The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane

Abstract

Cell invasion through basement membrane (BM) barriers is important in development, immune function, and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of C. elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including TCTP (translationally controlled tumor protein). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling, and ribosome biogenesis analysis revealed a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands prior to invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

De novo lipid synthesis and polarized prenylation drives cell invasion through basement membrane

“…The AC expresses lipid anchored proteins that are enriched at the invasive plasma membrane and then are found in the protrusion, such as the matrix degrading GPI-anchored MMP ZMP-1, the prenylated Rho GTPases CED-10 (Rac) and MIG-2 (Rac-like), and the Ras-like GTPase RAP-1 (Fig. 1, B-D; previously quantified in (Costa et al, 2023; Hagedorn et al, 2013; Kelley et al, 2019; Naegeli et al, 2017; Wang et al, 2014a). How the AC synthesizes additional lipid membranes for protrusion formation and organizes production of lipid anchored proteins is unknown.…”

“…The AC is specified at the late larval L2 stage, just prior to the L2/L3 molt, and during the L3 larval stage expresses pro-invasive actin regulators and matrix remodeling proteins (P6.p 1-cell stage) until the early-to-mid L3 when the AC initiates BM beaching with a small F-actin rich invadosome protrusion that penetrates the BM (P6.p 2-cell; Fig. 1 A) (Costa et al, 2023; Hagedorn et al, 2013; Kimble, 1981). After an invadosome breaches the BM, the UNC-40 (DCC) receptor localizes to the breach site and directs lysosome exocytosis to form a large protrusion that transiently increases the size of the AC by as much as ∼40% in surface area (2-4-cell stage) (Morrissey et al, 2013; Naegeli et al, 2017).…”

“…The UNC-40 receptor and GPI-anchored proteins like ZMP-1 are partitioned to sphingolipid enriched lipid rafts (Herincs et al, 2005; Sangiorgio et al, 2004). In addition, C. elegans Rac and Ras-like GTPases that promote F-actin formation for invasive protrusion outgrowth are anchored and enriched in the invasive protrusion through C-terminal lipid prenylation (Costa et al, 2023; Hagedorn et al, 2013; Lohmer et al, 2016; Wang et al, 2014b). Previous work examining energy sources that fuel invasion revealed that the AC does not contain lipid stores nor require lipid import transporters (Garde et al, 2022; Zechner et al, 2017).…”

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis inC. elegans

“…The UPR is also activated in the anchor cell prior to invasion. Here, ribosome biogenesis is also upregulated to accommodate increased translation of transmembrane and secreted proteins (Costa et al, 2023). Although we do not find enrichment of ribosome biogenesis genes in TTM, we believe that the UPR is activated due to increased translation in the tail tip as well.…”

A Window into Mammalian Basement Membrane Development: Insights from themTurq2-Col4a1Mouse Model

“…The Vkg::GFP (collagen IVa2) Drosophila line, created as part of a protein-trap strategy (Morin et al, 2001, Buszczak et al, 2007), has been invaluable in studying dissemination of tumor cells through the BM, as well as in BM-controlled shaping of growing organs during development (Tamori et al, 2016, Rei et al, 2021, Harmansa et al, 2023, Page-McCaw et al, 2007, Page-McCaw, 2008). The stereotyped invasion of the C. elegans anchor cell through the uterine and vulval basement membranes during development, together with the successful endogenous tagging of over 60 C. elegans BM proteins has greatly increased our understanding of BM composition, dynamics and the cellular and molecular mechanisms underpinning the invasion process (Schindler and Sherwood, 2013, Sherwood and Sternberg, 2003, Costa et al, 2023, Kelley et al, 2017, Keeley et al, 2020, Kelley et al, 2019, Kenny-Ganzert and Sherwood, 2023).…”