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

Reversible 26S proteasome disassembly upon mitochondrial stress

In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging

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

PDZ-RhoGEF ubiquitination by Cullin3–KLHL20 controls neurotrophin-induced neurite outgrowth

Ubiquitin-dependent degradation of PDZ-RhoGEF restricts RhoA activity to facilitate neurite outgrowth

A Novel Complex of Nucleoporins, Which Includes Sec13p and a Sec13p Homolog, Is Essential for Normal Nuclear Pores

AbstractIn a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p–ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis

SUS1, a functional component of the SAGA transcription complex and the mRNA export machinery.

Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery

Molecular Architecture of the Yeast Nuclear Pore Complex: Localization of Nsp1p Subcomplexes

The nuclear pore complex (NPC), a supramolecular assembly of ∼100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the ∼12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket

TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export

The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA-binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA-binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 markedly reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA

Comparative genomics of proteins involved in RNA nucleocytoplasmic export

Background: The establishment of the nuclear membrane resulted in the physical separation of transcription and translation, and presented early eukaryotes with a formidable challenge: how to shuttle RNA from the nucleus to the locus of protein synthesis. In prokaryotes, mRNA is translated as it is being synthesized, whereas in eukaryotes mRNA is synthesized and processed in the nucleus, and it is then exported to the cytoplasm. In metazoa and fungi, the different RNA species are exported from the nucleus by specialized pathways. For example, tRNA is exported by exportin-t in a RanGTP-dependent fashion. By contrast, mRNAs are associated to ribonucleoproteins (RNPs) and exported by an essential shuttling complex (TAP-p15 in human, Mex67-mtr2 in yeast) that transports them through the nuclear pore. The different RNA export pathways appear to be well conserved among members of Opisthokonta, the eukaryotic supergroup that includes Fungi and Metazoa. However, it is not known whether RNA export in the other eukaryotic supergroups follows the same export routes as in opisthokonts. Methods: Our objective was to reconstruct the evolutionary history of the different RNA export pathways across eukaryotes. To do so, we screened an array of eukaryotic genomes for the presence of homologs of the proteins involved in RNA export in Metazoa and Fungi, using human and yeast proteins as queries. Results: Our genomic comparisons indicate that the basic components of the RanGTP-dependent RNA pathways are conserved across eukaryotes, and thus we infer that these are traceable to the last eukaryotic common ancestor (LECA). On the other hand, several of the proteins involved in RanGTP-independent mRNA export pathways are less conserved, which would suggest that they represent innovations that appeared later in the evolution of eukaryotes. Conclusions: Our analyses suggest that the LECA possessed the basic components of the different RNA export mechanisms found today in opisthokonts, and that these mechanisms became more specialized throughout eukaryotic evolution