Alex Sigal

Oscillations and variability in the p53 system

Understanding the dynamics and variability of protein circuitry requires accurate measurements in living cells as well as theoretical models. To address this, we employed one of the best‐studied protein circuits in human cells, the negative feedback loop between the tumor suppressor p53 and the oncogene Mdm2. We measured the dynamics of fluorescently tagged p53 and Mdm2 over several days in individual living cells. We found that isogenic cells in the same environment behaved in highly variable ways following DNA‐damaging gamma irradiation: some cells showed undamped oscillations for at least 3 days (more than 10 peaks). The amplitude of the oscillations was much more variable than the period. Sister cells continued to oscillate in a correlated way after cell division, but lost correlation after about 11 h on average. Other cells showed low‐frequency fluctuations that did not resemble oscillations. We also analyzed different families of mathematical models of the system, including a novel checkpoint mechanism. The models point to the possible source of the variability in the oscillations: low‐frequency noise in protein production rates, rather than noise in other parameters such as degradation rates. This study provides a view of the extensive variability of the behavior of a protein circuit in living human cells, both from cell to cell and in the same cell over time.

Dynamic Proteomics of Individual Cancer Cells in Response to a Drug

Why do seemingly identical cells respond differently to a drug? To address this, we studied the dynamics and variability of the protein response of human cancer cells to a chemotherapy drug, camptothecin. We present a dynamic-proteomics approach that measures the levels and locations of nearly 1000 different endogenously tagged proteins in individual living cells at high temporal resolution. All cells show rapid translocation of proteins specific to the drug mechanism, including the drug target (topoisomerase-1), and slower, wide-ranging temporal waves of protein degradation and accumulation. However, the cells differ in the behavior of a subset of proteins. We identify proteins whose dynamics differ widely between cells, in a way that corresponds to the outcomes—cell death or survival. This opens the way to understanding molecular responses to drugs in individual cells.

Variability and memory of protein levels in human cells

Protein expression is a stochastic process that leads to phenotypic variation among cells. The cell–cell distribution of protein levels in microorganisms has been well characterized but little is known about such variability in human cells. Here, we studied the variability of protein levels in human cells, as well as the temporal dynamics of this variability, and addressed whether cells with higher than average protein levels eventually have lower than average levels, and if so, over what timescale does this mixing occur. We measured fluctuations over time in the levels of 20 endogenous proteins in living human cells, tagged by the gene for yellow fluorescent protein at their chromosomal loci. We found variability with a standard deviation that ranged, for different proteins, from about 15% to 30% of the mean. Mixing between high and low levels occurred for all proteins, but the mixing time was longer than two cell generations (more than 40 h) for many proteins. We also tagged pairs of proteins with two colours, and found that the levels of proteins in the same biological pathway were far more correlated than those of proteins in different pathways. The persistent memory for protein levels that we found might underlie individuality in cell behaviour and could set a timescale needed for signals to affect fully every member of a cell population.

Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy

Latency and ongoing replication have both been proposed to explain the drug-insensitive human immunodeficiency virus (HIV) reservoir maintained during antiretroviral therapy. Here we explore a novel mechanism for ongoing HIV replication in the face of antiretroviral drugs. We propose a model whereby multiple infections per cell lead to reduced sensitivity to drugs without requiring drug-resistant mutations, and experimentally validate the model using multiple infections per cell by cell-free HIV in the presence of the drug tenofovir. We then examine the drug sensitivity of cell-to-cell spread of HIV, a mode of HIV transmission that can lead to multiple infection events per target cell. Infections originating from cell-free virus decrease strongly in the presence of antiretrovirals tenofovir and efavirenz whereas infections involving cell-to-cell spread are markedly less sensitive to the drugs. The reduction in sensitivity is sufficient to keep multiple rounds of infection from terminating in the presence of drugs. We examine replication from cell-to-cell spread in the presence of clinical drug concentrations using a stochastic infection model and find that replication is intermittent, without substantial accumulation of mutations. If cell-to-cell spread has the same properties in vivo, it may have adverse consequences for the immune system, lead to therapy failure in individuals with risk factors, and potentially contribute to viral persistence and hence be a barrier to curing HIV infection.

Collective and single cell behavior in epithelial contact inhibition

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as “contact inhibition.” The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

Dynamics and Variability of ERK2 Response to EGF in Individual Living Cells

Signal-transduction cascades are usually studied on cell averages, masking variability between individual cells. To address this, we studied in individual cells the dynamic response of ERK2, a well-characterized MAPK signaling protein, which enters the nucleus upon stimulation. Using fluorescent tagging at the endogenous chromosomal locus, we found that cells show wide basal variation in ERK2 nuclear levels. Upon EGF stimulation, cells show (1) a fold-change response, where peak nuclear accumulation of ERK2 is proportional to basal level in each cell; and (2) exact adaptation in nuclear levels of ERK2, returning to original basal level of each cell. The timing of ERK2 dynamics is more precise between cells than its amplitude. We further found that in some cells ERK2 exhibits a second pulse of nuclear entry, smaller than the first. The present study suggests that this signaling system compensates for natural biological noise: despite large variation in nuclear basal levels, ERK2s fold dynamics is similar between cells.

Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins

We examined cell cycle–dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized the cells in silico by aligning protein dynamics in each cell between consecutive divisions. We observed widespread (40%) cell-cycle dependence of nuclear protein levels and detected previously unknown cell cycle–dependent localization changes. This approach to dynamic proteomics can aid in discovery and accurate quantification of the extensive regulation of protein concentration and localization in individual living cells.

Change of the Death Pathway in Senescent Human Fibroblasts in Response to DNA Damage Is Caused by an Inability To Stabilize p53

ABSTRACT The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.

Integrity of the N-terminal transcription domain of p53 is required for mutant p53 interference with drug-induced apoptosis.

The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N‐terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug‐induced apoptosis, further modification at the N‐terminus abolished this blocking activity. Furthermore, expression of c‐myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53‐expressing cells, but was abolished in the presence of the transcription‐deficient p53 core mutant. In addition, wild‐type p53, mutated in the N‐terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N‐terminus is essential for both the activity of wild‐type p53 in apoptosis and for mutant p53‐mediated block of drug‐induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.

Generation of a fluorescently labeled endogenous protein library in living human cells

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100–200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3′ rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.