The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells.

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Intravital Imaging Reveals Motility of Adult Hematopoietic Stem Cells in the Bone Marrow Niche.

Adult mammalian hematopoietic stem cells (HSCs) reside in the bone marrow (BM) but can be mobilized into blood for use in transplantation. HSCs interact with BM niche cells that produce growth factor c-Kit ligand (Kitl/SCF) and chemokine CXCL12, and were thought to be static and sessile. We used two-photon laser scanning microscopy to visualize genetically labeled HSCs in the BM of live mice for several hours. The majority of HSCs showed a dynamic non-spherical morphology and significant motility, undergoing slow processive motion interrupted by short stretches of confined motion. HSCs moved in the perivascular space and showed intermittent close contacts with SCF-expressing perivascular stromal cells. In contrast, mobilization-inducing blockade of CXCL12 receptor CXCR4 and integrins rapidly abrogated HSC motility and shape dynamics in real time. Our results reveal an unexpectedly dynamic nature of HSC residence in the BM and interaction with the SCF+ stromal niche, which is disrupted during HSC mobilization.

Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices.

We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell's response to the cytokines-the phosphorylation of a transcription factor-that can be visualized through antibody staining. Our devices aim at mimicking conditions in dense tissues, such as lymph nodes. A small number of secreting cells is deposited on a polylysine-coated glass and covered by multiple layers of cytokine-consuming. The cells are left to communicate for 1 h, after which the top layers are removed and the bottom layer of cells is antibody labeled for the response to cytokines. Then a cross-section of cytokine fields can be visualized by standard fluorescence microscopy. This manuscript summarized our method to quantify the extent of cytokine-mediated cell-to-cell communications in dense collection of cells in vitro.

Actin Turnover in Lamellipodial Fragments.

Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively in vitro. However, a comprehensive picture of actin turnover in vivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of the fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and extraction experiments to show that about two-thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, whereas the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the in vitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a non-polymerizable "reserve" pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust yet rapidly adaptable and amenable to regulation.

Catch and Release of Cytokines Mediated by Tumor Phosphatidylserine Converts Transient Exposure into Long-Lived Inflammation.

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.

A Tunable Diffusion-Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell-to-Cell Communication in the Immune System.

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 µm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors.

Scanning fluorescence correlation spectroscopy as a versatile tool to measure static and dynamic properties of soft matter systems.

We present the formalism and experimental implementation of scanning fluorescence correlation spectroscopy (SFCS) for the measurements of soft matter system structure and dynamics. We relate the SFCS function Fourier transform to the system intermediate scattering function and demonstrate how SFCS can be combined with specific labelling to measure the desired statistical and kinetic features of the system. Using DNA as a model polymer, we demonstrate the application of SFCS to measure (1) the static structure factor of the system, (2) polymer end-to-end distance distribution, and (3) polymer segmental dynamics in dilute and in dense solutions. The measured DNA end-to-end distance distributions are close to Gaussian. Implementing SFCS we obtain reliable data on segmental mean-square displacement kinetics in dense solutions, where the static FCS approach fails because of dye photobleaching. For moderate concentrations in the semidilute regime (at ∼7 overlap concentrations) segmental dynamics exhibit only weak entanglements. Both of these experimental findings are consistent with theoretical predictions of the weakness of excluded interactions in semiflexible polymers.

T cells translate individual, quantal activation into collective, analog cytokine responses via time-integrated feedbacks.

Variability within isogenic T cell populations yields heterogeneous 'local' signaling responses to shared antigenic stimuli, but responding clones may communicate 'global' antigen load through paracrine messengers, such as cytokines. Such coordination of individual cell responses within multicellular populations is critical for accurate collective reactions to shared environmental cues. However, cytokine production may saturate as a function of antigen input, or be dominated by the precursor frequency of antigen-specific T cells. Surprisingly, we found that T cells scale their collective output of IL-2 to total antigen input over a large dynamic range, independently of population size. Through experimental quantitation and computational modeling, we demonstrate that this scaling is enforced by an inhibitory cross-talk between antigen and IL-2 signaling, and a nonlinear acceleration of IL-2 secretion per cell. Our study reveals how time-integration of these regulatory loops within individual cell signaling generates scaled collective responses and can be leveraged for immune monitoring. DOI: http://dx.doi.org/10.7554/eLife.01944.001.

Structure of DNA coils in dilute and semidilute solutions.

We apply scanning fluorescence correlation spectroscopy to study the structure of individual DNA coils in dilute and semidilute solutions. In dilute solutions, over two decades in length, from 0.6 to 46 µm, DNA behave as ideal chains, in agreement with theoretical predictions and in disagreement with prior experiments. In semidilute solutions, up to very high densities, the structures of individual DNA coils are independent of concentration, unlike flexible coils that shrink with increasing density. Our experimental findings are consistent with the marginal solution theory of semiflexible polymers.

Competition for IL-2 between Regulatory and Effector T Cells to Chisel Immune Responses.

In this review we discuss how the competition for cytokines between different cells of the immune system can shape the system wide immune response. We focus on interleukin-2 (IL-2) secretion by activated effector T cells (T(eff)) and on the competition for IL-2 consumption between T(eff) and regulatory T cells (T(reg)). We discuss the evidence for the mechanism in which the depletion of IL-2 by T(reg) cells would be sufficient to suppress an autoimmune response, yet not strong enough to prevent an immune response. We present quantitative estimations and summarize our modeling effort to show that the tug-of-war between T(reg) and T(eff) cells for IL-2 molecules can be won by T(reg) cells in the case of weak activation of T(eff) leading to the suppression of the immune response. Or, for strongly activated T(eff) cells, it can be won by T(eff) cells bringing about the activation of the whole adaptive immune system. Finally, we discuss some recent applications attempting to achieve clinical effects through the modulation of IL-2 consumption by T(reg) compartment.

Marginal nature of DNA solutions.

We adapt a scanning fluorescence correlation spectroscopy technique to measure the structure factor of complex fluid systems and present the first measurements of the structure of semidilute solutions of long DNA polymers. The measured structure factors exhibit screening effects which, as expected for semidilute polymer solutions, grow stronger with increasing DNA concentration c. The measured concentration dependence of the screening length xi proportional to c{0.53+/-0.02} is unusual, but can be understood within the framework of a marginal solutions theory for semiflexible polymers.

End-monomer Dynamics in Semiflexible Polymers.

Spurred by an experimental controversy in the literature, we investigate the end-monomer dynamics of semiflexible polymers through Brownian hydrodynamic simulations and dynamic mean-field theory. Precise experimental observations over the last few years of end-monomer dynamics in the diffusion of double-stranded DNA have given conflicting results: one study indicated an unexpected Rouse-like scaling of the mean squared displacement (MSD) 〈r(2)(t)〉 ~ t(1/2) at intermediate times, corresponding to fluctuations at length scales larger than the persistence length but smaller than the coil size; another study claimed the more conventional Zimm scaling 〈r(2)(t)〉 ~ t(2/3) in the same time range. Using hydrodynamic simulations, analytical and scaling theories, we find a novel intermediate dynamical regime where the effective local exponent of the end-monomer MSD, α(t) = d log〈r(2)(t)〉/d log t, drops below the Zimm value of 2/3 for sufficiently long chains. The deviation from the Zimm prediction increases with chain length, though it does not reach the Rouse limit of 1/2. The qualitative features of this intermediate regime, found in simulations and in an improved mean-field theory for semiflexible polymers, in particular the variation of α(t) with chain and persistence lengths, can be reproduced through a heuristic scaling argument. Anomalously low values of the effective exponent α are explained by hydrodynamic effects related to the slow crossover from dynamics on length scales smaller than the persistence length to dynamics on larger length scales.

Universality of persistence exponents in two-dimensional Ostwald ripening.

We measured persistence exponents theta(phi) of Ostwald ripening in two dimensions, as a function of the area fraction phi occupied by coarsening domains. The values of theta(phi) in two systems, succinonitrile and brine, quenched to their liquid-solid coexistence region, compare well with one another, providing compelling evidence for the universality of the one-parameter family of exponents. For small phi, theta(phi) approximately = 0.39phi, as predicted by a model that assumes no correlations between evolving domains. These constitute the first measurements of persistence exponents in the case of phase transitions with a conserved order parameter.

Dynamics of a fluorophore attached to superhelical DNA: FCS experiments simulated by Brownian dynamics.

We investigated the dynamics of a single-fluorophore-labeled pUC18 plasmid through a Brownian dynamics algorithm, followed by a simulation of the fluorescence correlation spectroscopy (FCS) process. Recent experimental FCS measurements indicated a sensitivity of the monomer mean square displacements in DNA circles towards superhelicity. Simulations with homogeneous DNA elasticity and local straight equilibrium are not sufficient to reproduce this observed behavior. But inserting permanently bent sequences into the DNA, which favor end loop formation, caused a dependence of the calculated FCS correlation curves on superhelical density. Furthermore, our simulations allow us to take into account the orientation of the fluorophore in polarized excitation, which might explain the observed appearance of a Rouse-like regime at intermediate time scales.

Nuclear export of Ho endonuclease of yeast via Msn5.

Exportin-5, an evolutionarily conserved nuclear export factor of the beta-karyopherin family, exports phosphorylated proteins and small noncoding RNAs. Msn5, the yeast ortholog, exports primarily phosphorylated cargoes including Ho endonuclease and a number of transcription factors and regulatory proteins. The Msn5-mediated nuclear export of Ho is dependent on phosphorylation of Thr225 by kinases of the DNA damage response pathway. Although Msn5 has been the object of many studies, no NES sequence capable of binding the exportin and/or of leading to Msn5-dependent export of a heterologous protein has been identified. Here we report identification of a 13-residue Ho sequence that interacts with Msn5 in vitro and directs Msn5-dependent nuclear export of GFP in vivo. A single point mutation in this 13-mer Ho NES abrogates both interaction with Msn5 and nuclear export of Ho and of GFP. However, this mutation, or of T225A, both of which abrogate nuclear export of Ho, does not interfere with its interaction with Msn5 implying that the exportin makes multiple contacts with its cargo. This can explain the lack of a conserved NES in Msn5 cargoes. Our results identify essential criteria for Msn5-mediated nuclear export of Ho: phosphorylation on HoT225, and interaction with the 13-mer Ho NES sequence.

Internal dynamics of superhelical DNA.

We present the first data on the temporal kinetics of monomer mean square displacements in DNA circles with defined degrees of superhelicity. The segmental dynamics of specifically labeled DNA plasmids with superhelical densities between 0 and -0.016 was assessed by fluorescence correlation spectroscopy. Introduction of superhelicity leads to progressively faster dynamics in the long time regime corresponding to the coil diffusion as observed previously by Langowski et al. [Biopolymers 34, 639 (1994)10.1002/bip.360340506], but also in the short time range corresponding to the segmental motion within the DNA coil.

Breathing dynamics in heteropolymer DNA.

While the statistical mechanical description of DNA has a long tradition, renewed interest in DNA melting from a physics perspective is nourished by measurements of the fluctuation dynamics of local denaturation bubbles by single molecule spectroscopy. The dynamical opening of DNA bubbles (DNA breathing) is supposedly crucial for biological functioning during, for instance, transcription initiation and DNA's interaction with selectively single-stranded DNA binding proteins. Motivated by this, we consider the bubble breathing dynamics in a heteropolymer DNA based on a (2+1)-variable master equation and complementary stochastic Gillespie simulations, providing the bubble size and the position of the bubble along the sequence as a function of time. We utilize new experimental data that independently obtain stacking and hydrogen bonding contributions to DNA stability. We calculate the spectrum of relaxation times and the experimentally measurable autocorrelation function of a fluorophore-quencher tagged basepair, and demonstrate good agreement with fluorescence correlation experiments. A significant dependence of opening probability and waiting time between bubble events on the local DNA sequence is revealed and quantified for a promoter sequence of the T7 phage. The strong dependence on sequence, temperature and salt concentration for the breathing dynamics of DNA found here points at a good potential for nanosensing applications by utilizing short fluorophore-quencher dressed DNA constructs.