scholarly journals Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

2015 ◽  
Vol 26 (10) ◽  
pp. 1845-1856 ◽  
Author(s):  
Andrew J. Holland ◽  
Rita M. Reis ◽  
Sherry Niessen ◽  
Cláudia Pereira ◽  
Douglas A. Andres ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Cytoplasmic Dynein ◽  
Molecular Replacement ◽  
Farnesyltransferase Inhibitors ◽  
Checkpoint Signaling ◽  
Cellular Processes ◽  
Chromosome Congression ◽  
Mitotic Abnormalities ◽  
Clinical Interest ◽  
Insight Into

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.

scholarly journals Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ivana Primorac ◽  
John R Weir ◽  
Elena Chiroli ◽  
Fridolin Gross ◽  
Ingrid Hoffmann ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Binding Mode ◽  
Signaling Networks ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Downstream Signaling ◽  
Signaling Components ◽  
Insight Into

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.

scholarly journals Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

2010 ◽  
Vol 21 (12) ◽  
pp. 1968-1981 ◽  
Author(s):  
Marin Barisic ◽  
Bénédicte Sohm ◽  
Petra Mikolcevic ◽  
Cornelia Wandke ◽  
Veronika Rauch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cytoplasmic Dynein ◽  
Mitotic Checkpoint ◽  
Deletion Mutants ◽  
Functional Component ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
A Cell ◽  
And Control

Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle–regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NΔ253), or the conserved Spindly box (ΔSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

scholarly journals Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

2010 ◽  
Vol 190 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Laura Hewitt ◽  
Anthony Tighe ◽  
Stefano Santaguida ◽  
Anne M. White ◽  
Clifford D. Jones ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Aurora B ◽  
Core Complex ◽  
Obvious Effect ◽  
Checkpoint Signaling ◽  
Mitotic Entry ◽  
The Core ◽  
Chromosome Congression ◽  
Chromosome Alignment

Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.

scholarly journals Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in Drosophila

2005 ◽  
Vol 16 (7) ◽  
pp. 3107-3116 ◽  
Author(s):  
Anindya Ghosh-Roy ◽  
Bela S. Desai ◽  
Krishanu Ray
Keyword(s):  
Light Chain ◽  
Cytoplasmic Dynein ◽  
Actin Dynamics ◽  
Dynein Light Chain ◽  
Actin Assembly ◽  
Cellular Functions ◽  
Directed Movement ◽  
Cellular Processes ◽  
Dynactin Complex

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

A Screen for Dynein Synthetic Lethals in Aspergillus nidulans Identifies Spindle Assembly Checkpoint Genes and Other Genes Involved in Mitosis

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 101-116
Author(s):  
Vladimir P Efimov ◽  
N Ronald Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Spindle Assembly Checkpoint ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Spindle Assembly ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Vesicle Transport ◽  
Synthetic Lethal ◽  
Checkpoint Genes

Abstract Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

scholarly journals A Conserved Domain Is Crucial for Acceptor Substrate Binding in a Family of Glucosyltransferases

2014 ◽  
Vol 197 (3) ◽  
pp. 510-517 ◽  
Author(s):  
Fan Zhu ◽  
Hua Zhang ◽  
Hui Wu
Keyword(s):  
Bacterial Adhesion ◽  
Substrate Binding ◽  
Molecular Replacement ◽  
Bacterial Sepsis ◽  
Content Type ◽  
Acceptor Substrate ◽  
Loop Region ◽  
Conserved Domain ◽  
Substrate Binding Domain ◽  
Insight Into

Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis.Streptococcus agalactiaeis the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP fromS. agalactiaestrain COH1, has been implicated in bacterial virulence. Four genes (gtfA,gtfB,gtfC, and gtfD) located downstream ofsrr2share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously thatgtfAandgtfBencode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.

scholarly journals Author response: Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

2013 ◽  
Author(s):  
Ivana Primorac ◽  
John R Weir ◽  
Elena Chiroli ◽  
Fridolin Gross ◽  
Ingrid Hoffmann ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Author Response ◽  
Checkpoint Signaling

BIOMIMETIC SYSTEMS SHED LIGHT ON ACTIN-BASED MOTILITY DOWN TO THE MOLECULAR SCALE

2009 ◽  
Vol 04 (01n02) ◽  
pp. 5-15 ◽  
Author(s):  
GUILLAUME ROMET-LEMONNE ◽  
EMMANUELE HELFER ◽  
VINCENT DELATOUR ◽  
BEATA BUGYI ◽  
MONTSERRAT BOSCH ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Molecular Mechanisms ◽  
Directed Assembly ◽  
Cellular Processes ◽  
Self Organized ◽  
Filament Assembly ◽  
Physical Measurements ◽  
Minimum Number ◽  
Broad Variety ◽  
Insight Into

Cell motility, one of the modular activities of living cells, elicits the response of the cell to extra-cellular signals, to move directionally, feed, divide or transport materials. The combined actions of molecular motors and re-modeling of the cytoskeleton generate forces and movement. Here we describe mechanistic approaches of force and movement produced by site-directed assembly of actin filaments. The insight derived from a biochemical analysis of the protein machineries involved in "actin-based motile processes" like cell protrusions, invaginations, organelle propulsion, is used to build reconstituted assays that mimic cellular processes, using several protein machineries known to initiate filament assembly by different mechanisms. Reconstitution of complex self-organized systems presents a broad variety of interests. Reconstituting actin-based movement of a functionalized particle from a minimum number of pure proteins, first used to prove the general thermodynamic principles at work in motility, then was the basis for fully controlled physical measurements of forces produced by polymerization of actin against an obstacle and of the mechanical properties of the resulting polymer arrays. In addition, measurements at the mesoscopic scale (trajectories, velocity, polymer mechanics, fluorescence of specifically labeled components of the actin array, use of mutated proteins) can provide further insight into the molecular mechanisms underlying motility.

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