Ron Milo

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.

Multiple sclerosis: Geoepidemiology, genetics and the environment

Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the central nervous system characterized by relapses and remissions. The risk of acquiring this complex disease is associated with exposure to environmental factors in genetically susceptible individuals. The epidemiology of MS has been extensively studied. We review the geographic epidemiology of the disease, the influence of immigration, age at immigration, clustering and epidemics. Various presumptive risk factors are discussed such as ultraviolet radiation, vitamin D, Epstein-Barr virus and infectious mononucleosis, other infectious agents and non-infectious factors. Two different hypotheses, the hygiene hypothesis and the prevalence hypothesis, were proposed to explain these environmental risk factors for MS. The epidemiological data, combined with pathological and immunological data, may contribute to the debate whether MS is an autoimmune disease, a latent or persistent viral disease, or a neurodegenerative disease.

BioNumbers—the database of key numbers in molecular and cell biology

BioNumbers (http://www.bionumbers.hms.harvard.edu) is a database of key numbers in molecular and cell biology—the quantitative properties of biological systems of interest to computational, systems and molecular cell biologists. Contents of the database range from cell sizes to metabolite concentrations, from reaction rates to generation times, from genome sizes to the number of mitochondria in a cell. While always of importance to biologists, having numbers in hand is becoming increasingly critical for experimenting, modeling, and analyzing biological systems. BioNumbers was motivated by an appreciation of how long it can take to find even the simplest number in the vast biological literature. All numbers are taken directly from a literature source and that reference is provided with the number. BioNumbers is designed to be highly searchable and queries can be performed by keywords or browsed by menus. BioNumbers is a collaborative community platform where registered users can add content and make comments on existing data. All new entries and commentary are curated to maintain high quality. Here we describe the database characteristics and implementation, demonstrate its use, and discuss future directions for its development.

A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.

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.

Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans.

It is often presented as common knowledge that, in the human body, bacteria outnumber human cells by a ratio of at least 10:1. Revisiting the question, we find that the ratio is much closer to 1:1.

Design and analysis of synthetic carbon fixation pathways

Carbon fixation is the process by which CO2 is incorporated into organic compounds. In modern agriculture in which water, light, and nutrients can be abundant, carbon fixation could become a significant growth-limiting factor. Hence, increasing the fixation rate is of major importance in the road toward sustainability in food and energy production. There have been recent attempts to improve the rate and specificity of Rubisco, the carboxylating enzyme operating in the Calvin–Benson cycle; however, they have achieved only limited success. Nature employs several alternative carbon fixation pathways, which prompted us to ask whether more efficient novel synthetic cycles could be devised. Using the entire repertoire of approximately 5,000 metabolic enzymes known to occur in nature, we computationally identified alternative carbon fixation pathways that combine existing metabolic building blocks from various organisms. We compared the natural and synthetic pathways based on physicochemical criteria that include kinetics, energetics, and topology. Our study suggests that some of the proposed synthetic pathways could have significant quantitative advantages over their natural counterparts, such as the overall kinetic rate. One such cycle, which is predicted to be two to three times faster than the Calvin–Benson cycle, employs the most effective carboxylating enzyme, phosphoenolpyruvate carboxylase, using the core of the naturally evolved C4 cycle. Although implementing such alternative cycles presents daunting challenges related to expression levels, activity, stability, localization, and regulation, we believe our findings suggest exciting avenues of exploration in the grand challenge of enhancing food and renewable fuel production via metabolic engineering and synthetic biology.

eQuilibrator—the biochemical thermodynamics calculator

The laws of thermodynamics constrain the action of biochemical systems. However, thermodynamic data on biochemical compounds can be difficult to find and is cumbersome to perform calculations with manually. Even simple thermodynamic questions like ‘how much Gibbs energy is released by ATP hydrolysis at pH 5?’ are complicated excessively by the search for accurate data. To address this problem, eQuilibrator couples a comprehensive and accurate database of thermodynamic properties of biochemical compounds and reactions with a simple and powerful online search and calculation interface. The web interface to eQuilibrator (http://equilibrator.weizmann.ac.il) enables easy calculation of Gibbs energies of compounds and reactions given arbitrary pH, ionic strength and metabolite concentrations. The eQuilibrator code is open-source and all thermodynamic source data are freely downloadable in standard formats. Here we describe the database characteristics and implementation and demonstrate its use.