Tomo-seq: A method to obtain genome-wide expression data with spatial resolution.

To improve our understanding of pattern formation during development and disease we heavily rely on the identification of novel regulators and pathways. While RNA sequencing yields genome-wide expression data that suit this purpose, it lacks spatial resolution. Such spatial resolution can be obtained by microscopy-based methods like in situ hybridization, but these fail to provide information on more than a few genes at a time. Here, we describe tomo-seq, a technique that combines the advantages of the above-mentioned approaches and provides genome-wide expression data with spatial information. The tomo-seq technique is based on cryosectioning of an embryo or tissue of interest and performing RNA-seq on individual sections. Using this method, we have generated genome-wide transcriptomics with high spatial resolution of the whole zebrafish embryo at various stages of development (Junker et al., 2014) and of adult zebrafish hearts after injury (Wu et al., 2016).

Inter- and intratumoral heterogeneity of BCL2 correlates with IgH expression and prognosis in follicular lymphoma.

Most follicular lymphomas (FLs) are genetically defined by the t(14;18)(q32;q21) translocation that juxtaposes the BCL2 gene to the immunoglobulin heavy chain (IgH) 3' regulatory regions (IgH-3'RRs). Despite this recurrent translocation, FL cases are heterogeneous in terms of intratumoral clonal diversity for acquired mutations and variations in the tumor microenvironment. Here we describe an additional mechanism that contributes to inter- and intratumoral heterogeneity in FLs. By applying a novel single-molecule RNA fluorescence-based in situ hybridization (FISH) technique to detect mRNA molecules of BCL2 and IgH in single cells, we found marked heterogeneity in the number of BCL2 mRNA transcripts within individual lymphoma cells. Moreover, BCL2 mRNA molecules correlated with IgH mRNA molecules in individual cells both in t(14;18) lymphoma cell lines and in patient samples. Consistently, a strong correlation between BCL2 and IgH protein levels was found in a series of 205 primary FL cases by flow cytometry and immunohistochemistry. Inter- and intratumoral heterogeneity of BCL2 expression determined resistance to drugs commonly used in FL treatment and affected overall survival of FL patients. These data demonstrate that BCL2 and IgH expressions are heterogeneous and coregulated in t(14;18)-translocated cells, and determine the response to therapy in FL patients.

Power-limited contraction dynamics of Vorticella convallaria: an ultrafast biological spring.

Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure-the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the viscosity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme. Microscopically, this scenario would arise if the mechanochemical wave that propagates along the spasmoneme is faster than the rate at which the cell body can respond due to its large hydrodynamic resistance. We corroborate this by using beads as markers on the stalk and find that the contraction starts at the cell body and proceeds down the stalk at a speed that exceeds the velocity of the cell body.

Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo.

We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.

Intrinsic noise in gene regulatory networks.

Cells are intrinsically noisy biochemical reactors: low reactant numbers can lead to significant statistical fluctuations in molecule numbers and reaction rates. Here we use an analytic model to investigate the emergent noise properties of genetic systems. We find for a single gene that noise is essentially determined at the translational level, and that the mean and variance of protein concentration can be independently controlled. The noise strength immediately following single gene induction is almost twice the final steady-state value. We find that fluctuations in the concentrations of a regulatory protein can propagate through a genetic cascade; translational noise control could explain the inefficient translation rates observed for genes encoding such regulatory proteins. For an autoregulatory protein, we demonstrate that negative feedback efficiently decreases system noise. The model can be used to predict the noise characteristics of networks of arbitrary connectivity. The general procedure is further illustrated for an autocatalytic protein and a bistable genetic switch. The analysis of intrinsic noise reveals biological roles of gene network structures and can lead to a deeper understanding of their evolutionary origin.

Cooperative symmetry-breaking by actin polymerization in a model for cell motility.

Polymerizing networks of actin filaments are capable of exerting significant mechanical forces, used by eukaryotic cells and their prokaryotic pathogens to change shape or to move. Here we show that small beads coated uniformly with a protein that catalyses actin polymerization are initially surrounded by symmetrical clouds of actin filaments. This symmetry is broken spontaneously, after which the beads undergo directional motion. We have developed a stochastic theory, in which each actin filament is modelled as an elastic brownian ratchet, that quantitatively accounts for the observed emergent symmetry-breaking behaviour. Symmetry-breaking can only occur for polymers that have a significant subunit off-rate, such as the biopolymers actin and tubulin.

Brownian ratchets: molecular separations in lipid bilayers supported on patterned arrays.

Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.

Motility of ActA protein-coated microspheres driven by actin polymerization.

Actin polymerization is required for the generation of motile force at the leading edge of both lamellipodia and filopodia and also at the surface of motile intracellular bacterial pathogens such as Listeria monocytogenes. Local catalysis of actin filament polymerization is accomplished in L. monocytogenes by the bacterial protein ActA. Polystyrene beads coated with purified ActA protein can undergo directional movement in an actin-rich cytoplasmic extract. Thus, the actin polymerization-based motility generated by ActA can be used to move nonbiological cargo, as has been demonstrated for classical motor molecules such as kinesin and myosin. Initiation of unidirectional movement of a symmetrically coated particle is a function of bead size and surface protein density. Small beads (