Differential adhesion regulates neurite placement via a retrograde zippering mechanism.

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the brain neuropil and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers.

Structural and developmental principles of neuropil assembly in C. elegans.

Neuropil is a fundamental form of tissue organization within the brain, in which densely packed neurons synaptically interconnect into precise circuit architecture. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation' to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold.

The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.

Background: Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans.

WormGUIDES: an interactive single cell developmental atlas and tool for collaborative multidimensional data exploration.

Background: Imaging and image analysis advances are yielding increasingly complete and complicated records of cellular events in tissues and whole embryos. The ability to follow hundreds to thousands of cells at the individual level demands a spatio-temporal data infrastructure: tools to assemble and collate knowledge about development spatially in a manner analogous to geographic information systems (GIS). Just as GIS indexes items or events based on their spatio-temporal or 4D location on the Earth these tools would organize knowledge based on location within the tissues or embryos.

Caenorhabditis elegans as a platform to study the mechanism of action of synthetic antitumor lipids.

Drugs capable of specifically recognizing and killing cancer cells while sparing healthy cells are of great interest in anti-cancer therapy. An example of such a drug is edelfosine, the prototype molecule of a family of synthetic lipids collectively known as antitumor lipids (ATLs). A better understanding of the selectivity and the mechanism of action of these compounds would lead to better anticancer treatments.

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex.

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton.

Stoichiometry of Nck-dependent actin polymerization in living cells.

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott-Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation.

The branched actin nucleator Arp2/3 promotes nuclear migrations and cell polarity in the C. elegans zygote.

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements.

New insights into the mechanisms and roles of cell-cell fusion.

Many types of eukaryotic cells can fuse together as part of their normal developmental program or life cycle. This review describes a diverse set of examples of such cell types and focuses attention on several molecules that appear intimately involved in the process of plasma membrane merger that lies at the crux of every cell-fusion event. Some of these examples come from experimental systems where the discovery of molecules essential for cell fusion is sped by the approachability of the experimental organism itself.

α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells.

The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian α-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel α-subunit, in body-wall muscle cells.

Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation.

Background: Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated.

Imaging embryonic development in Caenorhabditis elegans.

Embryos are remarkable for their combination of pluripotency, three-dimensionality, and swiftness of subcellular and developmental rearrangements. Embryogenesis in the nematode Caenorhabditis elegans is uniquely suited among model systems to high-resolution dynamic imaging. Within a single high-magnification, high-numerical aperture (NA) microscope field, at submicrometer resolution, it is possible to observe several entire animals taking form.

Suspended embryo mount for imaging Caenorhabditis elegans.

This protocol describes the preparation of Caenorhabditis elegans embryos for four-dimensional (4D) imaging. Embryos are excised from gravid hermaphrodites and allowed to adhere directly to a poly-L-lysine-coated coverslip. The coverslip is then used to create a slide chamber in which the embryos are suspended in buffer.

Measurement of muscle disease by quantitative second-harmonic generation imaging.

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function.

Quantitative assays for cell fusion.

Cell fusion would seem to be obviously recognizable upon visual inspection, and many studies employ a simple microscopic fusion index to quantify the rate and extent of fusion in cell culture. However, when cells are not in monolayers or when there is a large background of multinucleation through failed cytokinesis, cell-cell fusion can only be proven by mixing of cell contents. Furthermore, determination of the microscopic fusion index must generally be carried out manually, creating opportunities for unintended observer bias and limiting the numbers of cells assayed and therefore the statistical power of the assay.

Ultrastructural imaging of cell fusion in Caenorhabditis elegans.

Caenorhabditis elegans is a well-established model system particularly suited for studying cell-cell fusion because of its highly predictable and rapid development and its known cell lineage. This chapter focuses on understanding the ultrastructural components of cell fusion through the use of transmission electron microscopy (TEM). Published TEM studies have described the initial demonstration of syncytial cells in the worm, the vesiculation of the bilayers between cells during widening of the normal fusion aperture, and the appearance of microfusion intermediates in the membranes of cells with fusion-defective mutations.

Optical imaging of cell fusion and fusion proteins in Caenorhabditis elegans.

Cell fusion is a very dynamic process in which the entire membrane and cellular contents of two or more cells merge into one. Strategies developed to understand the component processes that make up a full fusion event require imaging to be performed over a range of space and time scales. These strategies must cover detection of nanometer-sized pores, monitoring cytoplasmic diffusion and the dynamic localization of proteins that induce fusion competence, and three-dimensional reconstruction of multinucleated cells.

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis.

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C.

Depolymerization-driven flow in nematode spermatozoa relates crawling speed to size and shape.

Cell crawling is an inherently physical process that includes protrusion of the leading edge, adhesion to the substrate, and advance of the trailing cell body. Research into advance of the cell body has focused on actomyosin contraction, with cytoskeletal disassembly regarded as incidental, rather than causative; however, extracts from nematode spermatozoa, which use Major Sperm Protein rather than actin, provide at least one example where cytoskeletal disassembly apparently generates force in the absence of molecular motors. To test whether depolymerization can explain force production during nematode sperm crawling, we constructed a mathematical model that simultaneously describes the dynamics of both the cytoskeleton and the cytosol.

Second harmonic generation imaging microscopy studies of osteogenesis imperfecta.

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues.

Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C.