). Putative Lap2β-EGFP-containing complexes are indicated by large arrows. (C) The high magnification of cropped in B (bottom panel) region. The multilamellar structures are indicated by large arrowheads. Insert: the unmodified ER cisternae (luminal spaces are indicated by asterisks). Scale bars: 10 µm (A), 1 µm (B) and 0.1 µm (C).

Fig. 2.. Localisation and dynamics of Lap2β-EGFP.

(A) Lap2β-EGFP is colocalised with calnexin, a resident ER protein (top panels), but is not colocalised with golgin 97, a Golgi marker protein (bottom panels). Green, Lap2β-EGFP; red, calnexin or golgin 97; blue, DNA. (B) Lap2β-EGFP is highly mobile within the vesicular structures (top panels and blue line in graph) and is exchanged between these structures and a cytoplasmic ER network (bottom panels and red line in graph). (C) Localisation of Lap2β-FLAG in cells with high level of expression. The FLAG epitope was detected with specific antibodies (green), DNA with DAPI (blue). Scale bars: 10 µm (A,C), 3 µm (B).