Microtubule-dependent apical polarization of basement membrane matrix mRNAs in mouse epithelial cells.

The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH). Moreover, electron microscopy and immunofluorescence revealed apical polarization of both the endoplasmic reticulum (ER) and Golgi apparatus, indicating that the site of BM component production was opposite to the site of deposition. At the apical side, BM mRNAs colocalized with ER, suggesting they may be co-translationally tethered. After microtubule inhibition, the BM mRNAs and ER became uniformly distributed rather than apically polarized, but they remained unchanged after inhibiting myosin II, ROCK, or F-actin, or after enzymatic disruption of the BM. Because Rab6 is generally required for Golgi-to-plasma membrane trafficking of BM components, we used lentivirus to express an mScarlet-tagged Rab6a in salivary gland epithelial cultures to visualize vesicle trafficking dynamics. We observed extensive bidirectional vesicle movements between Golgi at the apical side and the basal plasma membrane adjacent to the BM. Moreover, we showed that these vesicle movements depend on the microtubule motor kinesin-1 because very few vesicles remained motile after treatment with kinesore to compete for cargo-binding sites on kinesin-1. Overall, our work highlights the diverse strategies that different organisms use to secrete bulky matrix proteins: while Drosophila follicle epithelial cells strategically place their sites of BM protein production close to their deposition, mouse embryonic epithelial cells place their sites of production at the opposite end. Instead of spatial proximity, they use the microtubule cytoskeleton to mediate this organization as well as for the apical-to-basal transport of BM proteins.

Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics.

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.[Figure: see text].

Enhanced protein isoform characterization through long-read proteogenomics.

BACKGROUND: The detection of physiologically relevant protein isoforms encoded by the human genome is critical to biomedicine. Mass spectrometry (MS)-based proteomics is the preeminent method for protein detection, but isoform-resolved proteomic analysis relies on accurate reference databases that match the sample; neither a subset nor a superset database is ideal. Long-read RNA sequencing (e.g., PacBio or Oxford Nanopore) provides full-length transcripts which can be used to predict full-length protein isoforms. RESULTS: We describe here a long-read proteogenomics approach for integrating sample-matched long-read RNA-seq and MS-based proteomics data to enhance isoform characterization. We introduce a classification scheme for protein isoforms, discover novel protein isoforms, and present the first protein inference algorithm for the direct incorporation of long-read transcriptome data to enable detection of protein isoforms previously intractable to MS-based detection. We have released an open-source Nextflow pipeline that integrates long-read sequencing in a proteomic workflow for isoform-resolved analysis. CONCLUSIONS: Our work suggests that the incorporation of long-read sequencing and proteomic data can facilitate improved characterization of human protein isoform diversity. Our first-generation pipeline provides a strong foundation for future development of long-read proteogenomics and its adoption for both basic and translational research.