Microtubules are hollow protein cylinders of 25 nm diameter which are implicated in cytokinetics and proliferation in all eukaryotic cells. Here we demonstrate in vivo how multiwalled carbon nanotubes (MWCNTs) interact with microtubules in human cancer cells (HeLa) blocking mitosis and leading to cell death by apoptosis. Our data suggest that, inside the cells, MWCNTs display microtubule biomimetic properties, assisting and enhancing noncentrosomal microtubule polymerization and stabilization. These features might be useful for developing a revolutionary generation of chemotherapeutic agents based on nanomaterials.

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Morphological evidence of the potential for adsorptive transcytosis of protein through the mammalian blood-brain fluid barriers, first reported from this laboratory in the mouse, has been confirmed and expanded upon in rats injected intravenously or into the lateral cerebral ventricle/subarachnoid space with with exogenous lectin wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP). Blood-borne WGA-HRP rapidly enters cerebral endothelia by the process of adsorptive endocytosis and labels the vascular tree throughout the CNS. At 3 h post-injection and longer, WGA-HRP occupies the perivascular clefts and labels perivascular cells and basal lamina; this suspected transendothelial transfer of the lectin conjugate from blood to brain involves specific constituents of the endothelial endomembrane system of organelles (e.g., plasmalemma, vesicles, endosomes, Golgi complex). Within 6 h, reaction product is evident in extracellular clefts beyond the perivascular basal lamina and labels endocytic vesicles, endosomes, and dense bodies within cells and processes of the neuropil. Exposure of the abluminal surface of blood-brain barrier endothelia for 1-18 h to WGA-HRP delivered into the cerebral ventricles or subarachnoid space indicates blood-brain barrier endothelia do not engage in demonstrable adsorptive endocytosis at the abluminal surface. In this preparation, no endothelial organelles comparable to those sequestering blood-borne WGA-HRP are labelled with the lectin conjugate; hence, significant adsorptive transcytosis of WGA-HRP through cerebral endothelia from brain to blood is unlikely. The demonstrable difference in membrane internalization of the luminal versus abluminal plasmalemma of blood-brain barrier endothelia suggests the blood-brain barrier is polarized regarding adsorptive endocytosis of WGA-HRP. If adsorptive transcytosis of macromolecules through the blood-brain barrier does occur, the process appears unidirectional, from blood to brain but not from brain to blood. Absence of demonstrable endocytosis at the abluminal front is an enigma in the scheme of transcytosis through the blood-brain barrier from blood to brain insofar as exocytosis and endocytosis are complementary events in the cellular secretory process. This unconventional membrane behavior associated with the abluminal plasmalemma argues against a significant transcytosis of blood-borne protein through blood-brain barrier endothelia. The potential for transcytosis of macromolecules through the blood-cerebrospinal fluid barrier of choroid plexus epithelia is not as problemmatic as that through blood-brain barrier endothelia; additional evidence is provided to suggest choroid plexus epithelia participate in adsorptive endocytosis circumferentially and adsorptive transcytosis of WGA-HRP bidirectionally between the blood and cerebrospinal fluid.

Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO− present in TBCB, which is similar to the EEY/F-COO− element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) × C57BL/6)F1 mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW × C57BL/6)F1-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW × C57BL/6)F1-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.

Tubulin cofactors, initially identified as a-, b-tubulin folding proteins, are now believed to participate in the complex tubulin biogenesis and degradation routes, and thus to contribute to microtubule functional diversity and dynamics. However, a concrete role of tubulin cofactor B (TBCB) remains to be elucidated because this protein is not required for tubulin biogenesis, and it is apparently not essential for life in any of the organisms studied. In agreement with these data, here we show that TBCB localizes at the transition zone of the growth cones of growing neurites during neurogenesis where it plays a role in microtubule dynamics and plasticity. Gene silencing by means of small interfering RNA segments revealed that TBCB knockdown enhances axonal growth. In contrast, excess TBCB, a feature of giant axonal neuropathy, leads to microtubule depolymerization, growth cone retraction, and axonal damage followed by neuronal degeneration. These results provide an important insight into the understanding of the controlling mechanisms of growth cone microtubule dynamics. Keywords: axonal growth control, giant axonal neuropathy, microtubule dynamics, neurogenesis, tubulin cofactor B. The establishment of connections between neurons and their targets is of fundamental importance to the genesis of a functional nervous system. A goal of modern developmental neuroscience and cell biology is to understand how axons are extended and how this process is controlled. However, despite the great deal of progress that has been made in understanding the nature of the extracellular signals that induce axon growth, we still know relatively little about the intracellular molecular mechanisms that elicit neurite extension.A role for microtubules in the control of axon outgrowth is well supported by the literature. Changes in the dynamic properties of the neuroblast microtubule cytoskeleton are achieved by various mechanisms that rely on, among other processes, changes of the a-and b-tubulin isotype composition of the microtubules. Yet, the way in which specific aor b-tubulin varieties are combined and how these are incorporated into the existing tubulin pool during neuronal differentiation is still unknown. Tubulin cofactors, originally discovered as proteins required for proper tubulin folding and heterodimer formation (Campo et al.

Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.