Figure 7.

Excretion of LaminB1-Rich Buds following Autophagy and Golgi Impairment

(A) Representative image showing DRPLA (17) fibroblasts after 72 hr Rap treatment showing formation of buds at the plasma membrane containing LaminB1 and p62 puncta. High magnification of insets in the image is shown on the left. Scale bar, 25 μm.

(B) Still frames (taken form Movie S3) of confocal live imaging of a SK-N-BE(3) neuroblastoma cell expressing EGFP and mCherry-LaminB1 treated with BafA1 for 48 hr. Arrow points at a LaminB1 punctum detaching from the nucleus and getting slowly excreted over time. Note the misshapen nucleus and LaminB1 infolding. Scale bar, 5 μm.

(C) Analysis of extracellular LaminB1 in SK-N-BE(2) neuroblastoma cells co-expressing EGFP and mCherry-LaminB1 as well as siRNAs against ATG5 or ATG6, genes participating in autophagy and ATG-dependent secretion. Extracellular LaminB1-mCherry puncta were enriched in the surrounding region to EGFP positive cells void of EGFP signal. Automated quantification of mCherry signal intensity was performed using Opera Phenix high-content screening system and Columbus software. Mean ± SEM, one-way-ANOVA ^∗p < 0.05.

(D–G) Analysis of excretion of cellular component in the medium of human DRPLA (17) fibroblasts treated with BafA1 and BrefA as in Figure 6F. The medium supernatant was collected and subjected to ultracentrifugation, while the cells were lysed separating nuclear and cytoplasmic fractions. BafA1 and combined BafA1/BrefA treatment increase the amount of LaminB1 in the cytoplasmic fraction (D and E). BafA1 increased significantly the amount of LaminB1 (D and F) and p62 (D and G) in the medium supernatant fraction collected after 100,000 × g ultracentrifugation; addition of BrefA did not significantly reduce the amount of LaminB1 or p62 found in this fraction. Note that the LaminB1 band in cytoplasmic and medium supernatant fraction runs at a lower molecular weight of ∼45 kDa as opposed to the expected ∼70 kDa in the nuclear fraction, compatible with proteolytic cleavage. Densitometry of medium supernatant fractions was standardized over cytoplasmic tubulin representing the amount of cells and cytoplasm present in the culture. One sample t test, mean ± SEM, ^∗∗p < 0.01, ^∗p < 0.05.

(H) Representative image of SK-N-BE(2) neuroblastoma cells expressing EGFP, mCherry-LaminB1, and Atg6 siRNA shows the uptake of LaminB1 puncta by neighboring cells. Arrow points at mCherry-LaminB1 dots found inside non-transfected cells. Phalloidin is used to mark the cytoplasmic area of all cells. Scale bar, 10 μm.

See also Figure S7.