There is an increasing appreciation that the regulation of cell size plays critical roles in many biological phenomena. In mammals, changes in cell size rather than cell number underlie important physiological changes in organ size, such as heart enlargement induced by exercise or liver shrinking caused by starvation. During development changes in cell size are frequent, perhaps best exemplified by the massive increase in cell size that accompanies the differentiation and arborization of neurons. We now know that derangements of cell size underlie certain human diseases. These include tuberous sclerosis complex, a mental retardation and tumor-prone syndrome, as well as pathological cardiac hypertrophy.
We are studying at the biochemical, cellular and organismal level a signaling network called the mTOR pathway. This pathway is emerging as a critical integrator of growth signals in mammals and is under the control of nutrients, stress, and growth factors like insulin. We are interested in understanding how the mTOR pathway senses and integrates upstream signals and coordinates cell growth with the cell cycle. We are also studying the function of novel components of the pathway in mice.
Our recent work suggests that the mTOR pathway also regulates cell proliferation. Thus, mTOR is both a regulator of cell size and cell number and will likely have fundamental roles in setting organ and organism size.
mTOR signaling complex and pathway
The mammalian Target Of Rapamycin (mTOR) protein was discovered in studies into the mechanism of action of rapamycin, a macrolide antibiotic produced by a streptomyces species of bacteria. When rapamycin enters mammalian cells it binds to a small protein called FKBP12 to create a drug-receptor complex that interacts with mTOR, a large protein kinase related to PI3-kinase. Exactly how FKBP12-rapamycin perturbs mTOR function is not known. mTOR is evolutionarily conserved and integrates nutrient-and growth factor-derived signals to control the cell growth machinery. Within cells mTOR is part of a large protein complex that includes several other components including raptor and GbL, two novel proteins we have recently discovered. We seek to understand how nutrients and growth factors regulate the mTOR complex and to identify the functions of the different components at the cell and organism levels. In addition, we are interested in the potential roles of deregulated mTOR signaling in diabetes and cancer.
Recent work suggests that in addition to the raptor-mTOR-GbL complex (now called mTORC1, for mTOR complex 1), mTOR also exists in the cell in a distinct complex that instead of raptor contains the novel proteins rictor and mSin1 (this complex is called mTORC2, for mTOR complex 2). Interestingly, mTORC2 does not appear to be sensitive to rapamycin although we have recently shown that long term treatment with rapamycin can inhibit the assembly of mTORC2. This complex regulates the important cancer-related kinase called Akt/PKB that becomes hyperactive in cells mutated for the tumor suppressor PTEN.
We have many ongoing projects to understand the role of mTOR signaling in cancer using mouse models.
Because of the difficulty of identifying the components of signaling networks in mammalian cells, we are creating and using technologies that allow us to probe gene function in a highly parallel fashion. Our work has lead to the development of ‘cell-based microarrays’. The features (or spots) of these microarrays consist of clusters of mammalian cells that either over- or under-express a particular gene product or are under the influence of a small drug-like molecule. The features are only 100-250 microns in diameter and, thus, on a standard microscope slide we can create arrays containing thousands of individual cell clusters, each with a perturbation in a different gene.
With this technology we can rapidly identify candidate genes that may underlie phenotypes of interest in mammalian cells (e.g. cell size) as well perform synthetic effect type screens. To create cell-based microarrays we use a robot to print onto a surface compatible with cell attachment and proliferation nanoliters of biodegradable polymers mixed with reagents that perturb gene function. We then culture adherent cells on the biopolymer-containing spots. As the polymers degrade the reagents are released, affecting, without the need of wells to sequester the individual reagents, gene function in defined local areas of a cell monolayer. Using this approach we have locally introduced into mammalian cells cDNAs in expression vectors (through a process named ‘reverse transfection’), lentiviruses, siRNAs, and small molecules. We can examine the cells for alterations in particular phenotypes using techniques compatible with cells growing on a surface, such as immunofluorescence or in situ hybridization. We are also adapting the cell microarray concept for screening double-stranded RNAs that mediated RNAi in drosophila tissue culture cells.
Mammalian RNAi library
RNA inteference (RNAi) refers to a natural phenomenon in which the expression of a double stranded RNA leads to the degradation, through a complex process, of mRNAs to which one of its strands is complementary. Several years ago our lab along with several others, including those of William Hahn (DFCI), Eric Lander (Broad Institute), and Nir Hacohen (MGH), formed a consortium to generate an arrayed library of RNAi reagents that target every human gene and to develop the methodologies for using the library in a high-throughput fashion. This consortium, now called The RNAi Consortium (TRC), has generated, in a lentiviral vector, 5 distinct RNAi constructs for ost human genes. Each construct consists of a short hairpin RNA (shRNA) that directs the expression of a small interfering RNA (siRNA) that in turn causes the degradation of the mRNA which it recognizes. Our goal is to create constructs for every human and mouse gene for a total library size of about 300,000 constructs (5 constructs/gene for ~30,000 genes in each organism). The initial set of 160,000 shRNAs targets most annotated genes in the human genes. The consortium has a production team that generates the lentiviral plasmids encoding the shRNAs and can deliver high quality DNA for transfection into packaging cells for the generation of lentiviruses that express the shRNAs. These lentiviruses can efficiently transduce the shRNAs into the vast majority of human cells. We have set up a dedicated facility at the Whitehead Institute for the well-based high-throughput screening of this library and also collaborate with the RNAi platform of the Broad Institute to undertake screening in their facility.
Guertin, D.A. and Sabatini, D.M. Defining the role of mTOR in cancer. Cancer Cell 12: 9-22 (2007).
Sancak, Y., Thoreen, C.C., Peterson, T.R., Lindquist, R.A., Kang, S.A., Spooner, E., Carr, S.A., and Sabatini, D.M. PRAS40 Is an Insulin-Regulated Inhibitor of the mTORC1 Protein Kinase. Molecular Cell 25: 903-915 (2007).
Guertin, D.A., Stevens, D.M., Thoreen, C.C., Burds, A.A., Kalaany, N.Y., Moffat, J., Brown, M., Fitzgerald, K.J., Sabatini, D.M. Ablation in Mice of the mTORC Components raptor, rictor, or mLST8 Reveals that mTORC2 Is Required for Signaling to Akt-FOXO and PKCalpha, but Not S6K1. Developmental Cell 11:859-871 (2006).
Sarbassov, D.D., Ali, S,M, Sengupta, S., Sheen, J.-H., Hsu, P.P., Bagley, A.F., Markhard, A.L. and Sabatini, D,M. Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB. Molecular Cell 22: 159-168 (2006).
Moffat, J., Grueneberg, D.A., Yang, X., Kim, S.Y., Kloepfer, A.M., Hinkle, G., Piqani, B., Eisenhaure, T.M., Luo, B., Grenier, J.K., Carpenter, A.E., Foo, S.Y., Stewart, S.A., Stockwell, B.R., Hacohen, N., Hahn, W.C., Lander, E.S., Sabatini, D.M., and Root, D.E. A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell 124: 1283-1298 (2006).
Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR complex. Science 307: 1098-1101 (2005)
Wheeler, D.B., Bailey, S.N., Guertin, D.A., Carpenter, A.E., Higgins, C.O., Sabatini, D.M. RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells. Nature Methods 1:127-132 (2004).
Carpenter, A.E. and Sabatini, D.M. Systematic genome-wide screens of gene function. Nature Reviews Genetics 5: 11-22 (2004).
Kim, D.-H., Sarbassov, D.D., Ali, S.M., Latek, R.R., Guntur, K.V.P., Erdjument-Bromage, H., Tempst, P., and Sabatini, D.M. GbL: a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between mTOR and raptor. Molecular Cell 11: 895-904 (2003).
Kim, D.-H., Sarbassov, D.D., Ali, S.M., King, J.E., Latek, R.R., Erdjument-Bromage, H., Tempst, P., and Sabatini, D.M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the growth machinery. Cell 110: 163-175 (2002).
Ziauddin, J. and Sabatini, D.M.. Microarrays of cell expressing defined cDNAs. Nature 411:107-110 (2001).