Ian X. McLeod

The CENP-H–I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H–I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H–I complex proteins, indicating that the CENP-H–I complex may function, in part, as a marker directing CENP-A deposition to centromeres.

ZW10 links mitotic checkpoint signaling to the structural kinetochore

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80–Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1–Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived “stop anaphase” inhibitor.

A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1–depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

A Bir1-Sli15 Complex Connects Centromeres to Microtubules and Is Required to Sense Kinetochore Tension

Proper connections between centromeres and spindle microtubules are of critical importance in ensuring accurate segregation of the genome during cell division. Using an in vitro approach based on the sequence-specific budding yeast centromere, we identified a complex of the chromosomal passenger proteins Bir1 and Sli15 (Survivin and INCENP) that links centromeres to microtubules. This linkage does not require Ipl1/Aurora B kinase, whose targeting and activation are controlled by Bir1 and Sli15. Ipl1 is the tension-dependent regulator of centromere-microtubule interactions that ensures chromosome biorientation on the spindle. Elimination of the linkage between centromeres and microtubules mediated by Bir1-Sli15 phenocopies mutations that selectively cripple Ipl1 kinase activation. These findings lead us to propose that the Bir1-Sli15-mediated linkage, which bridges centromeres and microtubules and includes the Aurora kinase-activating domain of INCENP family proteins, is the tension sensor that relays the mechanical state of centromere-microtubule attachments into local control of Ipl1 kinase activity.

A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex.

Chromosome segregation requires stable bipolar attachments of spindle microtubules to kinetochores. The dynein/dynactin motor complex localizes transiently to kinetochores and is implicated in chromosome segregation, but its role remains poorly understood. Here, we use the Caenorhabditis elegans embryo to investigate the function of kinetochore dynein by analyzing the Rod/Zwilch/Zw10 (RZZ) complex and the associated coiled-coil protein SPDL-1. Both components are essential for Mad2 targeting to kinetochores and spindle checkpoint activation. RZZ complex inhibition, which abolishes both SPDL-1 and dynein/dynactin targeting to kinetochores, slows but does not prevent the formation of load-bearing kinetochore-microtubule attachments and reduces the fidelity of chromosome segregation. Surprisingly, inhibition of SPDL-1, which abolishes dynein/dynactin targeting to kinetochores without perturbing RZZ complex localization, prevents the formation of load-bearing attachments during most of prometaphase and results in extensive chromosome missegregation. Coinhibition of SPDL-1 along with the RZZ complex reduces the phenotypic severity to that observed following RZZ complex inhibition alone. We propose that the RZZ complex can inhibit the formation of load-bearing attachments and that this activity of the RZZ complex is normally controlled by dynein/dynactin localized via SPDL-1. This mechanism could coordinate the hand-off from initial weak dynein-mediated lateral attachments, which help orient kinetochores and enhance their ability to capture microtubules, to strong end-coupled attachments that drive chromosome segregation.

A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction.

G protein–gated potassium (Kir3) channels are important for controlling neuronal excitability in the brain. Using a proteomics approach, we have identified a unique rodent intracellular protein, sorting nexin 27 (SNX27), which regulates the trafficking of Kir3 channels. Like most sorting nexins, SNX27 possesses a functional PX domain that selectively binds the membrane phospholipid phosphatidylinositol-3-phosphate (PI3P) and is important for trafficking to the early endosome. SNX27, however, is the only sorting nexin to contain a PDZ domain. This PDZ domain discriminates between channels with similar class I PDZ-binding motifs, associating with the C-terminal end of Kir3.3 and Kir3.2c (−ESKV), but not with that of Kir2.1 (−ESEI) or Kv1.4 (−ETDV). SNX27 promotes the endosomal movement of Kir3 channels, leading to reduced surface expression, increased degradation and smaller Kir3 potassium currents. The regulation of endosomal trafficking via sorting nexins reveals a previously unknown mechanism for controlling potassium channel surface expression.

Molecular analysis of kinetochore architecture in fission yeast

Kinetochore composition and structure are critical for understanding how kinetochores of different types perform similar functions in chromosome segregation. We used affinity purification to investigate the kinetochore composition and assembly in Schizosaccharomyces pombe. We identified a conserved DASH complex that functions to ensure precise chromosome segregation. Unlike DASH in budding yeast that is localized onto kinetochores throughout the cell cycle, SpDASH is localized onto kinetochores only in mitosis. We also identified two independent groups of kinetochore components, one of which, the Sim4 complex, contains several novel Fta proteins in addition to known kinetochore components. DASH is likely to be associated with the Sim4 complex via Dad1 protein. The other group, Ndc80–MIND–Spc7 complex, contains the conserved Ndc80 and MIND complexes and Spc7 protein. We propose that fission yeast kinetochore is comprised of at least two major structural motifs that are biochemically separable. Our results suggest a high degree of conservation between the kinetochores of budding yeast and fission yeast even though many individual protein subunits do not have a high degree of sequence similarity.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Chromosomal instability by inefficient Mps1 auto-activation due to a weakened mitotic checkpoint and lagging chromosomes.

Background Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase. Principal Findings Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively. Conclusions/Significance We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability.

CTA4 Transcription Factor Mediates Induction of Nitrosative Stress Response in Candida albicans

ABSTRACT This work has identified regulatory elements in the major fungal pathogen Candida albicans that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of C. albicans inside the human body. Exposing C. albicans to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how C. albicans is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the YHB1 regulatory region. Deletion of CTA4 caused a small but significant decrease in virulence. A CTA4-dependent putative sulfite transporter encoded by SSU1 is also implicated in NO response, but C. albicans ssu1 mutants were not sensitive to NO, in contrast to findings in Saccharomyces cerevisiae. Cta4p is the first protein found to be necessary for initiating NO response in C. albicans.