A polarity pathway for exocyst-dependent intracellular tube extension

Lumen extension in intracellular tubes can occur when vesicles fuse with an invading apical membrane. Within theCaenorhabditis elegansexcretory cell, which forms an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane and is required for its outgrowth, suggesting that exocyst-targeted vesicles extend the lumen. Here, we identify a pathway that promotes intracellular tube extension by enriching the exocyst at the lumenal membrane. We show that PAR-6 and PKC-3/aPKC concentrate at the lumenal membrane and promote lumen extension. Using acute protein depletion, we find that PAR-6 is required for exocyst membrane recruitment, whereas PAR-3, which can recruit the exocyst in mammals, appears dispensable for exocyst localization and lumen extension. Finally, we show that CDC-42 and RhoGEF EXC-5/FGD regulate lumen extension by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a pathway that connects CDC-42, PAR proteins, and the exocyst to extend intracellular tubes.

A polarity pathway for exocyst-dependent intracellular tube extension

ABSTRACTLumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. Using acute protein depletion, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.

Cell Types, Morphology and Evolution of Animal Excretory Organs

Excretion and osmoregulation are fundamental processes of the organism, as they prevent the accumulation of toxic waste products in the body and control the osmotic differences between the cells and the environment. In most of the animals these phenomena are taking place through specialized organs, namely excretory organs, composed of diverse cell types that are performing tasks such as secretion and ultrafiltration. Although the morphology and embryology of excretory organs can differ dramatically, the common spatial arrangement of structural proteins and transporters as well as the similar transcriptional developmental programs underlying their formation suggests the homology of their cell types. In this chapter we discuss the current understanding of the evolution of excretory organsfrom a comparative morphological, developmental and functional perspective, flanked by an additional, cell-type perspective. We argue that a putative homologization of certain excretory cell types does not necessarily reflect the homology of the resulting organs, and that integrating all different levels of comparison is crucial for addressing evolutionary questions.

Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.