Transfer of polarity information via diffusion of Wnt ligands in C. elegans embryos.

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.

Wnt ligands regulate the asymmetric divisions of neuronal progenitors in C. elegans embryos.

Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.

Revised role for Hfq bacterial regulator on DNA topology.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency.

Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns.

Molecular clustering in the cell: from weak interactions to optimized functional architectures.

Molecular components of the cell, such as lipids, proteins or RNA molecules, can associate through weak interactions and form clusters. A growing number of studies have shown that clustering of molecules is crucial for cell functions such as signal optimization and polarization. Clustering provides an intermediate level of organization between the molecular and cellular scales. Here we review recent studies focusing on how molecular clustering functions in different biological contexts, the potential importance of clustering for information processing, as well as the physical nature of cluster formation. We mainly refer to literature focusing on clusters within cell membranes, but also report findings on clusters in the cytosol, emphasizing their ubiquitous role.

Oscillatory AAA+ ATPase Knk1 constitutes a novel morphogenetic pathway in fission yeast.

Cellular morphogenesis relies partly on cell polarization by the cytoskeleton. In the fission yeast Schizosaccharomyces pombe, it is well established that microtubules (MTs) deliver the spatial cue Tea1, a kelch repeat protein, to the tip regions to direct the growth machinery at the cell tips driving the linear extension of the rod-shaped organism to maintain a straight long axis. Here, we report the characterization of Knk1 (kink), a previously unidentified member of the superfamily of ATPases associated with various cellular activities (AAA(+)), whose deletion causes a unique morphological defect characterized by the formation of kinks close to cell tips. Through genetic analysis, we place Knk1 into a novel pathway controlling cell shape independently of MTs and Tea1. Knk1 localizes at cell tips. Its localization is mediated by the Knk1 N terminus and is enhanced upon ATP binding to the C-terminal ATPase domain. Furthermore, Knk1 tip recruitment is regulated by SRC-like adaptor 2 (Sla2) and cell division cycle 42 (Cdc42) independently of Sla2's role in endocytosis. Finally, we discovered that Knk1 shows an anticorrelated oscillatory behavior between the two cell tips at a periodicity that is different from the reported oscillatory Cdc42 dynamics.

Microfabricated polyacrylamide devices for the controlled culture of growing cells and developing organisms.

The ability to spatially confine living cells or small organisms while dynamically controlling their aqueous environment is important for a host of microscopy applications. Here, we show how polyacrylamide layers can be patterned to construct simple microfluidic devices for this purpose. We find that polyacrylamide gels can be molded like PDMS into micron-scale structures that can enclose organisms, while being permeable to liquids, and transparent to allow for microscopic observation. We present a range of chemostat-like devices to observe bacterial and yeast growth, and C. elegans nematode development. The devices can integrate PDMS layers and allow for temporal control of nutrient conditions and the presence of drugs on a minute timescale. We show how spatial confinement of motile C. elegans enables for time-lapse microscopy in a parallel fashion.

Dissecting spindle architecture with a laser.

Microtubules in spindles are too dense to resolve by light microscopy, even with super-resolution methods. Using a new method based on laser-ablation techniques, Brugués et al. present the first quantitative characterization of the vertebrate meiotic spindle and propose an assembly mechanism for building this architecture.

Linker histones incorporation maintains chromatin fiber plasticity.

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

A general method for manipulating DNA sequences from any organism with optical tweezers.

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein-DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from approximately 10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino-carboxyl linkage, which allows for reliable DNA overstretching, is also described.