Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons

Mechanochemical modeling of dynamic microtubule growth involving sheet-to-tube transition

Microtubule dynamics is largely influenced by nucleotide hydrolysis and the resultant tubulin configuration changes. The GTP cap model has been proposed to interpret the stabilizing mechanism of microtubule growth from the view of hydrolysis effects. Besides, the microtubule growth involves the closure of a curved sheet at its growing end. The curvature conversion also helps to stabilize the successive growth, and the curved sheet is referred to as the conformational cap. However, there still lacks theoretical investigation on the mechanical-chemical coupling growth process of microtubules. In this paper, we study the growth mechanisms of microtubules by using a coarse-grained molecular method. Firstly, the closure process involving a sheet-to-tube transition is simulated. The results verify the stabilizing effect of the sheet structure, and the minimum conformational cap length that can stabilize the growth is demonstrated to be two dimers. Then, we show that the conformational cap can function independently of the GTP cap, signifying the pivotal role of mechanical factors. Furthermore, based on our theoretical results, we describe a Tetris-like growth style of microtubules: the stochastic tubulin assembly is regulated by energy and harmonized with the seam zipping such that the sheet keeps a practically constant length during growth.Comment: 23 pages, 7 figures. 2 supporting movies have not been uploaded due to the file type restriction

A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity

Proteomic, localization, and enzymatic activity screens in fission yeast reveal how deubiquitinating enzyme localization and function are tuned

Chemokine receptor expression on monocytes from healthy individuals

Chronic immune mediated inflammation is characterized by continuous chemokine mediated recruitment and activation of pro-inflammatory cells, monocytes in particular. We believe that an evaluation of the recruitment profile of monocytes during healthy condition is essential for the understanding of cellular response in disease. For this, we have established normal reference values and 95% confidence intervals for receptor expression of 20 chemokine receptors on monocyte subsets; classical (CD14+ CD16−), non-classical (CD14+ CD16+) and HLA-DRhi monocytes from 20 healthy controls using flow cytometry. We demonstrate significant differences in the chemokine receptor expression profiles and high correlation between fraction of cells and level of expression. This is the first global approach to provide a platform for comparable evaluation of cell recruitment during normal and under inflammatory conditions. This will be useful when exploring chemokine–chemokine receptor interactions, inhibition of chemokine signaling and selective removal of migrating cells, which are new treatment strategies in immune mediated diseases

Influence of TNF-α and biomechanical stress on endothelial anti- and prothrombotic genes

Biomechanical stress modulates vascular tone, vascular remodelling and the spatial localisation of atherosclerotic plaques. Inflammatory cytokines, such as TNF-α, regulate expression of genes that impair the function of endothelial cells. This study investigates the combinatory effect of different biomechanical stresses and TNF-α on the expression of endothelial anti- and prothrombotic genes. Human umbilical vein endothelial cells were exposed to TNF-α and different levels of static/pulsatile tensile stress or shear stress. The response in endothelial cells to TNF-α was not modulated by tensile stress. However, shear stress was a more potent stimulus. Shear stress counteracted the cytokine-induced expression of VCAM-1, and the cytokine-suppressed expression of thrombomodulin and eNOS. Shear stress and TNF-α additively induced PAI-1, whereas shear stress blocked the cytokine effect on t-PA and u-PA. A flow profile characterized by high laminar shear stress seems to render the endothelial cell more resistant to inflammatory stress. © 2009 Elsevier Inc. All rights reserved

Motor protein driven microtubule transport on gold particle nanopatterns

Novel glass surfaces with quasi-hexagonally arranged gold nanoparticles to control motor protein immobilisation and motor protein dependent microtubule transport were applied. We first show that the kinesin-like motor protein Eg5 adsorbs efficiently and selectively to gold nanodots comprising a molecular motor nanopattern, while the glass surface between nanodots is passivated by a layer of polyethylene glycol in order to reduce non-specific interactions of individual motors with the substrate. We show that the motor nanopattern and density is indeed controlled by the gold nanodot density. We then use these motor protein arrays to investigate the kinetics of microtubule transport and find that the characteristics of the molecular motor nanopattern influence the characteristics of microtubule transport. This finding describes new biomimetic surfaces of molecularly controlled motor protein surface densities

The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals