Juan Carlos Zabala

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.

Tubulin cofactor B plays a role in the neuronal growth cone

Tubulin cofactors, initially identified as α‐, β‐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.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of α/β‐tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with β‐tubulin in a quasi‐native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF‐7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF‐7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements

SummaryA revised physical map of the α-haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.

Purification of α-hemolysin from an overproducing E. coli strain

SummaryThe genetic determinant of the α-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.α-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified α-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the α-hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.

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.

The β-tubulin monomer release factor (p14) has homology with a region of the DnaJ protein

p14 is a molecular chaperone involved in β‐tubulin folding which catalyzes the release of β‐tubulin monomers from intermediate complexes. Here we demostrate that active p14 protein which we have purified from an overproducing Escherichia coli strain can also release ‐tubulin monomers from tubulin dimers in the presence of an additional cofactor (Z). Analysis of p14 secondary structure suggests that this protein may belong to a family of conserved proteins which share structural similarities with the J‐domain of DnaJ. We have constructed deletions and site‐directed mutations in the p14 gene. A single D to E mutation in the region shown in DnaJ to be an essential loop for its function affected the monomer‐release activity of p14. These results support the hypothesis that this p14 loop interacts with β‐tubulin in a similar fashion as DnaJ interacts with DnaK and suggest a possible role of p14 in the folding process.

A 14 kDa release factor is involved in GTP‐dependent β‐tubulin folding

The tubulin folding pathway is a model system to understand protein folding in the cell. It involves the interaction of several chaperones, including TCP‐1 and other as yet uncharacterized factors. Release of tubulin monomers from folding intermediates (C900 and C300) and their incorporation into tubulin dimers is dependent on GTP hydrolysis, magnesium ions and release factors. In this work, we have purified to homogeneity the protein factor responsible for the release of β‐tubulin monomers from C300 complexes. It has an apparent molecular mass of 14 kDa (p14) as judged by SDS electrophoresis. The protein behaved as a dimer of about 28 kDa when analyzed by gel filtration chromatography. Furthermore, the p14‐dependent release of β‐tubulin monomers from C300 complexes takes place in the presence of GTP. These results suggest that p14 is a new chaperone that assists in tubulin folding by facilitating the acquisition of the native conformation.

Three-Dimensional Structure of Human Tubulin Chaperone Cofactor A

alpha and beta-Tubulin fold in a series of chaperone-assisted steps. At least five protein cofactors are involved in the post-chaperonin tubulin folding pathway and required to maintain the supply of tubulin; some of them also participate in microtubule dynamics. The first tubulin chaperone identified in the tubulin folding pathway was cofactor A (CoA). Here we describe the three-dimensional structure of human CoA at 1.7 A resolution, determined by multiwavelength anomalous diffraction (MAD). The structure is a monomer with a rod-like shape and consists of a three-alpha-helix bundle, or coiled coil, with the second helix kinked by a proline break, offering a convex surface at one face of the protein. The helices are connected by short turns, one of them, between alpha2 and alpha3, including a 3(10)-helix. Peptide mapping analysis and competition experiments with peptides show that CoA interacts with beta-tubulin via the three alpha-helical regions but not with the rod-end loops. The main interaction occurs with the middle kinked alpha2 helix, at the convex face of the rod. Strong 3D structural homology is found with the Hsp70 chaperone cofactor BAG domain, suggesting that these proteins define a family of cofactors of simple compact architecture. Further structural homology is found with alpha-spectrin/alpha-actinin repeats, all are rods of identical length of ten helical turns. We propose to call these three-helix bundles alpha ten modules.