Aida Flor A. de la Cruz

The Drosophila Cyclin D–Cdk4 complex promotes cellular growth

Mammalian cyclin D–Cdk4 complexes have been characterized as growth factor‐responsive cell cycle regulators. Their levels rise upon growth factor stimulation, and they can phosphorylate and thus neutralize Retinoblastoma (Rb) family proteins to promote an E2F‐dependent transcriptional program and S‐phase entry. Here we characterize the in vivo function of Drosophila Cyclin D (CycD). We find that Drosophila CycD–Cdk4 does not act as a direct G1/S‐phase regulator, but instead promotes cellular growth (accumulation of mass). The cellular response to CycD–Cdk4‐driven growth varied according to cell type. In undifferentiated proliferating wing imaginal cells, CycD–Cdk4 caused accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD–Cdk4 caused excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD–Cdk4 caused cell enlargement (hypertrophy) in post‐mitotic cells. Interaction tests with a Drosophila Rb homolog, RBF, indicate that CycD–Cdk4 can counteract the cell cycle suppressive effects of RBF, but that its growth promoting activity is mediated at least in part via other targets.

Intrinsic negative cell cycle regulation provided by PIP box- and Cul4Cdt2-mediated destruction of E2f1 during S phase.

E2F transcription factors are key regulators of cell proliferation that are inhibited by pRb family tumor suppressors. pRb-independent modes of E2F inhibition have also been described, but their contribution to animal development and tumor suppression is unclear. Here, we show that S phase-specific destruction of Drosophila E2f1 provides a novel mechanism for cell cycle regulation. E2f1 destruction is mediated by a PCNA-interacting-protein (PIP) motif in E2f1 and the Cul4(Cdt2) E3 ubiquitin ligase and requires the Dp dimerization partner but not direct Cdk phosphorylation or Rbf1 binding. E2f1 lacking a functional PIP motif accumulates inappropriately during S phase and is more potent than wild-type E2f1 at accelerating cell cycle progression and inducing apoptosis. Thus, S phase-coupled destruction is a key negative regulator of E2f1 activity. We propose that pRb-independent inhibition of E2F during S phase is an evolutionarily conserved feature of the metazoan cell cycle that is necessary for development.

Stepwise Evolution of Essential Centromere Function in a Drosophila Neogene

Essential Novelty The evolution of essential function for newly originated genes presents a conundrum, in that prior to the genes origin either the essential function was absent or else performed by another gene or set of genes. In order to better understand how new genes acquire essential function, Ross et al. (p. 1211) investigated the origin of the Drosophila gene Umbrea. Umbrea became an essential protein in certain Drosophila species through the gain of localization at the centromere and a role in chromosome segregation. How does a recently evolved gene come to encode an essential function? Evolutionarily young genes that serve essential functions represent a paradox; they must perform a function that either was not required until after their birth or was redundant with another gene. How young genes rapidly acquire essential function is largely unknown. We traced the evolutionary steps by which the Drosophila gene Umbrea acquired an essential role in chromosome segregation in D. melanogaster since the genes origin less than 15 million years ago. Umbrea neofunctionalization occurred via loss of an ancestral heterochromatin-localizing domain, followed by alterations that rewired its protein interaction network and led to species-specific centromere localization. Our evolutionary cell biology approach provides temporal and mechanistic detail about how young genes gain essential function. Such innovations may constantly alter the repertoire of centromeric proteins in eukaryotes.

Rheb-TOR signaling promotes protein synthesis, but not glucose or amino acid import, in Drosophila

BackgroundThe Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability.ResultsWe show that Rheb and TOR do not promote the import of glucose, bulk amino acids, or arginine in Drosophila S2 cells, but that both gene products are important regulators of ribosome biogenesis, protein synthesis, and cell size. S2 cell size, protein synthesis, and glucose import were largely insensitive to manipulations of insulin signaling components, suggesting that cellular energy levels and TOR activity can be maintained through insulin/PI3K-independent mechanisms in S2 cell culture. In vivo in Drosophila larvae, however, we found that insulin signaling can regulate protein synthesis, and thus may affect TOR activity.ConclusionRheb-TOR signaling controls S2 cell growth by promoting ribosome production and protein synthesis, but apparently not by direct effects on the import of amino acids or glucose. The effect of insulin signaling upon TOR activity varies according to cellular type and context.

Flow cytometric analysis of Drosophila cells.

Flow cytometry is a powerful technique that allows the researcher to measure fluorescence emissions on a per-cell basis, at multiple wavelengths, in populations of thousands of cells. In this chapter, we outline the use of flow cytometry for the analysis of cells from Drosophilas imaginal discs, which are developing epithelial organs that give rise to, but not exclusively, the wings, eyes, and legs of the adult. A variety of classical and transgenic genetic methods can be used to mark cells (e.g., mutant, or overexpressing a gene, or in a particular compartment) in these organs with green fluorescent protein (GFP), which is readily detected by flow cytometry. After dissecting an organ out of the animal and dissociating it into single cells, a flow cytometer can be used to assay the size, DNA content, and other parameters in GFP-marked experimental cells as well as GFP-negative control cells from the same sample. Specific marked cell populations can also be physically sorted, and then used in diverse biochemical assays. This chapter includes protocols for isolation and dissociation of larval imaginal discs and pupal appendages for flow cytometry, and as well as for flow cytometric acquisition and analysis. In addition, we present protocols for performing flow cytometry on fixed or live-cultured Drosophila S2 cells.

Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress

Drosophila cyclinD (CycD) is the single fly ortholog of the mammalian cyclin D1 and promotes both cell cycle progression and cellular growth. However, little is known about how CycD promotes cell growth. We show here that CycD/Cdk4 hyperactivity leads to increased mitochondrial biogenesis (mitobiogenesis), mitochondrial mass, NRF-1 activity (Tfam transcript levels) and metabolic activity in Drosophila, whereas loss of CycD/Cdk4 activity has the opposite effects. Surprisingly, both CycD/Cdk4 addition and loss of function increase mitochondrial superoxide production and decrease lifespan, indicating that an imbalance in mitobiogenesis may lead to oxidative stress and aging. In addition, we provide multiple lines of evidence indicating that CycD/Cdk4 activity affects the hypoxic status of cells and sensitizes animals to hypoxia. Both mitochondrial and hypoxia-related effects can be detected at the global transcriptional level. We propose that mitobiogenesis and the hypoxic stress response have an antagonistic relationship, and that CycD/Cdk4 levels regulate mitobiogenesis contemporaneous to the cell cycle, such that only when cells are sufficiently oxygenated can they proliferate.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila

Essential genes and species incompatibilities Crosses between two fruit fly species, Drosophila melanogaster and D. simulans, result in hybrid progeny that are all female. Although some of the genes responsible for this species barrier are known, the full complement of molecular determinants that lead to inviable males has remained mysterious. Phadnis et al. used mutagenesis and a sequencing-based genomic screen to link hybrid inviability to the cell cycle. The inviable males result from an interaction between three genes, one of which is essential, which precluded its identification with standard genetic screens. This strategy to identify speciation genes can be applied to other model and nonmodel systems. Science, this issue p. 1552 A hybrid incompatibility system in flies requires an essential cell cycle gene. Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle–regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems.

Mammalian cyclin D1/Cdk4 complexes induce cell growth in Drosophila

The Drosophila melanogaster cyclin dependent protein kinase complex CycD/Cdk4 has been shown to regulate cellular growth (accumulation of mass) as well as proliferation (cell cycle progression). In contrast, the orthologous mammalian complex has been shown to regulate cell cycle progression, but possible functions in growth control have not been addressed directly. To test whether mammalian Cyclin D1/Cdk4 complexes are capable of driving cell growth, we expressed such a complex in Drosophila. Using assays that distinguish between mass increase and cell cycle progression, we found that this complex stimulated cell growth, like its Drosophila counterpart. Furthermore, Hif-1 prolyl hydroxylase (Hph) is required for both complexes to drive growth. Our data suggest that the growth-specific function of CycD/Cdk4 is conserved from arthropods to mammals.

Changes in neuronal CycD/Cdk4 activity affect aging, neurodegeneration, and oxidative stress

Mitochondrial dysfunction has been implicated in human diseases, including cancer, and proposed to accelerate aging. The Drosophila Cyclin‐dependent protein kinase complex cyclin D/cyclin‐dependent kinase 4 (CycD/Cdk4) promotes cellular growth by stimulating mitochondrial biogenesis. Here, we examine the neurodegenerative and aging consequences of altering CycD/Cdk4 function in Drosophila. We show that pan‐neuronal loss or gain of CycD/Cdk4 increases mitochondrial superoxide, oxidative stress markers, and neurodegeneration and decreases lifespan. We find that RNAi‐mediated depletion of the mitochondrial transcription factor, Tfam, can abrogate CycD/Cdk4s detrimental effects on both lifespan and neurodegeneration. This indicates that CycD/Cdk4s pathological consequences are mediated through altered mitochondrial function and a concomitant increase in reactive oxygen species. In support of this, we demonstrate that CycD/Cdk4 activity levels in the brain affect the expression of a set of ‘oxidative stress’ genes. Our results indicate that the precise regulation of neuronal CycD/Cdk4 activity is important to limit mitochondrial reactive oxygen species production and prevent neurodegeneration.