Integrative approach of secretion pathways in the mammary epithelial cell: molecular dissection of milk fat adaptability – MilkChEST

On the sidelines of technology and more conventional and proven methods of Cell Biology (subcellular fractionation, electron microscopy) and biochemistry (electrophoresis, immunochemical assays) and enzymology, different approaches and strategies based on innovative and highly resolutive technologies such as laser microdissection (cell sorting under morphological control), high-throughput sequencing of RNAs (gene expression profiling), high performance liquid chromatography coupled with mass spectrometry (Golgi proteome, profiling of the major milk proteins and isoforms) or quantitative PCR using microfluidic flow (transcriptional expression of target genes) will be implemented. A non-invasive sampling method of RNA, from the milk fat globule, of which the validity has been proven will be exploited for population studies. The preparation and characterization of extracellular microvesicles present in milk (lactosomes) and produced by adipocytes require methodologies that will be developed in interaction between the teams concerned (INSERM and INRA). Global analyzes of the milk fat globules and microvesicles membranes (proteomics and lipidomics) will be outsourced to specialized platforms. Finally, the development of computational and statistical tools for the integration of data from multiple formats generated by these approaches is one of the major challenges of this program.

- A first deep transcriptome of the mammary epithelial cell was established, consisting of more than 17,000 genes.
- A validated and optimized method for isolating extracellular vesicles from milk has been developed.
- Similarly a method to isolate extracellular vesicles produced by adipocytes has been developed and implemented to characterize the production of EV adipocytes in an in vivo obesity context
- Identification and characterization of a reticulum stress associated with Unfolded Protein Response type response (UPR) in O / O genotype individuals locus CSN1S1

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The biosynthetic pathways responsible for the production of the main milk components are known in general outline but there is a gap in our knowledge of most of the molecular events occurring during assembly, intracellular transport and secretion of the two major constituents of milk: casein micelles and milk fat globules (MFG). Regarding the release of microparticles in milk, nothing is known. To deal with this complex research issue, four partners (three from INRA and one at Inserm) will collaborate, combining their highly qualified research experiences and core facilities in a multidisciplinary approach. The proposed research project is a continuation of the "Genomilk Fat" program funded by the ANR (Genanimal 2005) and APIS-Gène, and to which most of the partners have already collaborated. It is partly based on well-characterized biological materials and significant breakthroughs produced in this context, notably the demonstration that a close mechanistic relationship exists between biosynthesis and secretion pathways of casein micelles and MFG. The relevance of the goat model, with a “naturally occurring KO" for the gene (CSN1S1) that specifies alpha s1-casein was also further demonstrated. We have shown that in goats homozygous for a null allele at the CSN1S1 locus, there is a chronic endoplasmic reticulum (ER) stress following the accumulation of the other caseins in this compartment. This in turn induces an adaptive Unfolded Protein Response (UPR), combined with an oxidative stress. Analysis of defective vs. control goats allowed identifying genes that contribute to the establishment of these responses, as well as proteins implicated in protein transport between the ER and the Golgi apparatus, and enzymes involved in the biogenesis of lipid droplets, the MFG precursors. In the present proposal we intend to pursue the molecular dissection of the biosynthetic and secretion pathways of the major goat milk components, processes that determine their structure and meanwhile their biological and technological properties. We will also introduce pioneer studies on milk microparticles, structures potentially implicated in the development of the gastrointestinal tractus and/or immune systems of the neonate. Our main research goals will be: i) to achieve a better definition of the genome fraction which is expressed in goat MEC during lactation, ii) to obtain a better understanding of the role played by alpha s1-casein in the export of caseins from the ER to the Golgi, including the study of the UPR, iii) to identify factors that determine the fatty acid profile of milk fat, iiii) to go further into the molecular dissection of the mechanisms driving lipid droplets formation (which originate from the ER) and their interaction with the apical plasma membrane in the course of MFG secretion by MEC. In this context, we will conduct a comparative study of lipid droplets formation in MEC vs. adipocytes, the later cells producing large lipid droplets but lacking the machinery to secrete them. On the other hand, the study of the production of microparticles by adipocytes and their characterization in goat milks will be undertaken. In addition, we will focus on the consequences of the absence of alpha s1-casein on protein and fat secretory pathway, as well as fine milk composition, to decipher the mechanisms driving production of these constituents by MEC. The proposed studies are also dedicated to the setting up of tools and resources which will enhance our knowledge on genome expression and modulation, specifically in MEC, for a better management of milk composition and to provide innovative phenotyping methods based on a non invasive sampling technique of mammary RNA. The ultimate goal is to identify genes playing key roles in the production of milk fat by MEC on which selection and breeding programme might be engaged to modulate milk fat composition and to improve the genetics of dairy ruminants.