Fig. 1: Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. | Nature Communications

Fig. 1: Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle.

From: The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

Fig. 1

a Midbody remnant purification. HeLa cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were EDTA-treated (Total). After 70g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to flow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP-negative counterpart (MBR−). b Representative pseudo-colored profile of flow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR− (44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b. c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), flow cytometry-sorted MBR− and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6. d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunofluorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome. Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x-axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y-axis. color code: proteins significantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome. See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome. The size of each bar (x-axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1.

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