The role of cytoplasmic calcium gradients, calmodulin, and actin filaments in the early development of the fucoid alga, Pelvetia compressa

The mechanisms by which the cellular polarity was established, maintained and regulated in the zygotes of the marine brown alga, Pelvetia compressa , were investigated. The foci of attention were the involvement of cytoplasmic calcium (Ca2+) gradients, calmodulin and actin cytoskeleton. To establish the long predicted existence of cytoplasmic Ca 2+ gradients during Pelvetia polarization, a long excitation wavelength Ca2+ indicator, Calcium Crimson dextran, and a Ca2+-insensitive reference dye, Rhodamine B dextran, were microinjected into living zygotes separately. Ratios representing relative Ca2+ concentrations were calculated by dividing the averaged fluorescent pixel values of Calcium Crimson images by the averaged fluorescent pixel values of corresponding Rhodamine B images. A cytoplasmic Ca 2+ gradient was found to form within one hour of the exposure of the zygotes to unilateral blue light during the photosensitive period. Similarly, zygotes polarizing in the dark formed cytoplasmic Ca2+ gradients prior to germination as well. The injection site was the preferred germination site for dark-grown zygotes, and injection-induced cytoplasmic Ca2+ gradients could be redirected elsewhere. The region of high Ca 2+ formed during polarization was localized to and predictive of the site of future germination. This indirect ratiometric approach was validated by its application to the growing rhizoid, where the known tip-focused Ca 2+ gradient was clearly revealed. Microinjection of zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished when the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. Those results indicate that calmodulin is the cellular mediator of cytoplasmic Ca2+ gradients during both germination and rhizoidal growth. Calmodulin may exert its effect through the activation of Ca2+/calmodulin-dependent protein kinase II or its homologue. Cytoplasmic Ca2+ gradients did not form during photopolarization in the presence of a specific actin filament inhibitor, but then formed normally upon the removal of the inhibitor. The dynamics of actin filaments in living zygotes imaged with Alexa 568-conjugated phalloidin revealed that actin cytoskeleton reorganization was rapid upon light exposure. Actin may be involved in actively transporting plasma membrane Ca2+ channels to the future germination site

Independent research projects using protein extraction: Affordable ways to inquire, discover and publish for undergraduate students

This article describes how to use protein extraction, quantification, and analysis in the undergraduate teaching laboratory to engage students in inquiry-based, discoverydriven learning. Detailed instructions for obtaining proteins from animal tissues, using BCA assay to quantify the proteins, and data analysis are provided. The experimental procedure requires laboratory equipment and supplies that can be found in most biology teaching labs. Suggestions for successful implementation that can lead to original research published in peer-reviewed journals are outlined

Using a novel approach - recombineering - to generate odf2 null alleles

This article uses a real-life example to illustrate the concept and methodology of recombineering, a revolutionary genetic engineering technique based on phage-mediated homologous recombination. A step-by-step approach is presented along with a flow diagram, from obtaining gene-harboring BACs to the in vitro generation of a conditional null allele. This method can be used to target any gene at any position, without the knowledge or use of any restriction site. The extensive applicability of recombineering to gene manipulation is discussed. © 2012 Higher Education Press and Springer-Verlag Berlin Heidelberg

The involvement of Ca\u3csup\u3e2+\u3c/sup\u3e gradients, Ca\u3csup\u3e2+\u3c/sup\u3e fluxes, and CaM kinase II in polarization and germination of Silvetia compressa zygotes

Previous work has shown that distinct Ca2+ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca2+ gradients prior to germination and then germinate from the site of elevated Ca2+. In no case did germination occur without a prior formation of a Ca2+ gradient. Using the self-referencing Ca2+-selective probe, we measured highly localized influx of Ca2+ during photopolarization, indicating that extracellular stores supply at least some of the Ca2+ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca2+ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination

CaM kinase II isoforms are expressed at lower levels in brains of shiverer mutants

In the mammalian brain, the hippocampus is involved in memory formation and storage and has an enriched level of Ca2+/calmodulin-dependent protein kinase type II (CaM kinase II). CaM kinase II has a number of downstream targets and is shown to play a role in memory development, axonal transport, and signaling across the synapse. The shiverer mutant mouse is a knockout lacking myelin basic protein. As a result, the axons of the central nervous system (CNS) of the shiverer have no or very thin myelin sheath, neurons in their CNS have distorted shapes, and synaptic signaling is impaired. shiverer mice develop symptoms similar to those experienced by patients with multiple sclerosis. In this study, proteins from the hippocampus, cerebellum, pons, medulla, and olfactory bulbs of shiverer and wild-type mice were extracted. Western blot analysis was used to compare the expression levels of CaM kinase II in these regions of the two types of mice. Analysis shows that at least two (50 and 58-59 kDa) of the four CaM kinase II isoforms are expressed in the brain, with one isoform (50 kDa) expressed in all regions examined. shiverer brain contains a decreased level of the two isoforms of CaM kinase II, an indication that the cognitive function of these mice might also be impaired. Copyright © 2007 Humana Press Inc. All rights of any nature whatsoever are reserved

Science and human rights: A bridge towards benefiting humanity

Three case studies involving scientists from different disciplines that contribute their expertise to advancing human rights in the US and abroad are discussed. Scientific research can have a greater impact on society if directed towards solving problems relevant to human rights. Progress in science and technology can be abused, leading to violations of human rights but can also benefit humanity. Scientists have an opportunity to play an active role in preserving human rights. © 2010 by The Johns Hopkins University Press