Mónica L. Fanarraga

Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation.

Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and post‐translational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class‐III β‐tubulin (also known as β6‐tubulin). Both the upregulation and the post‐translational processing of class‐III β‐tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class‐III β‐tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U‐β6, specific to unphosphorylated class‐III β‐tubulin has been developed, characterized and compared with its commercial homologue TuJ‐1. Our experiments indicate that the two antibodies recognize different forms of class‐III β‐tubulin both in vitro and in vivo. Biochemical data revealed that U‐β6 bound unphosphorylated soluble class‐III β‐tubulin specifically, while TuJ‐1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U‐β6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti‐ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA‐NCAM), as well as differentiating neurons. These studies with U‐β6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U‐β6 +ve/TuJ‐1 –ve cells); a differentiation stage (U‐β6 +ve/TuJ‐1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class‐III β‐tubulin immunostaining levels. These investigations also conclude that U‐β6 is an earlier marker than TuJ‐1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.

Tubulin folding cofactor D is a microtubule destabilizing protein

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the αβ‐tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627–632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422–9426; Campo et al. (1994) FEBS Lett. 353, 162–166; Lewis et al. (1996) J. Cell Biol. 132, 1–4; Lewis et al. (1997) Trends Cell Biol. 7, 479–484; Tian et al. (1997) J. Cell Biol. 138, 821–823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327–332; Hirata et al. (1998) EMBO J. 17, 658–666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243–254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule‐destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe‐inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361–363; McNally (1999) Curr. Biol. 9, R274–R276]. Here we show that the tubulin folding cofactor D, although it does not co‐polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering β‐tubulin from GTP‐bound αβ‐heterodimers.

Multiwalled Carbon Nanotubes Display Microtubule Biomimetic Properties in Vivo, Enhancing Microtubule Assembly and Stabilization

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.

TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning

In animal cells the centrosome is positioned at the cell centre in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here, we report the first characterization of human TBCC‐domain containing 1 (TBCCD1), a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE‐1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1‐depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound‐healing assays. However, the major microtubule‐nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization.

Regulated expression of p14 (cofactor A) during spermatogenesis

The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.

TBCD Links Centriologenesis, Spindle Microtubule Dynamics, and Midbody Abscission in Human Cells

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.

Multiwalled Carbon Nanotubes Hinder Microglia Function Interfering with Cell Migration and Phagocytosis

The intranasal drug delivery route provides exciting expectations regarding the application of engineered nanomaterials as nano-medicines or drug-delivery vectors into the brain. Among nanomaterials, multiwalled CNTs (MWCNTs) are some of the best candidates for brain cancer therapy since they are well known to go across cellular barriers and display an intrinsic ability to block cancer cell proliferation triggering apoptosis. This study reveals that microglial cells, the brain macrophages and putative vehicles for MWCNTs into the brain, undergo a dose-dependent cell division arrest and apoptosis when treated with MWCNTs. Moreover, it is shown that MWCNTs severely interfere with both cell migration and phagocytosis in live microglia. These results lead to a re-evaluation of the safety of inhaled airborne CNTs and provide strategic clues of how to biocompatibilize MWCNTs to reduce brain macrophage damage and to develop new nanodrugs.

Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.

Multiwalled Carbon Nanotubes Inhibit Tumor Progression in a Mouse Model.

Understanding the molecular mechanisms underlying the biosynthetic interactions between particular nanomaterials with specific cells or proteins opens new alternatives in nanomedicine and nanotoxicology. Multiwalled carbon nanotubes (MWCNTs) have long been explored as drug delivery systems and nanomedicines against cancer. There are high expectations for their use in therapy and diagnosis. These filaments can translocate inside cultured cells and intermingle with the protein nanofilaments of the cytoskeleton, interfering with the biomechanics of cell division mimicking the effect of traditional microtubule-binding anti-cancer drugs such as paclitaxel. Here, it is shown how MWCNTs can trigger significant anti-tumoral effects in vivo, in solid malignant melanomas produced by allograft transplantation. Interestingly, the MWCNT anti-tumoral effects are maintained even in solid melanomas generated from paclitaxel-resistant cells. These findings provide great expectation in the development of groundbreaking adjuvant synthetic microtubule-stabilizing chemotherapies to overcome drug resistance in cancer.

Anti-Cancer Cytotoxic Effects of Multiwalled Carbon Nanotubes

Recent research has opened new alternatives to traditional chemotherapy treatments using nanomaterials as cytotoxic agents. Anti-cancer nanomedicines do not require specific target sites on key proteins or genes to kill cancer cells and have radically different mechanisms to interact with the living matter. Among 1D nanomaterials, multiwalled carbon nanotubes (MWCNTs) have the intrinsic ability to bind tubulin and interfere with microtubule dynamics, mimicking the effect of traditional cytotoxic microtubule-binding agents such as paclitaxel (taxol®). Here, we review the cytotoxic properties of MWCNTs and show a direct pro-apoptotic effect of these nanomaterials in vitro in different cancer cell lines and tumor cells obtained from surgical specimens. Understanding the bio-synthetic relationship between MWCNTs and microtubules could serve to improve these nanomaterials to be used as broad spectrum antineoplastic agents in combination to traditional microtubule-binding treatments, thus avoiding drug resistance mechanisms in cancer cells.