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. 2018 May 3;7:e32911. doi: 10.7554/eLife.32911

Figure 1. Inner NE proteins are sequestered into distinct microdomains.

(A) Representative super-resolution microscopy (structured illumination, SIM) image of a live cell harboring Bqt4-GFP. Lateral resolution is 120–150 nm. Arrows indicate regions with relatively low abundance of detectable fluorophore. Scale bars represent 2 µm in all images. (B) SIM images of representative fixed cells expressing Man1-GFP and Bqt4-mCherry, and (C) Lem2-GFP and Bqt4-mCherry. Colocalization quantitations and procedures are described in Figure 1—figure supplement 1A. (D) DeltaVision deconvolution fluorescence imaging of live cells harboring Lem2-GFP and Man1-tdTomato. Quantitation is shown in Figure 1—figure supplement 1C. (E) A representative single z-plane image of a live wt cell expressing Lem2-GFP and histone H3-mCherry. Lem2-GFP is detectable around the NE with an intense dot beneath the SPB. (F) In the absence of Bqt4, Lem2 fails to encircle the NE but remains beneath the SPB. Lateral resolution for (E–F) ~300 nm.

Figure 1.

Figure 1—figure supplement 1. Quantitation of NE protein colocalizations.

Figure 1—figure supplement 1.

(A) Pearson correlation coefficients derived from SIM imaging of fixed cells. Values indicate levels of overlap between Man1-GFP and Bqt4-mCherry or Lem2-GFP and Bqt4-mCherry were measured using Applied Precision softWorX software. Horizontal black lines indicate mean (Man1 Lem2: 0.143, n = 38; Lem2 Bqt4: 0.37, n = 22) and SD. (B) Additional examples of simultaneous live imaging of Man1-tdtomato and Lem2-GFP as in Figure 1. (C) Colocalization of the indicated protein pairs as assessed by live microscopy is quantified as in (A).
Figure 1—figure supplement 2. Bqt4 controls localization of Lem2 but not Man1, Nup107 or Cut11.

Figure 1—figure supplement 2.

(A) Bqt4 is required for the localization of Lem2 around the NE. Snapshots of WT and bqt4Δ cells expressing Lem2-GFP and Pcp1-RFP. The visible Lem2-GFP dot in bqt4Δ cells colocalizes with the SPB (marked by Pcp1-RFP). The images are maximal intensity projections of imaged cross-sections through the entire nucleus. (B) Deletion of Bqt4 does not affect localization of Man1-GFP, Nup107-GFP and Cut11-GFP. Live cell imaging was performed in cells expressing the indicated tagged membrane proteins in wt and bqt4Δ cells.