Jakub K. Famulski

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.

Aurora B Kinase-Dependent Recruitment of hZW10 and hROD to Tensionless Kinetochores

The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores (reviewed in [1, 2]). Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. Here, we report that hZW10 and hROD, two components of the evolutionarily conserved RZZ complex, accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, we showed that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. We also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, we show that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. We therefore conclude that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest.

Siah Regulation of Pard3A Controls Neuronal Cell Adhesion During Germinal Zone Exit

Exiting the Birthplace In the developing mammalian brain, new neurons are not always born where they are needed. In order for immature neurons of the mouse cerebellum to leave their birthplace in the germinal zone and find their functional niche in the brain, the neurons need to migrate. Famulski et al. (p. 1834, published online 25 November; see the Perspective by Métin and Luccardini) now show that ubiquitin-mediated protein degradation regulates development of specific cell adhesions that the neurons need in order to exit their birthplace en route to their final functional location. A ubiquitination cascade regulates formation of cell adhesions that immature neurons require in the developing mouse brain. The brain’s circuitry is established by directed migration and synaptogenesis of neurons during development. Although neurons mature and migrate in specific patterns, little is known about how neurons exit their germinal zone niche. We found that cerebellar granule neuron germinal zone exit is regulated by proteasomal degradation of Pard3A by the Seven in Absentia homolog (Siah) E3 ubiquitin ligase. Pard3A gain of function and Siah loss of function induce precocious radial migration. Time-lapse imaging using a probe to measure neuronal cell contact reveals that Pard3A promotes adhesive interactions needed for germinal zone exit by recruiting the epithelial tight junction adhesion molecule C to the neuronal cell surface. Our findings define a Siah-Pard3A signaling pathway that controls adhesion-dependent exit of neuronal progenitors or immature neurons from a germinal zone niche.

HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint

Accurate chromosomal segregation is monitored by the mitotic checkpoint, and an increased rate of chromosomal missegregation leads to chromosomal instability (CIN). Here, we demonstrate that the HBV X protein (HBx) binds BubR1, a component of the mitotic checkpoint complex and co-localizes with BubR1 at the kinetochores. HBx binding to BubR1 attenuates the association between BubR1 and CDC20, an activator of the anaphase-promoting complex/cyclosome (APC/C) and induces slippage of mitotic arrest in the presence of microtubule poisons. In addition, HBx binding to BubR1 results in the accumulation of lagging chromosomes and chromosome bridges. In contrast, a C-terminally truncated HBx mutant (HBx1–100) fails to bind BubR1 and does not cause aberrant chromosomal segregation. This provides a novel mechanism for dysregulation of the mitotic checkpoint by a viral pathogen linking it to the accumulation of chromosomal instability in HBV-associated hepatocarcinogenesis.

Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint

The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD–ZW10–Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1–noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein–hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

Contribution of growth differentiation factor 6-dependent cell survival to early-onset retinal dystrophies

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

Dynein/Dynactin-Mediated Transport of Kinetochore Components off Kinetochores and onto Spindle Poles Induced by Nordihydroguaiaretic Acid

The mitotic checkpoint functions to ensure accurate chromosome segregation by regulating the progression from metaphase to anaphase. Once the checkpoint has been satisfied, it is inactivated in order to allow the cell to proceed into anaphase and complete the cell cycle. The minus end-directed microtubule motor dynein/dynactin has been implicated in the silencing of the mitotic checkpoint by “stripping” checkpoint proteins off kinetochores. A recent study suggested that Nordihydroguaiaretic acid (NDGA) stimulates dynein/dynactin-mediated transport of its cargo including ZW10 (Zeste White 10). We analyzed the effects of NDGA on dynein/dynactin dependent transport of the RZZ (Zeste White 10, Roughdeal, Zwilch) complex as well as other kinetochore components from kinetochores to spindle poles. Through this approach we have catalogued several kinetochore and centromere components as dynein/dynactin cargo. These include hZW10, hZwilch, hROD, hSpindly, hMad1, hMad2, hCENP-E, hCdc27, cyclin-B and hMps1. Furthermore, we found that treatment with NDGA induced a robust accumulation and complete stabilization of hZW10 at spindle poles. This finding suggests that NDGA may not induce dynein/dynactin transport but rather interfere with cargo release. Lastly, we determined that NDGA induced accumulation of checkpoint proteins at the poles requires dynein/dynactin-mediated transport, hZW10 kinetochore localization and kinetochore-microtubule attachments but not tension or Aurora B kinase activity.

A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores

The mitotic checkpoint protein Spindly is farnesylated in vivo and this modification is required for its interaction with the RZZ complex and its localization to kinetochores.

Evolution of and Evolutionary Relationships between Extant Vaccinia Virus Strains

ABSTRACT Although vaccinia virus (VACV) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of VACV are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. Here, we sequenced additional VACV strains that either represent relatively pristine examples of old vaccines (e.g., Dryvax, Lister, and Tashkent) or have been subjected to additional laboratory passage (e.g., IHD-W and WR). These genome sequences were compared with those previously reported for other VACVs as well as other orthopoxviruses. These extant VACVs do not always cluster in simple phylogenetic trees that are aligned with the known historical relationships between these strains. Rather, the pattern of deletions suggests that all existing strains likely come from a complex stock of viruses that has been passaged, distributed, and randomly sampled over time, thus obscuring simple historical or geographic links. We examined surviving nonclonal vaccine stocks, like Dryvax, which continue to harbor larger and now rare variants, including one that we have designated “clone DPP25.” DPP25 encodes genes not found in most VACV strains, including an ankyrin-F-box protein, a homolog of the variola virus (Bangladesh) B18R gene which we show can be deleted without affecting virulence in mice. We propose a simple common mechanism by which recombination of a larger and hypothetical DPP25-like ancestral strain, combined with selection for retention of critically important genes near the terminal inverted repeat boundaries (vaccinia virus growth factor gene and an interferon alpha/beta receptor homolog), could produce all known VACV variants. IMPORTANCE Smallpox was eradicated by using a combination of intensive disease surveillance and vaccination using vaccinia virus (VACV). Interestingly, little is known about the historical relationships between different strains of VACV and how these viruses may have evolved from a common ancestral strain. To understand these relationships, additional strains were sequenced and compared to existing strains of VACV as well as other orthopoxviruses by using whole-genome sequence alignments. Extant strains of VACV did not always cluster in simple phylogenetic trees based on known historical relationships between these strains. Based on these findings, it is possible that all existing strains of VACV are derived from a single complex stock of viruses that has been passaged, distributed, and sampled over time.

New spin on an old transition: epithelial parallels in neuronal adhesion control

During histogenesis of the vertebrate central nervous system (CNS), neuronal progenitors must interact with germinal zone (GZ) niches, differentiate, and morphologically mature, and neurons must migrate to their final positions. The extrinsic cues that control neurogenesis, specify neurons, and guide their movement are relatively well understood. However, less is known about how neurons spatiotemporally modify cell-cell interactions and cell polarization to navigate through complex, distinct cellular environments during neuronal circuit formation. Here we examine the parallels between the mechanisms controlling epithelial morphogenesis and the cell adhesion events by which neural cells organize GZ niches and direct neuronal migration. We focus on the emerging relationship between neuronal adhesive interactions and conserved cell-polarity signaling cascades.