Academic journal article Genetics

Caenorhabditis Elegans Num-1 Negatively Regulates Endocytic Recycling

Academic journal article Genetics

Caenorhabditis Elegans Num-1 Negatively Regulates Endocytic Recycling

Article excerpt

ABSTRACT

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AT::GFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.

ENDOCYTOSIS is of vital importance to eukaryotic cells for their ability to take up nutrients from their environment and for the proper subcellular distribution of cell membrane proteins and lipids (BRODSKY et al. 2001; POLO and DI FIORE 2006). In neurons, endocytosis is also required for the retrieval of synaptic vesicle components after their release upon nerve stimulation (WU 2004). Critical for the maintenance of proper plasma membrane composition and for the correct expression of many cell surface molecules is the rapid transport of a subset of internalized proteins and lipids back to the plasma membrane by endocytic recycling (MAXFIELD and McGRAW 2004). It is thought that, in many cells, as much as 50% of proteins, lipids, and fluids taken into the cell are recycled back to the cell surface within a few minutes (MAXFIELD and McGRAW 2004). In polarized epithelial cells, endocytic recycling is also thought to be essential for maintaining the correct apical-basal polarity (WANG et al. 2000).

While the different compartments within the endosomal system have been well described at a morphological level (MAXFIELD and McGRAW 2004), many of the proteins and lipids that are required for correct transport between different endosomal organelles have yet to be identified. This is particularly true for trafficking during endocytic recycling for which only a small number of molecules have been characterized to date (MAXFIELD and McGRAW 2004).

Following internalization, transmembrane receptors that are destined to be recycled back to the cell membrane are often first separated from their respective ligands in sorting endosomes. Recycling of receptors and lipids from the sorting endosome to the plasma membrane occurs by either of two different routes: directly or via a long-lived, specialized part of the endosomal system, the endosome recycling compartment (ERC) (MAXFIELD and McGRAW 2004). It is thought that a large proportion of the molecules that are recycled is transported via the ERC (HAO and MAXFIELD 2000), which is a collection of tubular-vesicular organelles that are associated with microtubules (HOPKINS 1983; YAMASHIRO et al. 1984). However, it is not understood how trafficking between the sorting endosome and the ERC and between the ERC and the plasma membrane is regulated.

Numb is a protein implicated in the regulation of Notch pathway signaling activity by endocytosis ( JAN and Jan 1998; LE BORGNE 2006). Immuno-electronmicroscopic studies of cultured mammalian cells have revealed that Numbis associated with the endosomal system (SANTOLINI et al. 2000). Furthermore, mammalian Numb binds the endocytic protein Eps15 in vitro (SALCINI et al. …

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