Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities.

In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo Localization of P granules and their physical nature remain poorly understood Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension As with other liquids, P granules rapidly dissolved and condensed Localization occurred by a biased increase in P granule condensation at the posterior This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm

Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.

Mammalian Rho GTPases: new insights into their functions from in vivo studies.

The Snail superfamily of zinc-finger transcription factors is involved in processes that imply pronounced cell movements, both during embryonic development and in the acquisition of invasive and migratory properties during tumour progression. Different family members have also been implicated in the signalling cascade that confers left–right identity, as well as in the formation of appendages, neural differentiation, cell division and cell survival.

The snail superfamily of zinc-finger transcription factors.

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

https://www.pnas.org/content/pnas/107/11/4872.full.pdf

Geometric cues for directing the differentiation of mesenchymal stem cells.

Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

/pdf/engineering-the-caenorhabditis-elegans-genome-using-cas9-4sn8szqvlo.pdf

Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

The PAR Proteins: Fundamental Players in Animal Cell Polarization

A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.

https://www.genetics.org/content/genetics/200/4/1035.full.pdf

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

http://labs.bio.unc.edu/Goldstein/Sawyeretal10.pdf

Apical constriction: a cell shape change that can drive morphogenesis.

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis

/pdf/apical-constriction-themes-and-variations-on-a-cellular-5cv43xpkdd.pdf

Bob Goldstein

Papers

Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

The PAR Proteins: Fundamental Players in Animal Cell Polarization

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

Apical constriction: a cell shape change that can drive morphogenesis.

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis