Methods in Cell Biology

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ISSN / EISSN : 0091-679X / 0091-679X
Published by: Elsevier BV (10.1016)
Total articles ≅ 139
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Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 43-45; doi:10.1016/bs.mcb.2019.03.006

Abstract:
A retrospective of an academic career. The article poses the question about the values involved in a life in the academy, starting with the role of hemoglobin in the chick embryo and ending with the role of calcium in the sea urchin spine.
Lauren T. Smith,
Published: 1 January 2019
Methods in Cell Biology, Volume 150, pp 251-268; doi:10.1016/bs.mcb.2019.01.001

Abstract:
During development metazoan embryos have to establish the molecular coordinates for elaboration of the embryonic body plan. Typically, bilaterian (bilaterally symmetric animals) embryos establish anterior-posterior (AP) and dorsal-ventral (DV) axes, and in most cases the AP axis is established first. For over a century it has been known that formation of the AP axis is strongly influenced by the primary axis of the egg, the animal-vegetal (AV) axis. The molecular basis for how the AV axis influences AP polarity remains poorly understood, but sea urchins have proven to be important for elucidating the molecular basis for this process. In fact, it is the first model system where a critical role for Wnt signaling in specification and patterning the AV and AP axis was first established. One current area of research is focused on identifying the maternal factors that regulate localized activation of Wnt/β-catenin signaling at the vegetal pole during development. An essential tool for this work is the means to identify the AV polarity in oocytes and eggs. This permits investigation into how polarity is established and allows development of experimental strategies to identify maternal factors that contribute to and control axial polarity. This chapter provides protocols to accomplish this in sea urchin eggs and early embryos. We describe simple methods to visualize polarity including direct observation of eggs and oocytes, using a microscope for overt morphological signs of polarity, and more extensive methods involving localization of known factors indicative of inherent embryonic polarity, such as the upstream regulators of the Wnt/β-catenin pathway.
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 49-54; doi:10.1016/bs.mcb.2019.03.007

Abstract:
Studies using sea urchins have contributed substantially to our understanding of how a fertilized egg is transformed during embryonic development. This brief review provides a personal perspective of the remarkable advances that have occurred over the past 45 years in our understanding of how urchin embryos work.
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 37-41; doi:10.1016/bs.mcb.2019.03.005

Abstract:
This perspective describes how our understanding of sea urchin development has been enabled by advances in technology. The early conceptual discoveries that put the sea urchin embryo on the research map had to wait until technologies were available to explain how those concepts worked. The explanatory phase continues as a number of mechanisms continue to be understood in ever greater detail, all made possible by further technical advances.
Nahomie Rodriguez-Sastre, Christopher F. Thomas,
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 459-472; doi:10.1016/bs.mcb.2019.01.007

Abstract:
Homeostasis of charged particles in biological systems is fundamental for life. Indeed, the efficient synthesis of ATP is predicated on an electrochemical gradient. Ion pumps and channels act as conduits that regulate membrane potential by controlling ion flux. This phenomenon is critical for the generation of action potentials in excitable cells such as neurons and muscle fibers, and for acidification of lysosomes in all cells. However, the production of action potentials or pH differentials is merely one facet of bioelectricity. Increasing evidence has shown that ion channels and pumps also play critical roles in other cellular processes: cell cycle regulation, wound healing, regeneration, and symmetry breaking during development. In recent years, the functional roles of ion channels have been explored in echinoderm development. The application of fluorescence-based ion- and voltage-sensitive dyes allows for live measurements of ion concentrations with both spatial and temporal resolution. In this chapter, we describe the use of such dyes for interrogating and visualizing ion and voltage gradients in sea urchin embryos.
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 519-526; doi:10.1016/bs.mcb.2018.10.010

Abstract:
Fluorescent calcium sensors provide a means of detecting and analyzing cytoplasmic calcium levels in embryos and larvae. Conventional RNA injection of eggs results in expression of protein sensors throughout larval tissues. Larvae are immobilized for wide field or confocal recordings and video records reveal recurrent fluctuations in cytoplasmic calcium levels in several cell types. Neurons can be identified by location and form, and continuous records made of their activity. Confocal image stacks are registered and Z-axis, fluorescence intensity profiles of individual neurons generated to provide time/activity plots. These optogenetic methods enable analysis in intact larvae of the activity of identified neurons or effectors, such as muscles or ciliary band cells.
, Christine Byrum, Pei Yun Lee, Robert L. Morris
Published: 1 January 2019
Methods in Cell Biology, Volume 150, pp 449-469; doi:10.1016/bs.mcb.2018.11.014

Abstract:
It is important to provide undergraduate students with research experiences so that they obtain essential problem-solving skills and come to appreciate the process of science whether or not they pursue graduate study. However, such experiences can be difficult to achieve at a primarily undergraduate institution where time and resources are limited. One strategy is to incorporate research into the laboratory component of courses, with students having input into the specific topic being investigated. In this chapter, we present a series of activities that can be brought together as a semester or year-long project after students select a gene with the potential to be analyzed in a novel species of echinoderm. Students become acquainted with important databases, software programs, and online tools as they clone their gene, confirm its identity through alignment with homologous sequences, and characterize its expression through both qPCR and WMISH. We provide streamlined protocols that allow the work to be accomplished in an efficient manner, and conclude with ideas for assignments that can be completed in parallel to improve students' writing and oral communication skills in preparation for any career.
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 55-61; doi:10.1016/bs.mcb.2019.03.008

Abstract:
At the most fundamental level, the genome is the basis for questions about the mechanisms of development: how it works. This perspective provides a brief historical review of the sequencing of the echinoderm genome and the progress in answering this complex question, which depends on technological advances as well as intellectual ones.
Catherine S. Schrankel, Tufan Gökirmak, Chang-Wook Lee, Geoffrey Chang,
Published: 1 January 2019
Methods in Cell Biology, Volume 151, pp 353-376; doi:10.1016/bs.mcb.2018.11.005

Abstract:
Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~ 15 kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.
Estafania Paredes, , Julien Vignier
Published: 1 January 2019
Methods in Cell Biology, Volume 150, pp 47-69; doi:10.1016/bs.mcb.2018.11.008

Abstract:
Sea urchins are excellent model organisms useful for several lines of biotechnological research. Sea urchins are typically collected from the sea and kept in research facilities all over the world for such purposes. Cryopreservation can be a very powerful tool to enhance the use of sea urchins as a model species for research. The development of cryopreservation protocols for different sea urchin gametes, embryos, and larvae allows year round access to high quality material outside the natural reproductive season. It also reduces the uncertainty and variability that may be caused by changing oceanic, meteorological, and environmental conditions. Access to cryopreserved gametes and embryos will allow using these model organisms in laboratories all around the world, regardless of their facilities or their proximity to a natural population of sea urchins. Cryopreservation is a very useful tool for aquaculture production, fisheries conservation, and wild stock enhancement allowing spat supply all year round without the need of conditioning broodstock for out of season reproduction—which is expensive, time consuming, and often unfruitful. It will also provide flexibility for selective breeding programs by allowing crossings of individuals with different reproductive seasons. Although cryopreservation protocols have been successfully developed for many valuable fisheries species such as sturgeons, salmonid fishes, and other marine invertebrates (e.g., oysters), only a small amount of research has been carried out regarding sea urchin cryopreservation. In this chapter, we outline protocols for the cryopreservation of sea urchin cells.
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