Organellar Contacts of Milk Lipid Droplets

Milk-secreting epithelial cells of the mammary gland are functionally specialized for the synthesis and secretion of large quantities of neutral lipids, a major macronutrient in milk from most mammals. Milk lipid synthesis and secretion are hormonally regulated and secretion occurs by a unique apocrine mechanism. Neutral lipids are synthesized and packaged into perilipin-2 (PLIN2) coated cytoplasmic lipid droplets within specialized cisternal domains of rough endoplasmic reticulum (ER). Continued lipid synthesis by ER membrane enzymes and lipid droplet fusion contribute to the large size of these cytoplasmic lipid droplets (5–15 μm in diameter). Lipid droplets are directionally trafficked within the epithelial cell to the apical plasma membrane. Upon contact, a molecular docking complex assembles to tether the droplet to the plasma membrane and facilitate its membrane envelopment. This docking complex consists of the transmembrane protein, butyrophilin, the cytoplasmic housekeeping protein, xanthine dehydrogenase/oxidoreductase, the lipid droplet coat proteins, PLIN2, and cell death-inducing DFFA-like effector A. Interactions of mitochondria, Golgi, and secretory vesicles with docked lipid droplets have also been reported and may supply membrane phospholipids, energy, or scaffold cytoskeleton for apocrine secretion of the lipid droplet. Final secretion of lipid droplets into the milk occurs in response to oxytocin-stimulated contraction of myoepithelial cells that surround milk-secreting epithelial cells. The mechanistic details of lipid droplet release are unknown at this time. The final secreted milk fat globule consists of a triglyceride core coated with a phospholipid monolayer and various coat proteins, fully encased in a membrane bilayer

Organellar Contacts of Milk Lipid Droplets

Milk-secreting epithelial cells of the mammary gland are functionally specialized for the synthesis and secretion of large quantities of neutral lipids, a major macronutrient in milk from most mammals. Milk lipid synthesis and secretion are hormonally regulated and secretion occurs by a unique apocrine mechanism. Neutral lipids are synthesized and packaged into perilipin-2 (PLIN2) coated cytoplasmic lipid droplets within specialized cisternal domains of rough endoplasmic reticulum (ER). Continued lipid synthesis by ER membrane enzymes and lipid droplet fusion contribute to the large size of these cytoplasmic lipid droplets (5–15 μm in diameter). Lipid droplets are directionally trafficked within the epithelial cell to the apical plasma membrane. Upon contact, a molecular docking complex assembles to tether the droplet to the plasma membrane and facilitate its membrane envelopment. This docking complex consists of the transmembrane protein, butyrophilin, the cytoplasmic housekeeping protein, xanthine dehydrogenase/oxidoreductase, the lipid droplet coat proteins, PLIN2, and cell death-inducing DFFA-like effector A. Interactions of mitochondria, Golgi, and secretory vesicles with docked lipid droplets have also been reported and may supply membrane phospholipids, energy, or scaffold cytoskeleton for apocrine secretion of the lipid droplet. Final secretion of lipid droplets into the milk occurs in response to oxytocin-stimulated contraction of myoepithelial cells that surround milk-secreting epithelial cells. The mechanistic details of lipid droplet release are unknown at this time. The final secreted milk fat globule consists of a triglyceride core coated with a phospholipid monolayer and various coat proteins, fully encased in a membrane bilayer

Comparative proteomic analysis of human milk fat globules and paired membranes and mouse milk fat globules identifies core cellular systems contributing to mammary lipid trafficking and secretion

Introduction: Human milk delivers critical nutritional and immunological support to human infants. Milk fat globules (MFGs) and their associated membranes (MFGMs) contain the majority of milk lipids and many bioactive components that contribute to neonatal development and health, yet their compositions have not been fully defined, and the mechanisms responsible for formation of these structures remain incompletely understood.Methods: In this study, we used untargeted mass spectrometry to quantitatively profile the protein compositions of freshly obtained MFGs and their paired, physically separated MFGM fractions from 13 human milk samples. We also quantitatively profiled the MFG protein compositions of 9 pooled milk samples from 18 lactating mouse dams.Results: We identified 2,453 proteins and 2,795 proteins in the majority of human MFG and MFGM samples, respectively, and 1,577 proteins in mouse MFGs. Using paired analyses of protein abundance in MFGMs compared to MFGs (MFGM-MFG; 1% FDR), we identified 699 proteins that were more highly abundant in MFGMs (MFGM-enriched), and 201 proteins that were less abundant in MFGMs (cytoplasmic). MFGM-enriched proteins comprised membrane systems (apical plasma membrane and multiple vesicular membranes) hypothesized to be responsible for lipid and protein secretion and components of membrane transport and signaling systems. Cytoplasmic proteins included ribosomal and proteasomal systems. Comparing abundance between human and mouse MFGs, we found a positive correlation (R2 = 0.44, p < 0.0001) in the relative abundances of 1,279 proteins that were found in common across species.Discussion: Comparative pathway enrichment analyses between human and mouse samples reveal similarities in membrane trafficking and signaling pathways involved in milk fat secretion and identify potentially novel immunological components of MFGs. Our results advance knowledge of the composition and relative quantities of proteins in human and mouse MFGs in greater detail, provide a quantitative profile of specifically enriched human MFGM proteins, and identify core cellular systems involved in milk lipid secretion

Dynamics and Molecular Determinants of Cytoplasmic Lipid Droplet Clustering and Dispersion

<div><p>Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.</p></div

Phosphorylation on Plin1ser492 precedes CLD cluster dispersion.

<p>(A) Representative images of Plin1 cells immunostained for Plin1 (red) and phosphorylated Plin1ser492 (phospho-Plin1S492, green) following stimulation for the indicated times with 10 µg/ml isoproterenol. Hoechst-stained nuclei are shown in blue. The size bar is 10 µm. (B) The time courses of CLD dispersion (blue line) and phosphorylation of Plin1S492 (red line). CLD dispersion is shown as the fraction of the total number of Plin1 objects/cell observed at 45 minutes. The extent of Plin1S492 phosphorylation is shown as a relative ratio of phospho-Plin1ser492 fluorescence to total Plin1 fluorescence. The values are mean ± SEM for 6 experiments with the evaluation of 60–100 cells per time point in each experiment. Statistical significances of Plin1 phosphorylation are indicated by lower case letters a and b: a, values differ from the 0 minute value (p<0.001); b, values differ from 0 minutes value (p<0.01) but not from 1 or 5 minute time point values. A 1-way ANOVA of Plin1 objects/cell yields a p<0.0001 and R² = 0.7507, a post test for linear trends was statistically positive with p = 0.0207.</p

Moesin is required for Plin1-CLD clustering.

<p>(A) Immunolocalization of Plin1CLD (red) and moesin or ezrin (green) in cells transfected with scrambled oligonucleotides (scrambled Si) or siRNA oligonucleotides to moesin (SiMoesin) or ezrin (SiEzrin) and exposed to vehicle (Control) or 10 µg/mL isoproterenol for 1 hour (Isoproterenol). Hoechst-stained nuclei are shown in blue. The size bar is 10 µm. (B and C) CLD dispersion as determined by the number of Plin1 objects/cell in cultures transfected with scrambled siRNA (Scrm) or with siMoesin (B, SiMoe) or siEzrin (C, SiEzr) oligonucleotides for 48 hours and then incubated for 30 minutes with control medium (Co) or medium containing 10 µg/mL isoproterenol isoproterenol (Iso). The values are mean ± SEM for 3 experiments performed in duplicate. The lower case letter a above the bars indicates values that differ from their respective controls (p<0.001). (D) The effects of moesin or ezrin knockdown on CLD distribution determined by morphological analysis. The average CLD distribution among Stages 1–3 is shown for cultures transfected with scrambled (Scrm), siMoesin and siEzrin oligonucleotides for 48 hours and then exposed to vehicle (Control) or isoproterenol (Isoproterenol) as described above in B and C. The values are averages of 3 replicate experiments in which 60–80 cells per condition were analyzed per experiment for each condition. Statistical significance is indicated by lower case letters a and b: a, CLD distribution in vehicle-treated cultures transfected with siMoesin oligonucleotides was significantly different (p<0.001) from that found in cultures transfected with scrambled or siEzrin oligonucleotides; b, CLD distributions in isoproterenol treated cultures were significantly different from that found in the respective control cultures (p<0.001). (E) The effects of moesin knockdown on CLD reclustering. The average CLD distribution among Stages 1–3 is shown for cultures transfected with scrambled (Scrm) or siMoesin oligonucleotides (siMoesin) following isoproterenol dispersion for one hour, washing twice, and then incubation in control medium for 16 hours to allow reclustering. The values are averages of 3 replicate experiments in which 60–80 cells per condition were analyzed per experiment for each condition. The lower case letter c indicates CLD distributions in scrambled oligonucleotide transfected cultures that are significantly different (p<0.001) from those transfected with siMoesin oligonucleotides.</p

Plin1-coated CLD cluster close to the MTOC and associate with motor proteins.

<p>(A) Representative immunofluorescence images showing the localization of Plin1-coated CLD (red) and <b>γ</b>-Tubulin (green). Hoechst-stained nuclei are shown in blue. The size bar is 10 µm. (B) Kinesin and dynein co-localization with Plin1. Representative immunofluorescence images depicting the localizations of Plin1 and dynein (Dynein+Plin1) or kinesin-5 family members (Kinesin+Plin1) in Plin1-expressing cells are shown. The panels show merged (red = Plin1; green = dynein or kinesins), and Plin1- and dynein- or kinesin-specific images (monochrome). Arrows point to areas of immunofluorescence overlap seen as a yellow color in the composite. Hoechst-stained nuclei are shown in blue. The size bar is 10 µm. (C) Cross-channel Pearson’s correlation coefficients for the overlap of dynein- or kinesin-immunofluorescence with that of Plin1 in control or isoproterenol-treated (Isopro) cells.</p

CLD clustering and dispersion mechanisms.

<p>Tight clusters of Plin1-coated CLD (red spheres) form near the MTOC (green stars) by moesin- and microtubule dependent processes in unstimulated cells (Stage 1). PKA activation induces rapid phosphorylation (less than a minute) of Plin1S492 (small purple ovals on red spheres), initiating processes that lead to disruption of CLD interactions and their separation into smaller clusters over a 4–10 minute period (Stage 2). (Stage 3), Mictroubule-movement of fully- or partially-disaggregated CLD leads to their dispersion over the course of 20–40 minutes. CLD movement on microtubules is not dependent on phosphorylation of Plin1 on S492 (indicated by red spheres with out purple ovals in the Stage 3 cell). Dispersed CLD undergo reclustering to Stage 2 and Stage 1 by kinetically distinct mictrotubule- and moesin-dependent processes.</p