Analysis of cell proliferation during C.elegans intestine development

The precise co-ordination of cell proliferation and developmental pathways is essential for the development of multicellular organisms and the maintenance of tissue homeostasis. The intestine (endoderm) of the nematode Caenorhabditis elegans is used as a model system to study the control of cell proliferation during development, because it consists of only 20 cells. These cells are generated in the embryo by a precise division-pattern that is largely invariant between different animals. Previously, a cdc-25.1 (ij48) gain-of-function allele has been identified that produces increased numbers of intestinal cells. CDC-25.1 belongs to the eukaryotic CDC25 family of positive-acting cell cycle regulators. Intriguingly, in cdc-25.1 (ij48) mutants, proliferation of other tissues is unaffected, but knockdown of CDC-25.1 by RNAi produces reduced cell divisions in most lineages. Thus, there is a general requirement for cdc-25.1 function in all embryonic blastomeres, but the cdc-25.1(ij48) mutant primarily affects proliferation of the intestine. It is therefore interesting to elucidate the mechanism underlying this tissue-specific phenotype. The ij48 lesion in CDC-25.1 constitutes a serine to phenylalanine mutation (CDC- 25.1(S46F)) in a highly conserved putative DSG consensus site, which may act as a site of negative regulation of CDC-25.1. In mammalian cells, the DSG motif of CDC25A acts as a recruitment site for the ubiquitin ligase component beta-TrCP, mediating ubiquitin-dependent degradation of CDC25A. However, to date no difference in the abundance or localisation of CDC-25.1(S46F) was identified. In this thesis, I set out to identify negative regulators of CDC-25.1 that control CDC-25.1 through S46, possibly in the intestine. Compelling evidence is provided demonstrating that LIN-23, the C. elegans orthologue of human beta-TrCP, negatively regulates the abundance of CDC-25.1 through S46 in C. elegans, specifically in early embryos. Surprisingly, the control of CDC-25.1 abundance is not restricted to intestinal cells, suggesting that the intestinal cell proliferation is more sensitive to elevated CDC-25.1 protein levels than other cell types. In a search for other molecules that may regulate the DSG site, GSK-3, APR-1 and WRM-1 were found to also cause excess intestinal cells. Intriguingly, their function is independent of S46 in CDC-25.1, because gsk-3, apr-1 or wrm-1 RNAi produce a synergistic increase in intestinal cells when combined with the cdc-25.1(ij48) allele. Thus, this thesis provides new insights to further our understanding of how the multicellular organism C. elegans controls proliferation of an entire tissue, the intestine

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Think locally: control of ubiquitin-dependent protein degradation in neurons

The nervous system coordinates many aspects of body function such as learning, memory, behaviour and locomotion. Therefore, it must develop and maintain an intricate network of differentiated neuronal cells, which communicate efficiently with each other and with non-neuronal target cells. Unlike most somatic cells, differentiated neurons are post-mitotic and characterized by a highly polarized morphology that determines the flow of information. Among other post-translational modifications, the ubiquitination of specific protein substrates was recently shown to have a crucial role in the regulation of neuronal development and differentiation. Here, we review recent findings that illustrate the mechanisms that mediate the temporal and spatial control of neuronal protein turnover by the ubiquitin–proteasome system (UPS), which is crucial for the development and function of the nervous system

A Screenable in vivo Assay to Study Proteostasis Networks in Caenorhabditis elegans

In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis as it regulates the turnover of damaged proteins. However, it is still unclear how the UPS integrates intrinsic and environmental challenges to promote organismal development and survival. Here, we set up an in vivo degradation assay to facilitate the genetic identification of ubiquitin-dependent proteolysis pathways in the multicellular organism Caenorhabditis elegans. Using this assay, we found that mild induction of protein-folding stress, which is nontoxic for wild-type worms, strongly reduces ubiquitin-dependent protein turnover. Ubiquitin-mediated degradation is also reduced by metabolic stress, which correlates with life-span extension. Unlike other stress conditions, however, acute heat stress results in enhanced rather than reduced proteolysis. Intriguingly, our study provides the first evidence for the existence of tissue-specific degradation requirements because loss of key regulators of the UPS, such as proteasomal subunits, causes accumulation of the model substrate, depending on the tissue type. Thus, here we establish a screenable degradation assay that allows diverse genetic screening approaches for the identification of novel cell-type-specific proteostasis networks important for developmental processes, stress response, and aging, thereby substantially extending the work on recently described mechanistic UPS reporter studies