Fig 1: Milk fat globule membrane (MFGM) protein composition. (A) Volcano plots showing log2 fold change in normalized spectral abundance factor (NSAF) ratios for proteins in WT and Plin2-Null MFGM versus -log10 of their q-values, multiple unpaired t tests with FDR = 1%. For purposes of display, proteins present in WT MFGM but absent in Plin2-Null MFGM were assigned NSAF ratios of 1,000 (Log2 = 10). Proteins present in Plin2-Null MFGM but absent in WT MFGM were assigned NSAF ratios of 0.001 (Log2 = -10). Proteins with q-values = 0.000001 are assigned values of 0.000001 (log10 = 6). WT enriched MFGM proteins are right and Plin2-Null enriched MFGM proteins are left of the vertical dotted line. Proteins found at LD-APM contact sites, BTN, XOR, Cidea and Plin2, are indicated by green diamond symbols. The horizontal line indicates q = 0.01. (B) STRING networks of proteins elevated (q > 0.01) in WT MFGM were identified using Cytoscape 3.9.1. Proteins forming a network with Plin2 include those implicated in formation of LD-APM contacts (XOR, BTN and Cidea) and Rab18 and FABP3. (C) Average ±SEM XOR:BTN and Cidea:BTN NSAF ratios in WT and Plin2-Null MFGM. Asterisk indicates p < 0.05, Students t-test. (D) Average ±SEM Rab18:BTN and Fabp3:BTN NSAF ratios in WT and Plin2-Null MFGM. Asterisk indicates p < 0.05, Students t-test. (E) STRING networks of proteins elevated (q < 0.01) in Plin2-Null MFGM are enriched in ER/chaperone and lipid metabolism proteins.
Fig 2: Glycocalyx remodeling at LD-APM contacts. (A) Representative 3D-projection images of mammary gland sections prepared from WT or Plin2-Null dams following removal of their litters for 2 h at L10 to inhibit milk secretion. The sections were immunostained with antibodies to BTN and XOR to identify LD-APM contacts and with Alexa633-tagged WGA (fl-WGA) to identify the glycocalyx. The images show exclusion of fl-WGA (blue green) from a BTN and XOR positive APM domain in WT sMEC, and partial exclusion of fl-WGA from a BTN and XOR positive APM domain in a Plin2 sMEC. Scale bars are 2 µm. (B,C) Pearson’s correlation coefficients describing the overlap between fl-WGA and BTN (B) or XOR (C) in 40–60 sMEC/animal from WT and Plin2-Null dams prepared as described in (A). Median values are indicated by white bars and average medians ±SEM are shown above each group. p-values for group differences were determined by nested t-test and are shown at the top of each figure. Pearson’s coefficients of 1, 0 and -1 represent perfect correlation (Corr), no correlation (Uncorr.) and perfect negative correlation (Anti-corr.) respectively as shown in each figure.
Fig 3: Model of LD-APM interactions and proposed roles of Plin2 in apocrine lipid secretion. Apocrine lipid secretion is proposed to involve four distinct steps: (1) LD transport to the APM; (2) formation of LD-APM contacts mediated by interactions between BTN and XOR (and possibly Cidea) and stabilized by Plin2; (3) Plin2 regulated envelopment of APM-bound LD and glycocalyx remodeling; (4) oxytocin dependent secretion of APM-enveloped LD to form milk fat globules (MFG).
Fig 4: Molecular interactions at LD-APM contacts. Representative images of XOR (green/monochrome) or Cidea (green/monochrome) and BTN (red/monochrome) immunofluorescence (A) or Rab18 (green/monochrome) and BTN (red/monochrome) immunofluorescence (D) at LD-APM contacts in WT mammary glands following litter removal for 2 h at L10 to inhibit milk secretion. LD in images are indicated by asterisks and outlined by dotted white lines. Blue fluorescence in D is Alexa633-tagged wheatgerm agglutinin staining of the APM glycocalyx. Bar is 10 µ. (B,C,E) Pearson’s correlation coefficients describing immunofluorescence overlap between BTN and XOR (B), BTN and Cidea (C), or BTN and Rab18 (E) in 40–60 sMEC/animal from 3–4 WT and Plin2-Null dams following litter removal for 2 h at L10. Median values are indicated by white bars and average medians ±SEM are shown above each group. p-values for group differences were determined by nested t-test and are shown at the top of each figure. Pearson’s coefficients of 1, 0 and -1 respectively represent perfect positive correlation (Corr), no correlation (Uncorr.) and perfect negative correlation (Anti-corr.).
Fig 5: Mutations in XOR influence uric acid (UA) production. Panel (A) shows UA levels in the XDH WT and mutant cell lines at baseline relative to total protein concentration or following normalization to XOR expression (B) determined by Western blotting of the same sample. Panels C–H show the absolute UA levels generated from 4 × 106 million cells incubated with xanthine 20–100 μM. Data are shown as mean ± SEM. Statistical analysis was conducted using one-way ANOVA with Dunnett's post-hoc analysis for comparison to hXDH WT of n = 8 independent experiments. *for p < 0.05, **for P < 0.005, ***P < 0.001 and **** for P < 0.0001.
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