A microtubule interactome: complexes with roles in cell cycle and mitosis.

addresses: Department of Zoology, University of Oxford, Oxford, United Kingdom.notes: PMCID: PMC2323305types: Journal Article; Research Support, Non-U.S. Gov'tCopyright: © 2008 Hughes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division

CD28 between tolerance and autoimmunity: The side effects of animal models [version 1; referees: 2 approved]

Regulation of immune responses is critical for ensuring pathogen clearance and for preventing reaction against self-antigens. Failure or breakdown of immunological tolerance results in autoimmunity. CD28 is an important co-stimulatory receptor expressed on T cells that, upon specific ligand binding, delivers signals essential for full T-cell activation and for the development and homeostasis of suppressive regulatory T cells. Many in vivo mouse models have been used for understanding the role of CD28 in the maintenance of immune homeostasis, thus leading to the development of CD28 signaling modulators that have been approved for the treatment of some autoimmune diseases. Despite all of this progress, a deeper understanding of the differences between the mouse and human receptor is required to allow a safe translation of pre-clinical studies in efficient therapies. In this review, we discuss the role of CD28 in tolerance and autoimmunity and the clinical efficacy of drugs that block or enhance CD28 signaling, by highlighting the success and failure of pre-clinical studies, when translated to humans

A Model of Curvature-Induced Phase Transitions in Inflationary Universe

Chiral phase transitions driven by space-time curvature effects are investigated in de Sitter space in the supersymmetric Nambu-Jona-Lasinio model with soft supersymmetry breaking. The model is considered to be suitable for the analysis of possible phase transitions in inflationary universe. It is found that a restoration of the broken chiral symmetry takes place in two patterns for increasing curvature : the first order and second order phase transition respectively depending on initial settings of the four-body interaction parameter and the soft supersymmetry breaking parameter. The critical curves expressing the phase boundaries in these parameters are obtained. Cosmological implications of the result are discussed in connection with bubble formations and the creation of cosmic strings during the inflationary era.Comment: 12 pages, 3 figures, REVTe

Curvature-induced phase transitions in the inflationary universe - Supersymmetric Nambu-Jona-Lasinio Model in de Sitter spacetime -

The phase structure associated with the chiral symmetry is thoroughly investigated in de Sitter spacetime in the supersymmetric Nambu-Jona-Lasinio model with supersymmetry breaking terms. The argument is given in the three and four space-time dimensions in the leading order of the 1/N expansion and it is shown that the phase characteristics of the chiral symmetry is determined by the curvature of de Sitter spacetime. It is found that the symmetry breaking takes place as the first order as well as second order phase transition depending on the choice of the coupling constant and the parameter associated with the supersymmetry breaking term. The critical curves expressing the phase boundary are obtained. We also discuss the model in the context of the chaotic inflation scenario where topological defects (cosmic strings) develop during the inflation.Comment: 29 pages, 6 figures, REVTe

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells