Golgi localization of the LIN-2/7/10 complex points to a role in basolateral secretion of LET-23 EGFR in the Caenorhabditiselegans vulval precursor cells.

The evolutionarily conserved LIN-2 (CASK)/LIN-7 (Lin7A-C)/LIN-10 (APBA1) complex plays an important role in regulating spatial organization of membrane proteins and signaling components. In Caenorhabditiselegans, the complex is essential for the development of the vulva by promoting the localization of the sole Epidermal growth factor receptor (EGFR) ortholog LET-23 to the basolateral membrane of the vulva precursor cells where it can specify the vulval cell fate. To understand how the LIN-2/7/10 complex regulates receptor localization, we determined its expression and localization during vulva development. We found that LIN-7 colocalizes with LET-23 EGFR at the basolateral membrane, whereas the LIN-2/7/10 complex colocalizes with LET-23 EGFR at cytoplasmic punctae that mostly overlap with the Golgi. Furthermore, LIN-10 recruits LIN-2, which in turn recruits LIN-7. We demonstrate that the complex forms in vivo with a particularly strong interaction and colocalization between LIN-2 and LIN-7, consistent with them forming a subcomplex. Thus, the LIN-2/7/10 complex forms on the Golgi on which it likely targets LET-23 EGFR trafficking to the basolateral membrane rather than functioning as a tether.

LIN-10 can promote LET-23 EGFR signaling and trafficking independently of LIN-2 and LIN-7.

During Caenorhabditis elegans larval development, an inductive signal mediated by the LET-23 EGFR (epidermal growth factor receptor), specifies three of six vulva precursor cells (VPCs) to adopt vulval cell fates. An evolutionarily conserved complex consisting of PDZ domain-containing scaffold proteins LIN-2 (CASK), LIN-7 (Lin7 or Veli), and LIN-10 (APBA1 or Mint1) (LIN-2/7/10) mediates basolateral LET-23 EGFR localization in the VPCs to permit signal transmission and development of the vulva. We recently found that the LIN-2/7/10 complex likely forms at Golgi ministacks; however, the mechanism through which the complex targets the receptor to the basolateral membrane remains unknown. Here we found that overexpression of LIN-10 or LIN-7 can compensate for loss of their complex components by promoting LET-23 EGFR signaling through previously unknown complex-independent and receptor-dependent pathways. In particular, LIN-10 can independently promote basolateral LET-23 EGFR localization, and its complex-independent function uniquely requires its PDZ domains that also regulate its localization to Golgi. These studies point to a novel complex-independent function for LIN-7 and LIN-10 that broadens our understanding of how this complex regulates targeted sorting of membrane proteins.

C. elegans Vulva Induction: An In Vivo Model to Study Epidermal Growth Factor Receptor Signaling and Trafficking.

Epidermal growth factor receptor (EGFR)-mediated activation of the canonical Ras/MAPK signaling cascade is responsible for cell proliferation and cell growth. This signaling pathway is frequently overactivated in epithelial cancers; therefore, studying regulation of this pathway is crucial not only for our fundamental understanding of cell biology but also for our ability to treat EGFR-related disease. Genetic model organisms such as Caenorhabditis elegans, a hermaphroditic nematode, played a vital role in identifying components of the EGFR/Ras/MAPK pathway and delineating their order of function, and continues to play a role in identifying novel regulators of the pathway. Polarized activation of LET-23, the C. elegans homolog of EGFR, is responsible for induction of the vulval cell fate; perturbations in this signaling pathway produce either a vulvaless or multivulva phenotype. The translucent cuticle of the nematode facilitates in vivo visualization of the receptor, revealing that localization of LET-23 EGFR is tightly regulated and linked to its function. In this chapter, we review the methods used to harness vulva development as a tool for studying EGFR signaling and trafficking in C. elegans.

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling.

Epidermal Growth Factor Receptor (EGFR) signaling is essential for animal development and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus-end directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell, genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants consistent with mammalian cell studies whereby dynein has been shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking.

Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types.

Mesostoma ehrenbergii spermatocytes are uniquely useful to study various aspects of cell division. Their chromosomes are large in size and few in number, with only three bivalent and four univalent chromosomes. During prometaphase, bipolar bivalents oscillate regularly to and from the poles for 1-2 hours. The univalents remain at the poles but occasionally move from one pole to the other. In addition, a precocious cleavage furrow forms during prometaphase and remains partially constricted until anaphase. Attempts to rear these animals indefinitely in laboratory conditions, however, have been mostly unsuccessful because of their reproductive strategy. M. ehrenbergii are hermaphroditic flatworms that can produce viviparous offspring (termed S eggs) and/or diapausing eggs (termed D eggs) and they follow either one of two reproductive patterns: (1) they first form S eggs and following the delivery of these eggs produce D eggs, or (2) they only produce D eggs. When only D eggs are formed, which is common under laboratory conditions, the stocks die out until the D eggs hatch, which is irregular and creates unpredictable wait times. Consequently, in order to maintain M. ehrenbergii stocks to study their spermatocytes, we examined various factors that might influence egg-type production. Feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 53rd generation. We herein describe our rearing and dissection methods, and some experiments which led to our present rearing methods.