Chromatin shapes the mitotic spindle.

In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA.

Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move toward chromatinized DNA structures. The velocity and directionality profiles suggest a random-walk with drift directed toward DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally, our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions.

The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes.

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

Negative regulation of cell-cycle progression by RINGO/Speedy E.

Cell-cycle transitions are controlled by CDKs (cyclin-dependent kinases), whose activation is usually associated with the binding of cyclins. RINGO/Speedy proteins can also bind to and activate CDKs, although they do not have amino acid sequence homology with cyclins. The RINGO/Speedy family members studied so far positively regulate cell-cycle progression. In the present paper, we report the biochemical and functional characterization of RINGO/Speedy E. We show that RINGO/Speedy E is a functionally distant member of this protein family that negatively affects cell-cycle progression. RINGO/Speedy E overexpression inhibits the meiotic progression in Xenopus oocytes as well as the proliferation of mammalian cells. RINGO/Speedy E can bind to endogenous CDK1 and CDK2 in both cellular systems. However, the RINGO/Speedy E-activated CDKs have different substrate specificity than the CDKs activated by other RINGO/Speedy proteins, which may account for their different effects on the cell cycle. Our results indicate that, although all RINGO/Speedy family members can activate CDKs, they may differently regulate cell-cycle progression.

Characterization of a new family of cyclin-dependent kinase activators.

Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53-68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases.

Cell cycle regulation of the mammalian CDK activator RINGO/Speedy A.

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), whose activation requires the binding of regulatory subunits named cyclins. RINGO/Speedy A is a mammalian protein that has no amino acid sequence homology with cyclins but can activate CDKs. Here we show that RINGO/Speedy A is a highly unstable protein whose expression and phosphorylation are periodically regulated during the cell cycle. RINGO/Speedy A is degraded by the proteasome and the process involves the ubiquitin ligase SCF(Skp2). Overexpression of a stabilized RINGO/Speedy A form results in the accumulation of high levels of RINGO/Speedy A at late stages of mitosis, which interfere with cytokinesis and chromosome decondensation. Our data show that tight regulation of RINGO/Speedy A is important for the somatic cell cycle.

Regulation of microtubule dynamics by reaction cascades around chromosomes.

During spindle assembly, chromosomes generate gradients of microtubule stabilization through a reaction-diffusion process, but how this is achieved is not well understood. We measured the spatial distribution of microtubule aster asymmetry around chromosomes by incubating centrosomes and micropatterned chromatin patches in frog egg extracts. We then screened for microtubule stabilization gradient shapes that would generate such spatial distributions with the use of computer simulations. Only a long-range, sharply decaying microtubule stabilization gradient could generate aster asymmetries fitting the experimental data. We propose a reaction-diffusion model that combines the chromosome generated Ran-guanosine triphosphate-Importin reaction network to a secondary phosphorylation network as a potential mechanism for the generation of such gradients.

CIN85 participates in Cbl-b-mediated down-regulation of receptor tyrosine kinases.

The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.