The N-terminal tail of C. elegans CENP-A interacts with KNL-2 and is essential for centromeric chromatin assembly.

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.

Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans.

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1's kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.

A Cell Biologist's Field Guide to Aurora Kinase Inhibitors.

Aurora kinases are essential for cell division and are frequently misregulated in human cancers. Based on their potential as cancer therapeutics, a plethora of small molecule Aurora kinase inhibitors have been developed, with a subset having been adopted as tools in cell biology. Here, we fill a gap in the characterization of Aurora kinase inhibitors by using biochemical and cell-based assays to systematically profile a panel of 10 commercially available compounds with reported selectivity for Aurora A (MLN8054, MLN8237, MK-5108, MK-8745, Genentech Aurora Inhibitor 1), Aurora B (Hesperadin, ZM447439, AZD1152-HQPA, GSK1070916), or Aurora A/B (VX-680). We quantify the in vitro effect of each inhibitor on the activity of Aurora A alone, as well as Aurora A and Aurora B bound to fragments of their activators, TPX2 and INCENP, respectively. We also report kinome profiling results for a subset of these compounds to highlight potential off-target effects. In a cellular context, we demonstrate that immunofluorescence-based detection of LATS2 and histone H3 phospho-epitopes provides a facile and reliable means to assess potency and specificity of Aurora A versus Aurora B inhibition, and that G2 duration measured in a live imaging assay is a specific readout of Aurora A activity. Our analysis also highlights variation between HeLa, U2OS, and hTERT-RPE1 cells that impacts selective Aurora A inhibition. For Aurora B, all four tested compounds exhibit excellent selectivity and do not significantly inhibit Aurora A at effective doses. For Aurora A, MK-5108 and MK-8745 are significantly more selective than the commonly used inhibitors MLN8054 and MLN8237. A crystal structure of an Aurora A/MK-5108 complex that we determined suggests the chemical basis for this higher specificity. Taken together, our quantitative biochemical and cell-based analyses indicate that AZD1152-HQPA and MK-8745 are the best current tools for selectively inhibiting Aurora B and Aurora A, respectively. However, MK-8745 is not nearly as ideal as AZD1152-HQPA in that it requires high concentrations to achieve full inhibition in a cellular context, indicating a need for more potent Aurora A-selective inhibitors. We conclude with a set of "good practice" guidelines for the use of Aurora inhibitors in cell biology experiments.

Interaction of mammalian end binding proteins with CAP-Gly domains of CLIP-170 and p150(glued).

End binding proteins (EBs) track growing microtubule ends and play a master role in organizing dynamic protein networks. Mammalian cells express up to three different EBs (EB1, EB2, and EB3). Besides forming homodimers, EB1 and EB3 also assemble into heterodimers. One group of EB-binding partners encompasses proteins that harbor CAP-Gly domains. The binding properties of the different EBs towards CAP-Gly proteins have not been systematically investigated. This information is, however, important to compare and contrast functional differences. Here we analyzed the interactions between CLIP-170 and p150(glued) CAP-Gly domains with the three EB homodimers and the EB1-EB3 heterodimer. Using isothermal titration calorimetry we observed that some EBs bind to the individual CAP-Gly domains with similar affinities while others interact with their targets with pronounced differences. We further found that the two types of CAP-Gly domains use alternative mechanisms to target the C-terminal domains of EBs. We succeeded to solve the crystal structure of a complex composed of a heterodimer of EB1 and EB3 C-termini together with the CAP-Gly domain of p150(glued). Together, our results provide mechanistic insights into the interaction properties of EBs and offer a molecular framework for the systematic investigation of their functional differences in cells.

Molecular insights into mammalian end-binding protein heterodimerization.

Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo- versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150(glued) or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.

Mammalian end binding proteins control persistent microtubule growth.

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

ABA-responsive RNA-binding proteins are involved in chloroplast and stromule function in Arabidopsis seedlings.

The phytohormone abscisic acid (ABA) regulates essential growth and developmental processes in plants. Recently, RNA-binding proteins have been described as components of ABA signaling during germination. We have identified ten ABA-regulated RNA-binding proteins in Arabidopsis seedlings. Among those genes, AtCSP41B and cpRNP29 are highly expressed in seedlings. Using promoter:reporter gene analyses, we showed that both AtCSP41B and cpRNP29 were in particular expressed in photosynthetically active organs like green cotyledons, leaves, and petioles. The analysis of CFP-fusion proteins demonstrates that cpRNP29 localized to chloroplasts and AtCSP41B to chloroplasts and stromules. Whereas RNA-binding of cpRNP29 has previously been shown, we demonstrated through in vitro RNA-binding assays that recombinant AtCSP41B binds to RNA, and that chloroplast petD RNA can serve as a target of AtCSP41B. Developmental or environmental stimuli affected the expression of AtCSP41B and cpRNP29 in seedlings. Both genes were repressed during senescence, but only AtCSP41B was significantly repressed upon water stress. In addition, AtCSP41B and cpRNP29 exhibited low expression in etiolated seedlings compared to green seedlings, and cpRNP29 was regulated during the day photoperiod. Homozygous T-DNA insertion lines were isolated, characterized on the molecular level, and monitored for phenotypic changes. Taken together, the data show that both proteins are regulated during processes that are known to involve ABA signaling. Their localization in chloroplasts and RNA-binding activity suggest a role in chloroplast RNA metabolism in Arabidopsis seedlings.