Eli Zamir

Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts

Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain α5β1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

Transcription-dependent induction of G1 phase during the zebra fish midblastula transition.

The early development of the zebra fish (Danio rerio) embryo is characterized by a series of rapid and synchronous cell cycles with no detectable transcription. This period is followed by the midblastula transition (MBT), during which the cell cycle gradually lengthens, cell synchrony is lost, and zygotic transcription is initially detected. In this work, we examined the changes in the pattern of the cell cycle during MBT in zebra fish and whether these changes are dependent on the initiation of zygotic transcription. To characterize the pattern of the early zebra fish cell cycles, the embryonic DNA content was determined by flow cytometric analysis. We found that G1 phase is below detection levels during the first 10 cleavages and can be initially detected at the onset of MBT. Inhibition of zygotic transcription, by microinjection of actinomycin D, abolished the appearance of G1 phase at MBT. Premature activation of zygotic transcription, by microinjection of nonspecific DNA, induced G1 phase before the onset of MBT, while coinjection of actinomycin D and nonspecific DNA abolished this early appearance of G1 phase. We therefore suggest that during the early development of the zebra fish embryo, G1 phase appears at the onset of MBT and that the activation of transcription at MBT is essential and sufficient for the G1-phase induction.

Reverse engineering intracellular biochemical networks

Although much is known about the molecular components of cellular signaling pathways, very little is known about how these multicomponent biochemical machineries process complex extracellular signals to generate a consolidated cellular response. A newly developed theoretical approach for reverse engineering network structure—analyzing how perturbations propagate in a network—can be combined with chemical perturbations and quantitative detection approaches to reveal the causal connections within protein networks in cells. This information indicates the dynamic capabilities of a network and thereby its potential function.

Quantitative Multicolor Compositional Imaging Resolves Molecular Domains in Cell-Matrix Adhesions

Background Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined. Methodology/Principal Findings We present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition. Conclusions/Significance Multicolor compositional imaging resolves “molecular signatures” characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional “contents-resolved” dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.

Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photobleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system. DOI: http://dx.doi.org/10.7554/eLife.02257.001

Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics

We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.

Resolving and classifying haematopoietic bone-marrow cell populations by multi-dimensional analysis of flow-cytometry data

The study of normal or malignant haematopoiesis requires the analysis of heterogeneous cell populations using multiple morphological and molecular criteria. Flow cytometry has the capacity to acquire multi‐parameter information of large haematopoietic cell populations, utilizing various combinations of >200 molecular markers (clusters of differentiation, CD). However, current flow cytometry analyses are based on serial gating of two‐parametric scatter plots – a process that is inherently incapable to discriminate all subgroups of cells in the data. Here we studied the cellular diversity of normal bone marrows (BM) using multi‐dimensional cluster analysis of six‐parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on the myeloid lineage. Twenty‐three subclasses of cells were resolved, many of them inseparable even when examined in all possible two‐parametric scatter plots. The multi‐dimensional analysis could distinguish the haematopoietic progenitors according to International Society of Haematotherapy and Graft Engineering criteria from other types of immature cells. Based on the defined clusters, we designed a classifier that assigns BM cells in samples to subclasses based on robust six‐dimensional position and extended shape. The analysis presented here can manage successfully both the increasing numbers of haematopoietic cellular markers and sample heterogeneity. This should enhance the ability to study normal haematopoiesis, and to identify and monitor haematopoietic disorders.

Multiplexed imaging of intracellular protein networks.

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade‐offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed.

Efficiently mining protein interaction dependencies from large text corpora

Biochemical research has yielded an extensive amount of information about dependencies between protein interactions, as generated by allosteric regulations, steric hindrance and other mechanisms. Collectively, this information is valuable for understanding large intracellular protein networks. However, this information is sparsely distributed among millions of publications and documented as freely styled text meant for manual reading. Here we develop a computational approach for extracting information about interaction dependencies from large numbers of publications. First, keyword-based tokenization reduces full papers to short strings, facilitating an efficient search for patterns that are likely to indicate descriptions of interaction dependencies. Sentences that match such patterns are extracted, thereby reducing the amount of text to be read by human curators. Application of this approach to the integrin adhesome network extracted from 59,933 papers 208 short statements, close to half of which indeed describe interaction dependencies. We visualize the obtained hypernetwork of dependencies and illustrate that these dependencies confine the feasible mechanisms of adhesion sites assembly and generate testable hypotheses about their switchability.

Oncogenic Signaling from the Plasma Membrane

Signaling reactions on membranes play an important role in extracellular information processing by cells. The amount of signaling proteins on the plasma membrane is dynamically maintained by cells to tightly control the qualitative response properties of the signaling system. When oncogenic mutations occur in signaling proteins that are associated with the plasma membrane, the ensemble behavior of signaling molecules can change to a completely different response regime that changes the phenotype of the cell. In order to illuminate the relevance of this spatial dimension in signaling systems, we will first describe how the concentration of signaling proteins determines the qualitative response properties of simple reaction cycles in homogenous protein solutions. From there, we discuss how this concentration parameter is determined by the spatial distribution of proteins in cells and expand this to explain how the translocation of signaling proteins to membrane surfaces elicits a signaling response by changing their local concentration. Within this framework we then describe how an oncogene product’s interaction with its wild-type variant can lead to qualitatively different signaling behaviors that depend on their local concentration at membranes as maintained by spatially organizing reactions. We then argue that spatially organizing reaction systems provide an interesting target for cancer therapy.