Bruce A. Edgar

Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins

Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumour syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP, suggesting that a GTPase might be the physiological target of Tsc2. Here we show that the small GTPase Rheb (Ras homologue enriched in brain) is a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupted its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. Our studies identify Rheb as a molecular target of the TSC tumour suppressors.

Endoreplication cell cycles: more for less.

neatly aligned in parallel arrays (see Urata et al., 1995), but polytene chromosomes greater than 16,000C have been noted in another insect, Chironomus. The distinc-and various intermediate DNA configurations can occur, Seattle, Washington 98109 differing primarily in the degree of association between 2 Whitehead Institute and duplicated chromatids (Figure 2D, Hammond and Laird, Department of Biology 1985). For this reason the term polyploid is often used Massachusetts Institute of Technology to refer to endoreplicated cells with virtually any chro-Cambridge, Massachusetts 02142 mosomal configuration. In endoreplication cell cycles, or endocycles, S Molecular studies from the past decade have revealed phases alternate with distinct gap phases that lack DNA striking conservation in the mechanisms of eukaryotic replication, but there is no cell division (Figure 3). Few, cell cycle control. Yet before the advent of molecular if any, cases of polypoidy resulting from continuous DNA genetics, it was clear that eukaryotes possessed many replication have been reported. Some endocycling cell different cell cycle variations, and thus that there must types retain hallmarks of mitosis, but many examples be diversity in mechanisms of control. One common cell lack all vestiges of mitosis, including chromosome con-cycle variant is the endoreplication cycle, in which cells densation, nuclear envelope breakdown, and the reor-increase their genomic DNA content without dividing. ganization of microtubules that builds the spindle. The Although endocycles are sometimes dismissed as an term endomitosis initially referred to a rare cell cycle in evolutionary peculiarity, they are widespread in protists, which mitosis occurred without nuclear envelope break-plants, and many animals including arthropods, mol-down or cytokinesis (for review see Nagl, 1978). How-lusks, and mammals. Endocycling cells can become ever, now this term is more generally used to describe incredibly polyploid, with chromatin values (C values cycles that proceed through anaphase but lack nuclear denote DNA content as a multiple of the normal haploid division and cytokinesis. In yet another cell cycle variant, nuclear division occurs without cytokinesis, giving rise genome) as high as 24,000 reported in some plant endo-to multinucleate cells. Such cycles are seen in mamma-sperms (Traas et al., 1998). Because cell size for a given lian hepatocytes and osteoclasts, and also in syncytial cell type is generally proportional to the amount of nu-slime molds like Physarum and early insect embryos. clear DNA, endoreplication constitutes an effective The regulation of these cycles is thought to be similar strategy of cell growth, and it is often found …

Drosophila myc regulates cellular growth during development.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut

Cells in intestinal epithelia turn over rapidly due to damage from digestion and toxins produced by the enteric microbiota. Gut homeostasis is maintained by intestinal stem cells (ISCs) that divide to replenish the intestinal epithelium, but little is known about how ISC division and differentiation are coordinated with epithelial cell loss. We show here that when enterocytes (ECs) in the Drosophila midgut are subjected to apoptosis, enteric infection, or JNK-mediated stress signaling, they produce cytokines (Upd, Upd2, and Upd3) that activate Jak/Stat signaling in ISCs, promoting their rapid division. Upd/Jak/Stat activity also promotes progenitor cell differentiation, in part by stimulating Delta/Notch signaling, and is required for differentiation in both normal and regenerating midguts. Hence, cytokine-mediated feedback enables stem cells to replace spent progeny as they are lost, thereby establishing gut homeostasis.

Rheb promotes cell growth as a component of the insulin/TOR signalling network

Insulin signalling is a potent inhibitor of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin). Recent studies suggest a that the tuberous sclerosis complex Tsc1–Tsc2 may couple insulin signalling to Tor activity. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1–S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1–Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.

Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region

Glucocorticoid receptor protein stimulates transcription initiation within murine mammary tumor virus (MTV) DNA sequences in vivo, and interacts selectively with MTV DNA in vitro. We mapped and compared five regions of MTV DNA that are bound specifically by purified receptor; one resides upstream of the transcription start site, and the others are distributed within transcribed sequences between 4 and 8 kb from the initiation site. Each region contains at least two strong binding sites for receptor, which itself appears to be a tetramer of 94,000 dalton hormone-binding subunits. Three of the five binding regions contain nine nuclease footprints that lack extensive homology, although a family of related octanucleotides can be discerned. Receptor interacts with the different regions with similar efficiencies, suggesting that receptor affinity for upstream and internal regions may differ by less than one order of magnitude. Moreover, each region appears to be bound independent of the others. A restriction fragment containing four footprint sequences from one of the regions has previously been shown to act in vivo as a receptor-dependent transcriptional enhancer element, implying that the binding sites detected in vitro may be biologically functional.

Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions.

Studies in Drosophila have characterized insulin receptor/phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but its function during development has remained uncertain. Here we show that inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. Hence we surmise that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions.

Genetic control of cell division patterns in the Drosophila embryo

In Drosophila embryogenesis, mitotic control undergoes a significant transition during the 14th interphase. Mitoses before interphase 14 run on maternal products, and occur in metasynchronous waves. Mitoses after interphase 14 require zygotic transcription, and occur asyncronously in an intricate, highly ordered spatio-temporal pattern. Mutations at the string (stg) locus cause cell-cycle arrest during this transition, in G2 of interphase 14, yet do not arrest other aspects of development. This phenotype suggests that stg is required specifically for initiating mitosis. We describe the cloning of stg, and show that its predicted amino acid sequence is homologous to that of cdc25, a regular of mitotic initiation in the yeast S. pombe. In addition, we show that zygotic expression of stg mRNA occurs in a dynamic series of spatial patterns which anticipate the patterns of the zygotically driven cell divisions. Therefore we suggest that regulated expression of stg mRNA controls the timing and location of these embryonic cell divisions.

Developmental control of cell cycle regulators : A fly's perspective

During early development in many species, maternally supplied gene products permit the cell cycle to run at maximum velocity, subdividing the fertilized egg into smaller and smaller cells. As development proceeds, zygotic controls are activated that first limit divisions to defined spatial and temporal domains, coordinating them with morphogenesis, and then halt proliferation altogether, to allow cell differentiation. Analysis of the regulation of cyclin-dependent kinases (Cdks) in Drosophila has provided insights into how this embryonic program of cell proliferation is controlled at the molecular level and how it is linked to developmental cues. Recent studies have also begun to reveal how cell proliferation is controlled during the second phase of Drosophila development, which occurs in imaginal tissues. In contrast to their embryonic progenitors, imaginal cells proliferate with a cycle that requires cell growth and is linked to patterning processes controlled by secreted cell signaling molecules. The functions of these signaling molecules appear to be nearly as conserved between vertebrates and invertebrates as the cell cycle control apparatus itself, suggesting that the mechanisms that coordinate growth, patterning, and cell proliferation in developing tissues have ancient origins.

The Three Postblastoderm Cell Cycles of Drosophila Embryogenesis Are Regulated in G2 by string

The string (stg) locus of Drosophila encodes a factor that is thought to trigger mitosis by activating the p34cdc2 protein kinase. stg is required for mitosis early in development and is transcribed in a dynamic pattern that anticipates the pattern of embryonic cell divisions. Here we show that differential cell cycle regulation during postblastoderm development (cell cycles 14-16) occurs in G2. We demonstrate that stg mRNA expressed from a heat shock promotor triggers mitosis, and an associated S phase, in G2 cells during these cycles. Hence, differential cell cycle timing at this developmental stage is controlled by stg. Finally, we use heat-induced stg expression to alter the normal pattern of embryonic mitoses. Surprisingly, the complex mitotic pattern evident during normal development is not essential for many features of pattern formation or for viability.