Daniel Louvard

Notch signals control the fate of immature progenitor cells in the intestine

The Notch signalling pathway plays a crucial role in specifying cellular fates in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in intestinal development. Here, by modulating Notch activity in the mouse intestine, we directly implicate Notch signals in intestinal cell lineage specification. We also show that Notch activation is capable of amplifying the intestinal progenitor pool while inhibiting cell differentiation. We conclude that Notch activity is required for the maintenance of proliferating crypt cells in the intestinal epithelium.

The aminopeptidase from hog intestinal brush border

Abstract The aminopeptidase bound to the brush border membrane of porcine jejunum and ileum has been purified. After treatment of the mucosa with Triton X-100, the enzyme was found to be still attached to very small membrane fragments (the aminopeptidase complex) from which it was liberated by trypsin. The free enzyme could be further purified to homogeneity by using classical techniques. The high molecular weight complex was shown to contain, besides aminopeptidase, at least one other protein inactive on the substrates of this enzyme. The free aminopeptidase was observed to have a molecular weight of about 280 000, and to contain 23% sugars and 2 atoms of zinc per mole. Gel electrophoresis in sodium dodecyl sulfate showed 4 bands. One, however, is probably an artefact arising from trypsin treatment. The other 3 suggest the existence in aminopeptidase of 3 subunits with molecular weights of 130 000, 97 000, 49 000 and which were also obtained from the complex. The aminopeptidase which has been purified accounts for all, or almost all, of the peptidase activity of the brush border membrane. It also accounts for all of the arylamidase activity, all of the activity against an heptapeptide substrate, about half the tripeptidase activity and a small but apparently significantly part of the dipeptidase activity of the whole jejunal and ileal mucosa.

Wnt/β-Catenin Is Essential for Intestinal Homeostasis and Maintenance of Intestinal Stem Cells

ABSTRACT The Wnt signaling pathway is deregulated in over 90% of human colorectal cancers. β-Catenin, the central signal transducer of the Wnt pathway, can directly modulate gene expression by interacting with transcription factors of the TCF/LEF family. In the present study we investigate the role of Wnt signaling in the homeostasis of intestinal epithelium by using tissue-specific, inducible β-catenin gene ablation in adult mice. Block of Wnt/β-catenin signaling resulted in rapid loss of transient-amplifying cells and crypt structures. Importantly, intestinal stem cells were induced to terminally differentiate upon deletion of β-catenin, resulting in a complete block of intestinal homeostasis and fatal loss of intestinal function. Transcriptional profiling of mutant crypt mRNA isolated by laser capture microdissection confirmed those observations and allowed us to identify genes potentially responsible for the functional preservation of intestinal stem cells. Our data demonstrate an essential requirement of Wnt/β-catenin signaling for the maintenance of the intestinal epithelium in the adult organism. This challenges attempts to target aberrant Wnt signaling as a new therapeutic strategy to treat colorectal cancer.

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

Microtubules and intermediate filaments cooperate with actin and other components of filopodia during invadopodia maturation.

THE CO-WORKERS OF ACTIN FILAMENTS: FROM CELL STRUCTURES TO SIGNALS

Cells have various surface architectures, which allow them to carry out different specialized functions. Actin microfilaments that are associated with the plasma membrane are important for generating these cell-surface specializations, and also provide the driving force for remodelling cell morphology and triggering new cell behaviour when the environment is modified. This phenomenon is achieved through a tight coupling between cell structure and signal transduction, a process that is modulated by the regulation of actin-binding proteins.

Notch and Wnt signals cooperatively control cell proliferation and tumorigenesis in the intestine

Notch and Wnt signals play essential roles in intestinal development and homeostasis, yet how they integrate their action to affect intestinal morphogenesis is not understood. We examined the interplay between these two signaling pathways in vivo, by modulating Notch activity in mice carrying either a loss- or a gain-of-function mutation of Wnt signaling. We find that the dramatic proliferative effect that Notch signals have on early intestinal precursors requires normal Wnt signaling, whereas its influence on intestinal differentiation appears independent of Wnt. Analogous experiments in Drosophila demonstrate that the synergistic effects of Notch and Wnt are valid across species. We also demonstrate a striking synergy between Notch and Wnt signals that results in inducing the formation of intestinal adenomas, particularly in the colon, a region rarely affected in available mouse tumor models, but the primary target organ in human patients. These studies thus reveal a previously unknown oncogenic potential of Notch signaling in colorectal tumorigenesis that, significantly, is supported by the analysis of human tumors. Importantly, our experimental evidence raises the possibility that Notch activation might be an essential initial event triggering colorectal cancer.

Fascin, a Novel Target of β-Catenin-TCF Signaling, Is Expressed at the Invasive Front of Human Colon Cancer

Cancer cells become metastatic by acquiring a motile and invasive phenotype. This step requires remodeling of the actin cytoskeleton and the expression of exploratory, sensory organelles known as filopodia. Aberrant beta-catenin-TCF target gene activation plays a major role in colorectal cancer development. We identified fascin1, a key component of filopodia, as a target of beta-catenin-TCF signaling in colorectal cancer cells. Fascin1 mRNA and protein expression were increased in primary cancers in a stage-dependent manner. Fascin1 was exclusively localized at the invasive front of tumors also displaying nuclear beta-catenin. Forced expression of fascin1 in colorectal cancer cells increased their migration and invasion in cell cultures and caused cell dissemination and metastasis in vivo, whereas suppression of fascin1 expression by small interfering RNA reduces cell invasion. Although expression of fascin1 in primary tumors correlated with the presence of metastases, fascin1 was not expressed in metastases. Our studies show that fascin1 expression is tightly regulated during development of colon cancer metastases and is a novel target of beta-catenin-TCF signaling. We propose that transient up-regulation of fascin1 in colorectal cancer promotes the acquisition of migratory and invasive phenotypes that lead to metastasis. Moreover, the expression of fascin1 is down-regulated when tumor cells reach their metastatic destination where migration ceases and proliferation is enhanced. Although metastasis to vital organs is often the cause of mortality, only limited success has been attained in developing effective therapeutics against metastatic disease. We propose that genes involved in cell migration and invasion, such as fascin1, could serve as novel targets for metastasis prevention.

A monoclonal antibody against a 135-K Golgi membrane protein.

A monoclonal antibody (53FC3) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000‐10 000, whereas in the presence of Triton X‐100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4‐6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.

ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling

The ERM (ezrin, radixin and moesin) family of proteins are linkers that tether actin microfilaments to the plasma membrane. Merlin, the NF2 tumor suppressor gene product, is highly homologous to ERM proteins. In ERM proteins and merlin, interdomain binding promotes auto-inhibition and homo-oligomerization or hetero-oligomerization. Recent studies have revealed that ERM proteins transduce growth signals, and have shed new light on how merlin links cell growth to the cytoskeleton.