The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

The Rho subfamily of GTPases is involved in control of cell morphology in mammals and yeast. The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA did not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation was also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation.

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.

The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.

No abstract

Rho and Rac, two members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins, regulate a variety of signal transduction pathways in eukaryotic cells. Upon stimulation of phagocytic cells, Rac enhances the activity of the enzyme nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase, resulting in the production of superoxide radicals. Activation of the NADPH oxidase requires the assembly of a multimolecular complex at the plasma membrane consisting of two integral membrane proteins, gp91phox and p21phox, and two cytosolic proteins, p67phox and p47phox. Rac1 interacted directly with p67phox in a GTP-dependent manner. Modified forms of Rac with mutations in the effector site did not stimulate oxidase activity or bind to p67phox. Thus, p67phox appears to be the Rac effector protein in the NADPH oxidase complex.