Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

Tubulin is subject to a special cycle of detyrosination͞tyrosination in which the C-terminal tyrosine of ␣-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persists in TTL null tissues, being present mainly in dividing TTL null cells where it originates from tubulin synthesis, but it is lacking in postmitotic TTL null cells such as neurons, which is apparently deleterious because early death in TTL null mice is, at least in part, accounted for by a disorganization of neuronal networks, including a disruption of the cortico-thalamic loop. Correlatively, cultured TTL null neurons display morphogenetic anomalies including an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. These anomalies may involve a mislocalization of CLIP170, which we find lacking in neurite extensions and growth cones of TTL null neurons. Our results demonstrate a vital role of TTL for neuronal organization and suggest a requirement of Tyr-tubulin for proper control of neurite extensions.CLIP170 ͉ tubulin code

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G 2 phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-Trityl-L-cysteine is a tight binding inhibitor (estimation of K i,app <150 nM at 300 mM NaCl and 600 nM at 25 mM KCl). S-Trityl-L-cysteine binds more tightly than monastrol because it has both an ϳ8-fold faster association rate and ϳ4-fold slower release rate (6.1 M ؊1 s ؊1 and 3.6 s ؊1 for S-trityl-L- Kinesins form a superfamily of motor proteins with about 14 different subfamilies clearly identified so far. They play important roles in intracellular transport and at different stages of cell division. The driving force behind these processes is ATP hydrolysis.The roles of different kinesins during cell division make them highly important for understanding fundamental aspects of mitosis and meiosis. In recent years, some of them have appeared as potential targets for anti-cancer drugs (1-3). One of these mitotic kinesins, human Eg5 (HsEg5/KSP), a member of the kinesin-5 family (4), is responsible for the formation and maintenance of the bipolar spindle (5). Eg5 represents an especially attractive target because when inhibited by microinjection with suitable antibodies (5), by RNAi 2 (6), or by treating cells with specific Eg5 inhibitors (7), it displays a very characteristic mitotic arrest phenotype, i.e. a monoastral spindle with an array of microtubules (MTs) emanating from a pair of nonseparated centrosomes surrounded by chromosomes. Cell-based as well as in vitro assays have led to the discovery of a series of inhibitors that target Eg5 and lead to mitotic arrest and cell death. Among these inhibitors are monastrol, the first Eg5 inhibitor discovered (7), terpendole E, identified from a fungal strain (8), HR22C16, structurally related to monastrol (9), CK0106023, a quinazolinone analogue representing the most potent Eg5 inhibitor identified so far (10), dihydropyrazoles (11), and S-trityl-L-cysteine (STLC) (12). Several of these inhibitors are currently intensively studied as potential anticancer drugs, as tools for studying fundamental processes in mitosis and function of its target (chemical genetics) (13), or simply as a model to understand the mechanisms of inhibition of this important class of proteins (14 -17).By using two small, preselected libraries from the NCI, we have recently identified several new inhibitors of human Eg5 activity, ...

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

Introduction Successful human placentation depends on adequate transformation of the uteroplacental vasculature by extravillous trophoblast (EVT) following proliferation, migration and invasion of these cellsinto the maternal decidua [1,2]. This process of vascular remodelling rises to a peak by the end of the first trimester and declines rapidly thereafter [3]. At around 10-12 weeks of gestation (wg), cytotrophoblasts (CT) Abstract Pre-eclampsia (PE), the major cause of maternal morbidity and mortality, is thought to be caused by shallow invasion of the maternal decidua by extravillous trophoblasts (EVT). Data suggest that a fine balance between the expressions of pro-and anti-invasive factors might regulate EVT invasiveness. Recently, we showed that the expression of the new growth factor endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is high in early pregnancy

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (␣ or ␣) and two regulatory (␤) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic ␣ and regulatory ␤ subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2␣ or GFP-CK2␤ revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2␤, CK2␣ can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2␣ is dramatically changed by its association with CK2␤, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.Protein kinase CK2 is a ubiquitous serine/threonine protein kinase, generally described as a stable ␣ 2 ␤ 2 tetramer, where ␣ and ␤ are the catalytic and regulatory subunits, respectively (3). Although its signaling function has long remained obscure, the importance of CK2 is suggested by the evolutionary conservation of the enzyme and by the fact that the disruption of both Saccharomyces cerevisiae genes encoding CK2 catalytic subunits is a lethal event (29). In addition to its role in embryonic development and terminal differentiation, the enzyme is required for normal cell cycle progression (20,30). At last, a function of CK2 in cell survival has recently emerged (1).Many of the identified CK2 substrates that are critical for cell proliferation and viability are localized in different cellular compartments. However, there is controversy as to the localization of CK2 and where its substrates are phosphorylated. Although the current prevailing view of CK2 is a tetrameric enzyme, accumulating evidence also indicates that free populations of both CK2 subunits can exist and exert specific functions in the cell (18, 37). At least in vitro, CK2␤ exerts a central role in modulating the catalytic activity of CK2 (26). Consequently, it is suspected that in vivo, the substrate specificity of the enzyme is likely to be determined both by subcellular localization and by affinity for its regulatory subunit that brings the kinase in proximity to the substrate.In a previous study, the behavior of CK2 subunits fused to GFP was characterized in living cells (25). The expressed fusion proteins were functional and interacted with endogenous CK2. Both subunits were mostl...

The p160–Rho-associated coiled-coil–containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis.

These findings demonstrate the stress-protective role of PrP(C) during development, and propose PrP(C) dysregulation as a novel causative element of IUGR.