Peter K. Sorger

Cells on chips

Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world.

Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis

In microorganisms, noise in gene expression gives rise to cell-to-cell variability in protein concentrations. In mammalian cells, protein levels also vary and individual cells differ widely in their responsiveness to uniform physiological stimuli. In the case of apoptosis mediated by TRAIL (tumour necrosis factor (TNF)-related apoptosis-inducing ligand) it is common for some cells in a clonal population to die while others survive—a striking divergence in cell fate. Among cells that die, the time between TRAIL exposure and caspase activation is highly variable. Here we image sister cells expressing reporters of caspase activation and mitochondrial outer membrane permeabilization after exposure to TRAIL. We show that naturally occurring differences in the levels or states of proteins regulating receptor-mediated apoptosis are the primary causes of cell-to-cell variability in the timing and probability of death in human cell lines. Protein state is transmitted from mother to daughter, giving rise to transient heritability in fate, but protein synthesis promotes rapid divergence so that sister cells soon become no more similar to each other than pairs of cells chosen at random. Our results have implications for understanding ‘fractional killing’ of tumour cells after exposure to chemotherapy, and for variability in mammalian signal transduction in general.

MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells.

The mitotic checkpoint protein hsMad2 is required to arrest cells in mitosis when chromosomes are unattached to the mitotic spindle. The presence of a single, lagging chromosome is sufficient to activate the checkpoint, producing a delay at the metaphase–anaphase transition until the last spindle attachment is made. Complete loss of the mitotic checkpoint results in embryonic lethality owing to chromosome mis-segregation in various organisms. Whether partial loss of checkpoint control leads to more subtle rates of chromosome instability compatible with cell viability remains unknown. Here we report that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts. Checkpoint-defective cells show premature sister-chromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome mis-segregation events in the absence of these agents. Furthermore, Mad2+/- mice develop lung tumours at high rates after long latencies, implicating defects in the mitotic checkpoint in tumorigenesis.

Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation

Heat shock promoters contain one or more binding sites for a specific heat shock factor (HSF). We report the cloning and sequence of the gene encoding yeast HSF, and demonstrate that HSF is required for growth at normal temperatures (15 degrees C-30 degrees C). The activity of a promoter containing a synthetic HSF binding site varies over a 200-fold range between 15 degrees C and 39 degrees C (heat shock). This change in activity is accompanied by multiple changes in the phosphorylation state of HSF, but all forms of HSF are able to bind DNA. We propose that the expression of heat shock genes in yeast is modulated by phosphorylation of DNA-bound HSF, and that this leads to a more efficient interaction of the factor with other components of the transcriptional machinery.

A role for the Adenomatous Polyposis Coli protein in chromosome segregation

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of β-catenin (reviewed in refs 1–3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore–microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.

Heat shock factor and the heat shock response

Peter K. Sorger Department of Microbiology and Immunology University of California San Francisco, California 94143-0502 The induction of eukaryotic heat shock genes in response to a temperature upshift is mediated by the binding of a transcriptional activator, heat shock factor, to a short highly conserved DNA sequence known as the heat shock element. This review will discuss progress in several or- ganisms in characterizing heat shock factor and will sug- gest preliminary answers to the following questions. How is the DNA-binding activity and transcriptional potency of heat shock factor regulated? How is autoregulation of heat shock protein (hsp) synthesis achieved? What is the mech- anism by which heat shock is sensed? Heat Shock Elements and Their Interaction with Heat Shock Factor Heat shock elements are best described as contiguous arrays of variable numbers of the 5 bp sequence nGAAn arranged in alternating orientation (n denotes less strongly conserved nucleotides that nevertheless may be involved in important DNA-protein interactions; Figure 1; Xiao and Lis, 1988; Amin et al., 1988). At least two nGAAn units are needed for high affinity binding of heat shock factor in vitro, and these may be arranged either head-to-head (nGAAnnTTCn) or tail-to-tail (nTTCnnGAAn; Perisic et al., 1989). How can heat shock factor bind to these structurally distinct sites as well as to heat shock elements with larger numbers of 5 bp units? The answer may lie in the oligo- merit nature of the heat shock factor protein (Figure 2). Heat shock factor from both Saccharomyces cerevisiae (SC-HSF) and Drosophila (D-HSF) associates to form pro- tein trimers in solution and when bound to DNA (Perisic et al., 1989; Sorger and Nelson, 1989). It is not clear, how- ever, whether heat shock factor exists in vivo primarily as a trimeric, hexameric, or possibly even larger complex. Each subunit of a D-HSF multimer is thought to bind to a single nGAAn unit, and the binding to successive units is highly cooperative (Xiao et al., 1991). The coiled-coil structure that has been proposed to form the interface between heat shock factor monomers is inherently three- fold symmetric (see below). Thus, the binding of heat shock factor trimers to DNA (which is inherently two-fold symmetric) would require a flexible hinge between the tri- merization and DNA-binding surfaces of individual mono- mers. Flexibility at this hinge may also be exploited in the binding of subunits to differently oriented nGAAn units. The binding of trimers to adjacent sites is also highly cooperative (Shuey and Parker, 1986): in vitro, the dissoci- ation rate from DNA of a complex of two DNA-bound tri- mers is more than three orders of magnitude lower than that of a single trimer (Xiao et al., 1991). Thus, even when the molarity of sites exceeds that of the heat shock factor protein, heat shock factor preferentially forms large com-

Physicochemical modelling of cell signalling pathways

Physicochemical modelling of signal transduction links fundamental chemical and physical principles, prior knowledge about regulatory pathways, and experimental data of various types to create powerful tools for formalizing and extending traditional molecular and cellular biology.

A Systems Model of Signaling Identifies a Molecular Basis Set for Cytokine-Induced Apoptosis

Signal transduction pathways control cellular responses to stimuli, but it is unclear how molecular information is processed as a network. We constructed a systems model of 7980 intracellular signaling events that directly links measurements to 1440 response outputs associated with apoptosis. The model accurately predicted multiple time-dependent apoptotic responses induced by a combination of the death-inducing cytokine tumor necrosis factor with the prosurvival factors epidermal growth factor and insulin. By capturing the role of unsuspected autocrine circuits activated by transforming growth factor–α and interleukin-1α, the model revealed new molecular mechanisms connecting signaling to apoptosis. The model derived two groupings of intracellular signals that constitute fundamental dimensions (molecular “basis axes”) within the apoptotic signaling network. Projection along these axes captures the entire measured apoptotic network, suggesting that cell survival is determined by signaling through this canonical basis set.

Chromosome Missegregation and Apoptosis in Mice Lacking the Mitotic Checkpoint Protein Mad2

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.

Electronic detection of DNA by its intrinsic molecular charge.

We report the selective and real-time detection of label-free DNA using an electronic readout. Microfabricated silicon field-effect sensors were used to directly monitor the increase in surface charge when DNA hybridizes on the sensor surface. The electrostatic immobilization of probe DNA on a positively charged poly-l-lysine layer allows hybridization at low ionic strength where field-effect sensing is most sensitive. Nanomolar DNA concentrations can be detected within minutes, and a single base mismatch within 12-mer oligonucleotides can be distinguished by using a differential detection technique with two sensors in parallel. The sensors were fabricated by standard silicon microtechnology and show promise for future electronic DNA arrays and rapid characterization of nucleic acid samples. This approach demonstrates the most direct and simple translation of genetic information to microelectronics.