Lipocalins Are Required for Apical Extracellular Matrix Organization and Remodeling in Caenorhabditis elegans

Abstract

A lipid and glycoprotein-rich apical extracellular matrix (aECM) or glycocalyx lines exposed membranes in the body, and is particularly important to protect narrow tube integrity. Lipocalins ("fat cups") are small, secreted, cup-shaped proteins that bind and transport lipophilic cargo and are often found in luminal or aECM compartments such as mammalian plasma, urine, or tear film. Although some lipocalins can bind known aECM lipids and/or matrix metalloproteinases, it is not known if and how lipocalins affect aECM structure due to challenges in visualizing the aECM in most systems. Here we show that two lipocalins, LPR-1 and LPR-3, have distinct functions in the precuticular glycocalyx of developing external epithelia. LPR-1 moves freely through luminal compartments, while LPR-3 stably localizes to a central layer of the membrane-anchored glycocalyx, adjacent to the transient zona pellucida domain protein LET-653 Like LET-653 and other glycocalyx components, these lipocalins are required to maintain the patency of the narrow excretory duct tube, and also affect multiple aspects of later cuticle organization. mutants cannot maintain a continuous excretory duct apical domain and have misshapen cuticle ridges (alae) and abnormal patterns of cuticular surface lipid staining. mutants cannot maintain a passable excretory duct lumen, properly degrade the eggshell, or shed old cuticle during molting, and they lack cuticle barrier function. Based on these phenotypes, we infer that both LPR-1 and LPR-3 are required to build a properly organized aECM, while LPR-3 additionally is needed for aECM clearance and remodeling. The glycocalyx provides a powerful system, amenable to both genetic analysis and live imaging, for investigating how lipocalins and lipids affect aECM structure.

A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen inCaenorhabditis elegans

“…Other sheath proteins also have been observed within the vulva (Forman-Rubinsky et al, 2017;Gill et al, 2016), and the adult vulva becomes cuticle-lined in adults (Page & Johnstone, 2007;Sulston & Horvitz, 1977). These observations suggested to us that a sheath-like matrix may exist within the developing vulva lumen.…”

“…Each fusion protein initially appeared at around the L4.2 stage, and then exhibited a temporally and spatially distinct pattern over the course of L4 vulva morphogenesis ( Figure 3B-D). All six matrix proteins were transient and disappeared by the time that animals reached the adult stage, indicating that they are not components of the mature cuticle, but rather define a distinct matrix type present only during morphogenesis (Forman-Rubinsky et al, 2017;Gill et al, 2016) (and data not shown).…”

“…Here we show that a spatially and temporally dynamic aECM assembles and disassembles within the vulva lumen during morphogenesis. This aECM shares components with the glycocalyx-like sheath or pre-cuticle matrix that coats other apical surfaces in C. elegans prior to each round of cuticle secretion (Forman-Rubinsky, Cohen, & Sundaram, 2017;Gill et al, 2016;Katz, Maybrun, Maul-Newby, & Frand, 2018;Kelley et al, 2015;Labouesse, 2012;Lazetic & Fay, 2017;Mancuso et al, 2012;Priess & Hirsh, 1986;Vuong-Brender, Suman, & Labouesse, 2017). It contains both fibrillar and gel-like or granular components, and also extracellular vesicles, as observed at the ultrastructural level.…”

“…To examine sheath protein expression and localization in the vulva, we examined Superfolder (Sf) GFP or mCherry-based translational fusions generated by transgenic methods or (in most cases) by CRISPR-Cas9 genome editing of the endogenous loci (Materials and Methods). Six different sheath matrix proteins were assessed ( Figure 3A): the ZP proteins FBN-1 (Kelley et al, 2015), LET-653 (Gill et al, 2016) and NOAH-1 (Vuong-Brender et al, 2017), the lipocalin LPR-3 (Forman-Rubinsky et al, 2017), and the extracellular leucine-rich repeat only (eLRRon) proteins LET-4 (Mancuso et al, 2012) and SYM-1 (Davies, Spike, Shaw, & Herman, 1999;Vuong-Brender et al, 2017). We also examined a shortened version of LET-653 containing only the ZP domain (Gill et al, 2016) ( Figure 3A).…”

Feedback regulation of BMP signaling byC. eleganscuticle collagens

“…We have established C. elegans as an in vivo model to study the interplay between TGF-β signaling and ECM. The C. elegans ECM includes both basement membrane, which lines internal organs, and cuticle, which is secreted apically from hypodermal tissue to protect the organism and is shed at each molt [3][4][5]. The cuticle is composed of multiple collagen layers coated by a lipid-rich epicuticle [6,7].…”