Gene Families

The Interactive Fly

Centromere and Kinetochore Proteins

  • Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation
  • Sisters Unbound is required for meiotic centromeric cohesion in Drosophila melanogaster
  • Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division
  • The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs
  • Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster
  • The Drosophila histone variant H2A.V works in concert with HP1 to promote kinetochore-driven microtubule formation
  • Lateral and end-on kinetochore attachments are coordinated to achieve bi-orientation in Drosophila oocytes
  • Polar ejection forces promote the conversion from lateral to end-on kinetochore-microtubule attachments on mono-oriented chromosomes
  • Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function
  • Network of protein interactions within the Drosophila inner kinetochore
  • An essential step of kinetochore formation controlled by the SNARE protein Snap29
  • Simple and complex centromeric satellites in Drosophila sibling species
  • Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes
  • Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
  • Drosophila kinesin-8 stabilizes the kinetochore-microtubule interaction
  • Kinetochore proteins have a post-mitotic function in neurodevelopment
  • The modular mechanism of chromocenter formation in Drosophila
  • Islands of retroelements are major components of Drosophila centromeres
  • Centromere-proximal meiotic crossovers in Drosophila melanogaster are suppressed by both highly-repetitive heterochromatin and proximity to the centromere
  • Dynamic turnover of centromeres drives karyotype evolution in Drosophila
  • Asymmetric centromeres differentially coordinate with mitotic machinery to ensure biased sister chromatid segregation in germline stem cells
  • The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells
  • Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function
  • Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing
  • Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration

  • Centromere proteins
  • Kinetochore protein
  • Passenger proteins
  • Other

    Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation

    Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. This study exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis (Unhavaithaya, 2013).

    Localization studies demonstrated centromere pairing in yeast, Drosophila, and plants, and it showed that the centromeres cluster together in Drosophila meiosis I. This study has establish that centromere function is required for both pairing and clustering. Thus, centromeres are integrally involved in these two processes and not brought together solely by external factors. Because these events occur before assembly of the kinetochore, it is likely that the chromatin and associated proteins at the centromere are critical. The mutations in cenp-C reveal that functional CENP-C is necessary at a minimum for maintenance of centromere pairing and clustering in Drosophila oocytes. The noncomplementation between truncated CENP-C and CAL1 protein forms implicates CAL1 as also being crucial for centromere pairing and clustering. Given the role of CENP-C in recruiting proteins to the centromere, the requirement for this protein could reflect a direct role in centromere pairing and clustering or the need for a protein whose localization is dependent on CENP-C and/or CAL1. In the cenp-C mutant and the cenp-C cal1 double-heterozygous mutant, CID is still localized to the centromere, as evidenced by its presence at brightly DAPI-stained heterochromatin at levels that, by immunofluorescence, are not significantly lower than WT. Thus, CID presence is insufficient for centromere clustering and pairing. The reduced level of CID staining in the double-heterozygous mutant is nearly significant, however; thus, the possibility that reduced CID levels contribute to the mutant defects is not excluded (Unhavaithaya, 2013).

    The proteins at the centromere may interact with nuclear structures to promote centromere clustering. This study identifies the nucleolus as a likely candidate. The centromere clusters are associated with the nucleolus in WT oocytes, and this association requires cenp-C and cal1 function. In Drosophila female meiosis, the nucleolus may serve as an anchor site for centromeres throughout prophase I. The SC also may cluster centromeres. Clustering has been shown to be disrupted in mutants for the SC transverse and central elements. The observation that the SC protein C(3)G fails to be retained at the centromere in cenp-C and cal1 mutants raises the possibility that the failure of clustering in these centromere protein mutants is a consequence of the absence of the SC. The hypothesis of this causality is consistent with the timing of defects; as early as pachytene, both centromere SC and clustering are absent. It remains to be determined how the SC, a structure contained between pairs of homologs, could gather centromeres into a cluster. In c(3)g mutants, more than four CID foci can be observed, indicating that both centromere pairing and clustering can be affected. Thus, failure of centromere retention of the SC also could account for the pairing defects in the centromere protein mutants (Unhavaithaya, 2013).

    The allele-specific noncomplementation (type I second-site noncomplementation) between the mutations causing C-terminal truncations of CENP-C and CAL1 is unusual and informative. Such mutations that alter protein structure rather than simply reducing protein levels provide the opportunity to investigate genetic interactions. This allele-specific noncomplementation affects all the processes analyzed: centromere pairing, centromere clustering and nucleolar association, SC retention at the centromere, and meiotic segregation. The antagonistic genetic interaction requires the truncated protein forms, because deficiencies for each of the genes complement the truncation allele of the other for meiotic segregation and cause only slight defects in centromere pairing and clustering. This is also true for the cenp-CZ3-4375 allele that reduces protein levels. Thus, simply decreasing the levels of the proteins does not perturb these processes. The C-terminal region of CAL1 binds to CENPC, whereas the N terminus binds to CID; thus, the truncated form could have a dominant negative effect by binding CID and blocking its link to CENP-C. The C terminus of CENP-C is required for its localization to the centromere as well as binding to CAL1, whereas it binds the KNL-1/Mis12 complex/Ndc80 complex (KMN) kinetochore network via its N terminus. Thus, C-terminal truncated CENP-C also could act as a dominant negative to uncouple the KMN complex from a functional centromere association, particularly given that the N terminus alone can bind to kinetochore proteins but not to the centromere. Expression of the N terminus alone also can disrupt the spindle assembly checkpoint. The truncation alleles of cenp-C and cal1 each alone have slight semidominant effects on centromere pairing, clustering, and meiotic segregation, consistent with dominant negative activities. The combination of the two dominant negative effects could account for perturbation of the meiotic processes. It cannot be excluded, however, that these truncation alleles act as recessive neomorphs, conferring novel properties on the proteins (Unhavaithaya, 2013).

    A critical question is whether centromere clustering is required for proper meiotic segregation. It remains to be determined whether the meiotic nondisjunction that occurs in these centromere protein mutants is linked to the failure of centromere clustering and/or centromere pairing. The meiotic segregation errors in oocytes affect both the X chromosome, which undergoes recombination, and the 4th chromosome, which is achiasmate and lacks SC. One way that meiotic segregation of both types of chromosomes could be dependent on clustering would be if association with the nucleolus is necessary for proper assembly of the kinetochore later in prophase I. It is notable, however, that the meiotic segregation errors in oocytes assayed for the X chromosome occurred exclusively in meiosis I; thus, a defect in kinetochore function necessary for both meiosis I and II was not evident. There are known meiosis I-specific requirements of the kinetochore, such as the need for the two sister kinetochores to co-orient in meiosis I, and establishment of these may require centromere clustering and/or nucleolar association (Unhavaithaya, 2013).

    This proposal is consistent with the demonstrated effects of cenp-C mutants in meiosis in Saccharomyces pombe. An alternative possibility is that the centromere mutations have independent effects on centromere clustering and subsequent segregation. For example, the centromere clustering defects could result from failure to retain the SC at the centromere and the meiotic nondisjunction could be an independent consequence of improperly assembled kinetochores later in meiosis I. The centromere mutations clearly can affect meiotic segregation independent of centromere pairing and clustering, given the meiotic nondisjunction in males double-heterozygous for the cenp-C and cal1 alleles. In Drosophila male meiosis, centromere clustering, SC formation, and recombination do not occur (Unhavaithaya, 2013).

    Although observed in yeast, plants, and Drosophila, a role for intrinsic centromere function in the nuclear localization of centromeres and associations between centromeres in meiosis has not yet been defined. The demonstration that proper centromere architecture is necessary for these interactions opens a path to define the molecular basis of centromere pairing and clustering across these species in meiosis (Unhavaithaya, 2013).

    Sisters Unbound is required for meiotic centromeric cohesion in Drosophila melanogaster

    Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two Structural Maintenance of Chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. This study identified a novel Drosophila gene called sisters unbound (sunn) (CG32088), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of nondisjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO (Sisters on the loose/Vasa) in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit Stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, these data show that SUNN is an essential meiosis-specific Drosophila cohesion protein (Krishnan, 2014).

    Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division

    Chromosome segregation requires centromeres on every sister chromatid to correctly form and attach the microtubule spindle during cell division. Even though centromeres are essential for genome stability, the underlying centromeric DNA is highly variable in sequence and evolves quickly. Epigenetic mechanisms are therefore thought to regulate centromeres. This study shows that the 359-bp repeat satellite III (SAT III), which spans megabases on the X chromosome of Drosophila melanogaster, produces a long noncoding RNA that localizes to centromeric regions of all major chromosomes. Depletion of SAT III RNA causes mitotic defects, not only of the sex chromosome but also in trans of all autosomes. It was furthermore found that SAT III RNA binds to the kinetochore component CENP-C, and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. In conclusion, these data reveal that SAT III RNA is an integral part of centromere identity, adding RNA to the complex epigenetic mark at centromeres in flies (Rosic, 2014).

    It is well-established that centromeric regions and their function are influenced by epigenetic mechanisms to maintain their identity throughout cell and organismal generations. The histone variant CENP-A has been singled out as a key player in determining centromeres in most organisms studied so far. However, diversity and differences within centromeres suggest that additional mechanisms also play a role in centromere determination. This study provides evidence that the SAT III transcripts from a highly repetitive region of the X chromosome of D. melanogaster are important to maintain correct centromeric function, and therefore normal chromosome segregation. SAT III RNA depletion causes severe chromosome segregation defects and a partial loss of essential kinetochore components that mediate the interaction with the mitotic spindle. Furthermore, SAT III RNA interacts with the inner kinetochore protein CENP-C. A model is proposed where SAT III RNA binds to CENP-C, which in turn is required to recruit or stabilize CENP-C and possibly CENP-C–interacting factors such as CENP-A at centromeres. When SAT III RNA is absent, the association of CENP-C with centromeres is destabilized or inhibited, which impairs the association of other proteins that are dependent on CENP-C for their centromeric localization. Reciprocally, in the absence of CENP-C, SAT III is absent from centromeres, which suggests an interdependence of SAT III RNA and CENP-C. CENP-C, together with CENP-A and CAL1, forms a platform for binding of KMN proteins (named for the Knl1 complex, the Mis12 complex and the Ndc80 complex), which are required for the attachment of chromosomes to the mitotic spindle. Therefore, it is proposed that as a consequence of the SAT III depletion, chromosome missegregation is caused by the destabilization of centromeric chromatin and therefore kinetochore formation during mitosis (Rosic, 2014).

    SAT III is transcribed in D. melanogaster embryos and adult flies (Usakin, 2007; Salvany, 2009). Long centromeric transcripts have been identified in other species as well. Even though long SAT III transcripts are predominantly detected, the existence of smaller transcripts cannot be excluded, as rapid centromeric transcript turnover has been described previously. In maize, centromeric transcripts remain bound to the kinetochore after transcription, and are thought to participate in stabilization of centromeric chromatin. Maize RNA binds to centromeric protein CENP-C transiently, and promotes its binding to DNA. Therefore, noncoding RNA may play a role similar to a protein chaperone. Once CENP-C is localized to centromeres, DNA binding is facilitated with the help from RNA to stabilize its position. During interphase, SAT III RNA localizes to the nucleus, and forms a cluster in proximity to sites of centromeric clusters, perhaps at its transcription site. During mitosis, SAT III RNA is present at centromeric regions. It is suggested that satellite transcripts function in stabilizing the centromeric positioning of CENP-C, thereby facilitating the building of kinetochore structures, and in turn require CENP-C to localize to centromeres. This mechanism may be evolutionarily conserved, as CENP-C has been described to bind RNA from centromeric repeats in maize. In addition to SAT III RNA present at centromeres, some SAT III RNA is also detectable at pericentromeres of mitotic chromosomes and is non-chromatin-associated. SAT III RNA that is present at pericentromeres might also contribute to overall kinetochore structure, and signals distant from chromatin might represent distinct ribonucleoprotein particles. However, additional work is required to address these questions (Rosic, 2014).

    Depletion of SAT III RNA in S2 cells caused severe mitotic defects, which indicates that SAT III RNA is crucial for cell division. The same phenotype was observed in vivo in D. melanogaster embryos. Importantly, flies carrying an X-Y translocation chromosome that has lost most of its SAT III DNA block do not transcribe any significant amount of SAT III RNA, and display segregation defects in early embryos similar to what what was described for S2 cells and SAT III LNA gapmer-injected embryos. Most of the Zhr1 flies are viable and fertile despite the segregation defects in early embryos. It is therefore suggeste that SAT III RNA function is only one part of a larger safeguard mechanism required for accurate chromosome segregation during mitosis. It has been shown that Zhr1 male flies rescue the female hybrid lethality in crosses between D. simulans females and D. melanogaster males. One of their hypotheses was that RNA originating from SAT III might be the cause of hybrid lethality in F1 daughters originating from these crosses. This study shows that Zhr1 flies do not have any SAT III transcripts, which indicates a possible incompatibility of SAT III RNA from wild-type D. melanogaster flies with either transcripts or the sequence of the X chromosome of D. simulans. However, this and other possibilities need to be tested in the future (Rosic, 2014).

    A previous study showed that transcription of SAT III depends on the homeobox-containing transcription factor Hth, and mutations of hth lead to abnormal distribution of CENP-A (Salvany, 2009). Similarly, inhibition of transcription during mitosis resulted in a decreased level of centromeric α-satellite transcripts in human cells, which in turn resulted in lagging chromosomes and a reduction of CENP-C. Inhibition of transcription or mutations of transcription factors may, however, cause pleiotropic effects in cells; together with the results presented from a direct depletion of SAT III transcripts, this study concludes that the SAT III RNA directly influences centromere function and that satellite transcripts may have a conserved function in kinetochore formation (Rosic, 2014).

    The inability of chromosomes to segregate properly in the absence of SAT III RNA is not restricted to chromosome X, the origin of SAT III transcripts. This indicates a trans-acting mechanism, as seen in dosage compensation and proposed for maize centromeric RNA. It has been suggested that each centromere is capable of producing RNA. Indeed, in D. melanogaster, active centromeric transcription by RNA polymerase II was observed on all chromosomes. This indicates that centromeric RNAs might have redundant functions, similar to what is described for the dosage compensation complex in Drosophila. Here, roX1 and roX2 RNA are required for spreading of the compensasome to the entire X chromosome. These two RNAs are redundant in their function, even though they have little sequence similarity. The presence of redundant RNAs may also explain why the majority of chromosomes usually segregate correctly upon SAT III RNA depletion, and why only some chromosomes are lagging (Rosic, 2014).

    This study shows that SAT III RNA function is independent of heterochromatin formation. In support of this, Usakin (2007) reported that many D. melanogaster pericentromeric transcripts participate in heterochromatin formation, but SAT III transcripts were not among the RNAs that had an effect on the formation of centromeric heterochromatin. The observed heterochromatin defects in hth mutant embryos (Salvany, 2009) are, therefore, possibly caused by additional effects of depleting this transcription factor. Pericentromeric heterochromatin is required for sister chromatid cohesion and bipolar orientation during mitosis. However, the levels of cohesion proteins, as well as the heterochromatin markers HP1 and H3 lysine 9 methylation, are unaffected in SAT III–depleted cells. It is therefore concluded that the observed chromosome segregation defects after SAT III depletion are unlikely to be caused by a loss of sister chromatid cohesion or heterochromatin integrity (Rosic, 2014).

    Levels of centromeric and kinetochore proteins were significantly reduced on mitotic chromosomes that failed to segregate properly in the absence of SAT III RNA, which implies a role of SAT III RNA in providing a competent centromere environment. Additionally, reducing the levels of CENP-C by RNAi caused a complete loss of SAT III from centromeres, which suggests that CENP-C and SAT III RNA are mutually dependent on each other for their centromeric localization. Because loading of CENP-C and CENP-A is mutually dependent as well, both proteins are reduced in the absence of SAT III, as expected. Spc105 is an essential component of Drosophila kinetochores; its localization is interdependent with MIS12 complex localization and required for localization of the NDC80 subcomplex, which directly binds microtubules. Hence, reduction of Spc105 protein at centromeres leads to severe defects in constructing a functional kinetochore, and provides an explanation for failures in chromosome segregation in the absence of SAT III RNA. Finally, SNAP tag experiments showed that loading of newly synthesized CENP-A and CENP-C proteins is also affected by the loss of SAT III, which suggests that SAT III plays an integral role in establishing and stabilizing centromeric chromatin. In conclusion, SAT III RNA was identified as an epigenetic factor involved in centromere regulation and function through interaction with the centromeric protein CENP-C, which suggests a vital and evolutionarily conserved role of noncoding RNAs in centromere determination and chromosome segregation (Rosic, 2014).

    The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs

    Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. This study shows the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. a four-stranded intercalated structure formed by the association of two parallel duplexes combined in an antiparallel fashion. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads to suggestion that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures (Garavís, 2015).

    Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. Previous work has shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. This report shows that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. These results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis (Carmena, 2014).

    The Drosophila histone variant H2A.V works in concert with HP1 to promote kinetochore-driven microtubule formation

    Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. This study shows that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impaired the formation of kinetochore-driven (k) fibers, which could account for defects in chromosome segregation. All defects were rescued by a transgene encoding H2A.V that lacked the H2A.x function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V was required for chromosome segregation. Loss of H2A.V weakened HP1 localization, specifically at the pericentric heterochromatin of metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in H2A.V null mutants, suggesting a role for HP1 in chromosome segregation. H2A.V precipitated HP1 from larval brain extracts indicating that both proteins were part of the same complex. Moreover, the overexpression of HP1 rescued chromosome missegregation and defects in the kinetochore-driven k-fiber regrowth of H2A.V mutants indicating that both phenotypes were influenced by unbalanced levels of HP1. Collectively, these results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth (Verní, 2015).

    This study provides compelling evidence that H2A.V, the Drosophila histone H2A variant, plays an important and unanticipated role during Drosophila mitosis. The cytological characterization of H2A.V810 mutant larval brain chromosomes revealed that loss of H2A.V has an impact on chromosome organization and cell proliferation, which is consistent with previous results on a role of this histone variant in chromatin remodeling and heterochromatin organization. This study also demonstrates that a significant proportion of H2A.V mutant cells fails to complete mitosis and contains chromosomes that remain scattered across the spindle (pseudo anaphase or PA) due to failed metaphase plate alignment. Similar effects have been previously described in Drosophila S2 cells depleted by RNAi of either kinetochore proteins, augmin components or splicing factors. However unlike those S2 interfered cells, which exhibit PAs with long spindles, H2A.V810 mutant cells have PA (premature- or pseudo-anaphase) spindles that appear similar to wild type anaphases. The reason why H2A.V mutant cells are not elongated is unclear, but it may depend on the different cellular systems employed in the different studies. Intriguingly, the presence of PAs in H2A.V810 mutants indicates for the first time that Drosophila H2A.V is also necessary for chromosome segregation growth (Verní, 2015).

    Interestingly, the results from both Dgt6 immunolocalization and spindle microtubule re-growth assay following cold-induced MT depolymerization in mitotic neuroblasts reveal that H2A.V might be involved in the organization of kinetochore-driven, k-fibers microtubule bundles that attach sister kinetochores to spindle poles. However, it is believed that defects in the organization of k-fibers are not a consequence of the reduction of Dgt6. Recent studies demonstrated that Wac, a newly discovered component of Augmin complex, is required for spindle formation in S2 cells but is dispensable for somatic mitosis. In fact, a wac deletion mutant was viable and displayed only weak defects in brain cell divisions, suggesting that the components of Augmin complex (including Dgt6) might have non essential roles in spindle assembly growth (Verní, 2015).

    It has been previously reported that defective k-fiber formation and elongation disrupt chromosome segregation and spindle formation in Drosophila cells. The results, which are in line with this finding, indicate that a specific chromatin organization is also necessary to ensure a proper spindle assembly. It is speculated that the observed PAs are a result of improper organization of k-fibers, and that PAs fail to complete mitosis, thus reducing in part the frequency of anaphases in H2A.V810 mutants. It is also plausible that persistent chromosome misalignment leads to a mitotic arrest of these cells, which in turn could explain the presence of H2A.V810 mutant cells with overcondensed chromosomes. However, while in a previous study, the presence of PAs was always associated to a strong increase of mitotic index (MI), the current mutants the MI did not change. One explanation is that the reduction of anaphase frequency in H2A.V810 mutants (20%) is not as dramatic as that reported for Dgt6-depleted S2 cells (50%) and therefore it unlikely affects mitotic progression. An alternative explanation is that loss of H2A.V might affect the regulation of G2-M and/or M-A cell cycle checkpoints thus preventing a metaphase arrest. Further investigations are required to verify this hypothesis. It is worth noting that, although a role for H2A.Z in chromosome segregation has been previously documented in human and yeast cells, the current data provide the first evidence of an potential involvement of H2A.Z in the organization of k-fibers growth (Verní, 2015).

    This study also provides unanticipated molecular evidence that H2A.V interacts directly or indirectly with HP1, confirming that both proteins are part of same complex. It is intriguing that the H2A.V-HP1 interaction depends on the HP1 CD domain, which also binds H3K9me2/3 and mediates heterochromatin formation. This supports the existence of a cascade of events that requires the recruitment of H2A.V and different histone modifications for the establishment of heterochromatin. Yet, the reason why depletion of H2A.V causes a direct loss of HP1 and particularly during mitosis is unclear. Nonetheless, as HP1 overexpression in H2A.V mutant cells prevents PA formation, it is speculated that a H2A.V-dependent stabilization/localization of HP1 at centromeric region is essential to ensure proper chromosomal behavior growth (Verní, 2015).

    Previous studies have shown that H2A.Z alters the nucleosomal surface, thus enabling preferential binding of HP1a to condensed higher chromatin structures 44RIDcit0044. It is conceivable then that the H2A.V-HP1 interaction is favored by the condensation of pericentric chromatin fiber in metaphase. Alternatively, these interactions may be encouraged by metaphase-specific posttranslational modifications of H2A.V, HP1 or other interacting proteins. Indeed, it has been proposed the mechanism underpinning HP1 recruitment on mitotic chromosomes might be different from that in interphase. Still, little is known about the factors required for specific localization of HP1 at mitotic centromeres save for a few discoveries. Human HP1α binding to INCENP, for instance, has been demonstrated as necessary for HP1α targeting to mitotic centromeres. It is believed that H2A.V may play a similar role in mediating HP1 binding, but how this takes place remains to be seen growth (Verní, 2015).

    This functional characterization of H2A.V has also unveiled the role of Drosophila HP1a in the assembly of the mitotic spindle. The results indicate that the loss of HP1 yields defects in the kinetochore-driven k-fiber organization, which can in turn compromise chromosome segregation thus generating PAs. Past studies have shown that HP1a contributes to chromosome segregation and centromere stability in a variety of organisms including mammals, but the mechanism is still not completely understood. HP1 is known to interact with components of the centromere and the kinetochore complex, providing targets to begin understanding how downregulation or mislocalization of HP1 result in mitotic defects. It has also been reported that in contrast to Swi6 in S. pombe, the correct localization of HP1 is not required for the recruitment of cohesins to centromeric regions in mammals. Yet, HP1a seems to help in protecting cohesins from degradation by recruiting the Shugoshin protein. This study has highlighted an additional function of HP1 during chromosome segregation, one which depends on interaction with H2A.V and is required to regulate k-fiber organization. These results thus provide further evidence of a functional versatility of HP1 that is likely conserved also in mammals growth (Verní, 2015).

    Lateral and end-on kinetochore attachments are coordinated to achieve bi-orientation in Drosophila oocytes

    In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation. This study examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. The initiation of spindle assembly was shown to result from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. It is proposed that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes (Radford, 2015).

    It is well established that oocyte spindle assembly in many organisms occurs in the absence of centrosomes. Instead, chromatin-based mechanisms play an important role in spindle assembly. The interactions between chromosomes and microtubules are paramount in oocytes, necessary for both the assembly of the spindle and the forces required for chromosome segregation. Less well understood, however, is the nature of the functional connections between chromosomes and microtubules in these cells. The role of the kinetochores, the primary site of interaction between chromosomes and microtubules, is poorly understood in acentrosomal systems. For example, spindles will assemble and chromatin will move without kinetochores in both Caenorhabditis elegans and mouse oocytes. In addition, both C. elegans and mouse oocytes experience a prolonged period during which chromosomes have aligned but end-on kinetochore-microtubule attachments have not formed. Previously shown that the central spindle, composed of antiparallel microtubules that assemble adjacent to the chromosomes, is important for spindle bipolarity and homolog bi-orientation. These studies suggest that lateral interactions between the chromosomes and microtubules drive homolog bi-orientation, but whether these interactions are kinetochore-based is not clear (Radford, 2015).

    There have been few studies directly analyzing kinetochore function in oocyte spindle assembly and chromosome segregation. Assembling a functional spindle requires the initiation of microtubule accumulation around the chromatin, the organization of microtubules into a bipolar structure, and the maturation of the spindle from promoting chromosome alignment to promoting segregation. Whether the kinetochores are required for spindle assembly or the series of regulated and directed movements chromosomes undergo to ensure their proper partitioning into daughter cells is not known. In Drosophila, the chromosomes begin the process within a single compact structure called the karyosome. Within the karyosome, centromeres are clustered prior to nuclear envelope breakdown (NEB). This arrangement, which is established early in prophase and maintained throughout diplotene/diakinesis, is also found in many other cell types. It is possible that the function of centromere clustering is to influence the orientation of the centromeres on the spindle independent of chiasmata. Following NEB, the centromeres separate. In Drosophila oocytes, centromere separation depends on the chromosomal passenger complex (CPC). Whether this movement depends on interactions between chromosomes and microtubules remains to be established (Radford, 2015).

    Following centromere separation, homologous centromeres move towards opposite spindle poles. During this time in Drosophila oocytes, the karyosome elongates and achiasmate chromosomes may approach the poles, separating from the main chromosome mass. As prometaphase progresses, the chromosomes once again contract into a round karyosome. These chromosome movements appear analogous to the congression of chromosomes to the metaphase plate that ultimately results in the stable bi-orientation of chromosomes. In mitotic cells, congression depends on lateral interactions between kinetochores and microtubules, and bi-orientation depends on the formation of end-on kinetochore-microtubule attachments. In oocytes, lateral chromosome-microtubule interactions have been suggested to be especially important, but how lateral and end-on kinetochore-microtubule attachments are coordinated to generate homolog bi-orientation has not been studied (Radford, 2015).

    To investigate the roles of lateral and end-on kinetochore-microtubule attachments in spindle assembly and prometaphase chromosome movements of acentrosomal oocytes, this study characterized Drosophila oocytes lacking kinetochore components. The KNL1/Mis12/Ndc80 (KMN) complex is at the core of the kinetochore, providing a link between centromeric DNA and microtubules. Both KNL1 and NDC80 bind to microtubules in vitro, but NDC80 is required specifically for end-on kinetochore-microtubule attachments. Therefore, this study examined oocytes lacking either NDC80 to eliminate end-on attachments or the Drosophila homolog of KNL1, SPC105R, to eliminate all kinetochore-microtubule interactions. Oocytes lacking the centromere-associated kinesin motor CENP-E because CENP-E promotes the movement of chromosomes along lateral kinetochore-microtubule attachments in a variety of cell types (Radford, 2015).

    This work has identified three distinct functions of kinetochores that lead to the correct orientation of homologs at meiosis I. First, SPC105R is required for the co-orientation of sister centromeres at meiosis I. This is a unique process that fuses sister centromeres, ensuring they attach to microtubules from the same pole at meiosis I. Second, lateral kinetochore-microtubule attachments are sufficient for prometaphase chromosome movements, which may be required for each pair of homologous centromeres to establish connections with microtubules from opposite poles. Third, end-on attachments are dispensable for prometaphase movement but are essential to stabilize homologous chromosome bi-orientation. Surprisingly, it was found that although Drosophila oocytes do not undergo traditional congression of chromosomes to the metaphase plate, CENP-E is required to prevent chromosomes from becoming un-aligned and to promote the correct bi-orientation of homologous chromosomes. It was also shown that the initiation of acentrosomal chromatin-based spindle assembly does not depend on kinetochores, suggesting the presence of important additional interaction sites between chromosomes and microtubules. The stability of the oocyte spindle, however, becomes progressively more dependent on kinetochores as the spindle transitions from prometaphase to metaphase. Overall, this work shows that oocytes integrate several chromosome-microtubule connections to promote spindle formation and the different types of chromosome movements that ensure the proper segregation of homologous chromosomes during meiosis (Radford, 2015).

    In acentrosomal oocytes, spindle assembly depends on the chromosomes. How the chromosomes can organize a bipolar spindle that then feeds back and drives processes like bi-orientation of homologous centromeres has been unclear. Previous studies have demonstrated that the central spindle is required for homolog bi-orientation. This study found that several types of functional chromosome-microtubule interactions exist in oocytes, and that each type participates in unique aspects of chromosome orientation and spindle assembly. A model for chromosome-based spindle assembly and chromosome movements in oocytes highlights the multiple and unappreciated roles played by kinetochore proteins such as SPC105R and NDC80, with implications for how homologous chromosomes bi-orient during meiosis I (Radford, 2015).

    While the spindle is assembling and becoming organized, the evidence suggests that the chromosomes undergo a series of movements that ultimately result in the bi-orientation of homologous chromosomes. The separation of clustered centromeres is CPC-dependent (Radford, 2012), but not kinetochore-dependent. One possibility is that the CPC-dependent interaction of microtubules with non-kinetochore chromatin drives centromere separation. An alternative is that CPC activity may result in a release of the factors that hold centromeres together in a cluster prior to NEB. A candidate for this factor is condensin, a known target of the CPC, that has been shown to promote the 'unpairing' of chromosomes in the Drosophila germline (Radford, 2015).

    Following separation of clustered centromeres, each pair of homologous centromeres bi-orients by separating from each other towards opposite poles. How bi-orientation is established in acentrosomal oocytes is poorly understood. Previous studies in C. elegans and mouse oocytes have suggested a combination of kinetochore-dependent and kinetochore-independent (e.g. involving chromokinesins and chromosome arms) microtubule interactions drive chromosome alignment and segregation. This study found that kinetochores play multiple roles, and the process of chromosome bi-orientation can be broken down into a series of chromosome movements that depend mostly on the kinetochores. First, the centromeres make an attempt at bi-orientation. In Drosophila oocytes, this results in the directed poleward movement of centromeres toward the edge of the karyosome and is accompanied by a stretching of the karyosome. Lateral kinetochore-microtubule attachments mediated by SPC105R are sufficient for this initial attempt at bi-orientation. End-on kinetochore-microtubule attachments via NDC80, however, are essential to maintain the bi-orientation of centromeres. Maintenance of centromere bi-orientation is associated with the stable positioning of the centromeres at the edges facing the poles (Radford, 2015).

    The lateral-based chromosome movements required for chromosome orientation are probably mediated by the meiotic central spindle, which have been shown to essential for chromosome segregation. In addition, recent reports in both mitotic and meiotic cells suggest that the initial orientation of chromosomes depends on the formation of a 'prometaphase belt' that likely brings centromeres into the vicinity of the central spindle. Therefore, it is proposed that the initial attempt at bi-orientation occurs during the period when both kinetochores and the central spindle are required for spindle stability. Then, as the oocyte progresses toward metaphase, and the central spindle decreases in importance, this reflects a trend toward the formation of stable end-on kinetochore-microtubule attachments that, in turn, stabilize the bipolar spindle. This model is also corroborated by evidence from mouse oocytes that stable end-on kinetochore-microtubule attachments form after a prolonged prometaphase (Radford, 2015).

    The data demonstrate that some chromosome movements, critical for bi-orientation, are dependent on lateral kinetochore-microtubule attachments. The kinetochore-associated kinesin motor CENP-E is thought to be responsible for chromosome movement along lateral kinetochore-microtubule attachments, resulting in chromosome alignment on the metaphase plate. However, because Drosophila meiotic chromosomes are compacted into a karyosome prior to NEB, they do not need to migrate in a plus-end-directed manner to achieve congression and alignment. Instead, centromeres must move toward the poles, perhaps in a minus-end directed manner, to achieve bi-orientation. Interestingly, this study found that CENP-E opposes this minus-end directed movement because in the absence of CENP-E, the karyosome split via lateral kinetochore-microtubule attachments. It is not yet clear what mediates the minus-end-directed movement, but the motors Dynein and NCD (the Drosophila kinesin-14 homolog) or microtubule flux are prime candidates (Radford, 2015).

    This study also observed that CMET (CENP-E) is required for the correct bi-orientation of homologous chromosomes. The function proposed in opposing minus-end directed movement may be required for making the correct attachments. As the centromere moves to the edge of the karyosome, CENP-E may not only prevent its separation from the karyosome, but could also force it back towards the opposite pole in cases where the homologs are not bi-oriented. A similar idea has been proposed for CENP-E in mouse oocytes. Alternatively, CENP-E has a second function in tracking microtubule plus-ends and regulating kinetochore-microtubule attachments. In fact, this study found that end-on kinetochore-microtubule stability is affected in the absence of CENP-E. Regulating the stability of microtubule plus-end attachments with kinetochores is critical for establishing correct bi-orientation of homologs. Therefore, both functions of CENP-E could contribute to the correct bi-orientation of centromeres in Drosophila oocytes (Radford, 2015).

    Loss of SPC105R has a more severe phenotype than loss of either NDC80 or CENP-E, consistent with a role as a scaffold. It recruits additional microtubule interacting proteins like NDC80 and CENP-E and also recruits checkpoint proteins such as ROD. In analyzing oocytes lacking SPC105R, another class of factors it may recruit was discovered: proteins required for co-orientation of sister centromeres during meiosis I. Co-orientation is a process that fuses the core centromeres and is important to ensure that two sister kinetochores attach to microtubules that are attached to the same spindle pole. Co-orientation could involve a direct linkage between sister kinetochores, as may be the case with budding yeast Monopolin or in maize, where a MIS12-NDC80 linkage may bridge sister kinetochores at meiosis I [56]. In contrast, in fission yeast meiosis I, cohesins are required for co-orientation. Cohesion is stably maintained at the core centromeres during meiosis I but not mitosis, and this depends on the meiosis-specific proteins Moa1 and Rec8. There is also evidence that Rec8 is required for co-orientation in Arabidopsis and this study found that loss of ORD, which is required for meiotic cohesion, also results in a loss of centromere co-orientation. Further studies, however, are necessary to determine if cohesins are required for co-orientation in Drosophila. Indeed, the proteins and mechanism that mediate this process in animals has not been known. Recently, however, the vertebrate protein MEIKIN has been found to provide a similar function to Moa1. Interestingly, both Moa1 and MEIKIN depend on interaction with CENP-C, but do not show sequence homology. Thus, Drosophila may have a Moa1/MEIKIN ortholog that has not yet been identified. In the future, it will be important to identify the proteins recruited by SPC105R and their targets in maintaining centromere co-orientation and how these interact with proteins recruited by CENP-C. The mechanism may involve the known activity of SPC105R in recruiting PP1, because PP1 has been shown to have a role in maintaining cohesion in meiosis I of C. elegans (Radford, 2015).

    This study's model for spindle assembly and chromosome orientation raises several important questions for future consideration. The CPC is required for spindle assembly in Drosophila oocytes and the current results highlight the importance of two CPC targets in homolog bi-orientation. One target is central spindle proteins, possibly through the CPC-dependent recruitment of spindle organization factors such as Subito. The CPC is also required for kinetochore assembly, similar to what has been shown in yeast, human cells, and Xenopus and consistent with the finding in human cells that Aurora B promotes recruitment of the KMN complex to CENP-C. It will be important to identify targets of the CPC that drive the initiation of spindle assembly, centromere separation, and bi-orientation. In addition, while this study has found that the CPC is required for kinetochore assembly, it is not known if the CPC promotes error correction by destabilizing kinetochore-microtubule attachments. The CPC may not promote kinetochore-microtubule detachment during meiosis because of the different spatial arrangement of sister centromeres during meiosis I. Indeed, it is not known what is responsible for correcting incorrect attachments at meiosis I or how they are differentiated from correct attachments (Radford, 2015).

    In prometaphase, the central spindle and kinetochores contribute to spindle stability. The current data suggests that the kinetochores increase in importance as the oocyte progresses to metaphase, perhaps as a result of the stabilization of end-on kinetochore-microtubule attachments as homologous chromosomes become bi-oriented. However, lateral kinetochore-microtubule interactions demonstrated some resistance to colchicine and allow bivalents to stretch in mouse oocytes. Thus, further studies are necessary to determine if lateral kinetochore-microtubule interactions also confer some stability. The current model also proposes that the transition from prometaphase to metaphase involves a switch from dynamic lateral kinetochore-microtubule interactions to stable end-on kinetochore-microtubule attachments. This transition involves the loss of central spindle microtubules, which occurs regardless of microtubule attachment status. Further studies will be necessary to determine if the prometaphase-to-metaphase transition is developmentally regulated rather than being controlled by the spindle assembly checkpoint. As proposed in mouse oocytes, this may contribute to the propensity for chromosome segregation errors in acentrosomal oocytes by closing the window of opportunity for error correction after key developmental milestones have been passed. Finally, one of the most poorly understood features of meiosis is co-orientation of sister centromeres at meiosis I. What SPC105R interacts with to mediate co-orientation will provide the first insights into the mechanism and regulation of this process in Drosophila (Radford, 2015).

    Polar ejection forces promote the conversion from lateral to end-on kinetochore-microtubule attachments on mono-oriented chromosomes

    Chromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs) were generated. SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation (Drpic, 2015).

    Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function

    Kinetochores allow attachment of chromosomes to spindle microtubules. Moreover, they host proteins that permit correction of erroneous attachments and prevent premature anaphase onset before bi-orientation of all chromosomes in metaphase has been achieved. Kinetochores are assembled from subcomplexes. Kinetochore proteins as well as the underlying centromere proteins and the centromeric DNA sequences evolve rapidly despite their fundamental importance for faithful chromosome segregation during mitotic and meiotic divisions. During evolution of Drosophila melanogaster, several centromere proteins were lost and a recent gene duplication has resulted in two Nnf1 paralogs, Nnf1a and Nnf1b, which code for alternative forms of a Mis12 kinetochore complex component. The rapid evolutionary divergence of centromere/kinetochore constituents in animals and plants has been proposed to be driven by an intragenome conflict resulting from centromere drive during female meiosis. Thus, a female meiosis-specific paralog might be expected to evolve rapidly under positive selection. While this characterization of the D. melanogaster Nnf1 paralogs hints at some partial functional specialization of Nnf1b for meiosis, no evidence was detected for positive selection in the analysis of Nnf1 sequence evolution in the Drosophilid lineage. Neither paralog is essential, even though some clear differences were found in subcellular localization and expression during development. Loss of both paralogs results in developmental lethality. It is therefore concluded that the two paralogs are still in early stages of differentiation (Blattner, 2016).

    Network of protein interactions within the Drosophila inner kinetochore

    The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. This study employed hydrogen-deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 (Nnf1a and Nnf1b) form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, it is concluded the organization of the Mis12-Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function (Richter, 2016).

    An essential step of kinetochore formation controlled by the SNARE protein Snap29

    The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. This study identified a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis-segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. It is proposed that part of the outer kinetochore is functionally similar to membrane fusion interfaces (Morelli, 2016).

    Cell division relies on organization of a microtubule (MT) spindle to which replicated chromosomes become attached for equal segregation. Defective MT attachment to kinetochores (KTs) leads to chromosome mis‐segregation and formation of fragmented nuclei after cell division. These events have been proposed to contribute to the genome instability observed in many cancers, indicating that control of MT attachment is a major tumor suppressing process (Morelli, 2016).

    The molecular nature of the outer KT, the structure that mediates MT attachment, has been studied extensively. MTs are engaged and stabilized by the Knl1, Mis12, and Ndc80 complexes, together referred to as the KMN network. The KMN network also holds in place the Rod‐Zw10‐Zwilch (RZZ) complex and spindle assembly checkpoint (SAC) proteins, which are important for signaling incomplete attachment. In mammalian cells, MT attachment is further assisted by the KNL1‐interactor ZWINT and by the SKA complex, which associates with curved MT ends at KTs (Morelli, 2016).

    In sheer contrast with the extensive molecular knowledge of the outer KT, much less is known about the steps that regulate its assembly. In Drosophila, a widely used metazoan model system for KT studies, part of the Mis12 complex resides at the KT throughout the cell cycle, while the rest of the outer KT is created de novo in early prophase, by stepwise addition of components. The earliest components added to the outer KT in early prophase appears to be Knl1, followed by the Ndc80 complex, both of which are recruited from unknown cellular locales, and SAC components, such as Mad1 and Mad2, which are recruited from nuclear pores (Morelli, 2016).

    Unexpectedly, it has been recently found that the Drosophila SNARE protein Snap29 can be isolated from cell extracts together with multiple components of the KMN network. These are the Drosophila Knl1 ortholog Spc105R, three out of four components of the Ndc80 complex (Nuf2 and the Drosophila Spc24 ortholog Kmn2) as well as three of the four subunits of the Mis12 complex (Mis12, Nnf1b, and the Drosophila Nsl1 ortholog Kmn1). SNARE (Soluble NSF Attachment REceptor) proteins (SNAREs) are part of the conserved coiled‐coil machinery that brings membranes in close proximity during trafficking, a prerequisite for most membrane fusion events. The Synaptosomal‐Associated Protein (SNAP) family of SNAREs in metazoans includes Snap25, Snap23, and Snap29, which are composed by two SNARE domains, separated by a linker region. The first two proteins are membrane‐associated and control synaptic transmission and a wide range of non‐neuronal membrane fusion processes, respectively. In contrast, Snap29 only transiently associates with membranes and contains an acidic NPF motif that mediates its association with endocytic factors. Such unconventional features, which are exclusive of Snap29 among the SNARE proteins, predict involvement in a versatile set of membrane trafficking processes, in line with reports in the literature. Consistent with this, Snap29 also controls fusion of autophagosomes with endo‐lysosomes in Drosophila and human cells, together with the SNAREs syntaxin17 (Syx17) and Vamp7 (VAMP8 in human cells) (Morelli, 2016).

    Despite involvement of Snap29 in multiple trafficking pathways in interphase, a possible function during cell division has not been explored. This study investigated whether Snap29 localizes and acts at the KT in cells and tissues. The data identify a novel step of KT formation that is conserved and supports tissue formation (Morelli, 2016).

    The data uncover an essential and conserved step of KT formation that occurs in prophase and requires Snap29. Such step controls localization of Knl1 (and ZWINT in human cells) to KTs. Snap29 and the RZZ components Rod and Zw10 are known to act in membrane transport between the Golgi apparatus and the ER, and the RZZ complex shares similarity with ER tethering complexes. Strikingly, the autophagosome, which depends on Snap29 for fusion to lysosomes, is formed de novo using Golgi and ER components and engages MTs for dynein‐directed transport to lysosomes, evoking tantalizing similarities between aspects of membrane trafficking and KT formation. Overall, the current data support the possibility that Snap29, Knl1, and the RZZ complex might act at the KT similarly to tethering and fusion complexes existing on membranes, which need to stabilize MTs during trafficking events. Such scenario might imply common ancestry, and underscored also the existence of protozoa that divide with KTs associated with the nuclear membrane that is itself attached to MT fibers (Gómez‐Conde, 2000) (Morelli, 2016).

    Ectopic recruitment of Knl1 and possibly RZZ and SKA complex components at sites of SNAP29 Q1 Q2 trapping predicts that the trafficking and KT functions of Snap29 are interconnected, perhaps because of the existence of a common cellular pool of Snap29. A possibility that awaits further investigation is that SNARE domains of Snap29 interact with KT proteins directly. Interestingly, the C‐terminal part of Knl1, Zwint, and Mis12 and Nnf1 all contain multiple coiled‐coil regions. These are placed at the interaction surface between the Mis12, Ndc80, and Knl1 complexes, exactly where Snap29 is seen located by super‐resolution microscopy. In mammalian cells, the Snap29 paralog Snap25 binds Zwint, and Snap29 has been found in Zwint immunoprecipitations. Thus, Snap29 could dock to the Mis12 complex with a SNARE domain and could stabilize interactions with Knl1 and Zwint with the second C‐terminal SNARE domain. Interestingly, no Zwint homolog has been found in Drosophila, suggesting that in flies Snap29 could substitute for Zwint. The ability of KNL1 to interact with a SNAP29 that cannot be released from SNARE complex, both suggest that the interaction of KNL1 with SNAP29 might occur on the side of the SNARE domain that is not occupied by a syntaxin or a Vamp. These data also suggest that SNAP29 could act on Knl1 also prior to nuclear entry and KT localization (Morelli, 2016).

    The evidence in vivo indicates that SNAP29 function could support tissue development by ensuring faithful chromosome segregation and that such activity is crucial in cells that become resistant to apoptosis. Based on this, it is predicted that loss of SNAP29 could be selected in cancers with highly unstable genomes. In addition, rare congenital syndromes such as CEDNIK, Roberts syndrome, and primary microcephaly (MCPH) are caused by mutations in Snap29 and other genes encoding proteins that regulate mitosis, respectively, overall suggesting that ability to cope with defective mitotic cells is major process for tissue development and homeostasis (Morelli, 2016).

    Simple and complex centromeric satellites in Drosophila sibling species

    Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats, often 100-400 bp in length, found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. This study use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5-bp and 10-bp repeats, but in closely related D. simulans, more complex repeats comprise the centromeres. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster (Talbert, 2018).

    Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes

    Sister kinetochores are connected to the same spindle pole during meiosis I and to opposite poles during meiosis II. The molecular mechanisms controlling the distinct behavior of sister kinetochores during the two meiotic divisions are poorly understood. To study kinetochore behavior during meiosis, time lapse imaging was optimized with Drosophila spermatocytes, enabling kinetochore tracking with high temporal and spatial resolution through both meiotic divisions. The correct bipolar orientation of chromosomes within the spindle proceeds rapidly during both divisions. Stable bi-orientation of the last chromosome is achieved within ten minutes after the onset of kinetochore-microtubule interactions. Analyses of something that sticks like glue (snama or mini-me) and teflon tef mutants, where univalents instead of bivalents are present during meiosis I, indicate that the high efficiency of normal bi-orientation depends on pronounced stabilization of kinetochore attachments to spindle microtubules by the mechanical tension generated by spindle forces upon bi-orientation. Except for occasional brief separation episodes, sister kinetochores are so closely associated that they cannot be resolved individually by light microscopy during meiosis I, interkinesis and at the start of meiosis II. Permanent evident separation of sister kinetochores during M II depends on spindle forces resulting from bi-orientation. In mnm and tef mutants, sister kinetochore separation can be observed already during meiosis I in bi-oriented univalents. Interestingly, however, this sister kinetochore separation is delayed until the metaphase to anaphase transition and depends on the Fzy/Cdc20 activator of the anaphase-promoting complex/cyclosome. It is proposed that univalent bi-orientation in mnm and tef mutants exposes a release of sister kinetochore conjunction that occurs also during normal meiosis I in preparation for bi-orientation of dyads during meiosis II (Chaurasia, 2018).

    Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

    Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. This study shows that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. Both SFs were demonstrated to bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells (Pellacani, 2018).

    Drosophila kinesin-8 stabilizes the kinetochore-microtubule interaction

    Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. This study finds that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end-stabilizing and -destabilizing activity, in addition to kinesin-8's commonly observed MT plus end-directed motility and tubulin-binding activity in vitro. This study further shows that Klp67A is required for stable kinetochore-MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an "end-on" fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end-stabilizing factor CLASP, but not by artificial shortening of MTs. Human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore-MT attachment (Edzuka, 2018).

    Kinetochore proteins have a post-mitotic function in neurodevelopment

    The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. This study shows that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. It is concluded that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals (Zhao, 2019).

    The modular mechanism of chromocenter formation in Drosophila

    A central principle underlying the ubiquity and abundance of pericentromeric satellite DNA repeats in eukaryotes has remained poorly understood. It has been proposed that the interchromosomal clustering of satellite DNAs into nuclear structures known as chromocenters ensures encapsulation of all chromosomes into a single nucleus. Chromocenter disruption led to micronuclei formation, resulting in cell death. This study shows that chromocenter formation is mediated by a 'modular' network, where associations between two sequence-specific satellite DNA-binding proteins, D1 and Prod, bound to their cognate satellite DNAs, bring the full complement of chromosomes into the chromocenter. D1 prod double mutants die during embryogenesis, exhibiting enhanced phenotypes associated with chromocenter disruption, revealing the universal importance of satellite DNAs and chromocenters. Taken together, it is proposed that associations between chromocenter modules, consisting of satellite DNA binding proteins and their cognate satellite DNA, package the Drosophila genome within a single nucleus (Jagannathan, 2019).

    Islands of retroelements are major components of Drosophila centromeres

    Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in understanding of their functional organization and evolution. This study identified the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, this study demonstrates that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. A specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. This work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA (Chang, 2019).

    Centromere-proximal meiotic crossovers in Drosophila melanogaster are suppressed by both highly-repetitive heterochromatin and proximity to the centromere

    Crossovers are essential in meiosis of most organisms to ensure the proper segregation of chromosomes, but improper placement of crossovers can result in nondisjunction and aneuploidy in progeny. In particular, crossovers near the centromere can cause nondisjunction. Centromere-proximal crossovers are suppressed by what is termed the centromere effect, but the mechanism is unknown. This study investigated contributions to centromere-proximal crossover suppression in Drosophila melanogaster. A large number of centromere-proximal crossovers were mapped; crossovers were found to be essentially absent from the highly repetitive (HR)heterochromatin surrounding the centromere but occur at a low frequency within the less-repetitive (LR)heterochromatic region and adjacent euchromatin. Previous research suggested that flies that lack the Bloom syndrome helicase (Blm) lose meiotic crossover patterning, including the centromere effect. Mapping of centromere-proximal crossovers in Blm mutants reveals that the suppression within the HRheterochromatin is intact, but the distance-dependent centromere effect is lost. It is concluded that centromere-proximal crossovers are suppressed by two separable mechanisms: an HR-heterochromatin effect that completely suppresses crossovers in the HRheterochromatin, and the centromere effect, which suppresses crossovers with a dissipating effect with distance from the centromere (Hartmann, 2019).

    Dynamic turnover of centromeres drives karyotype evolution in Drosophila

    Centromeres are the basic unit for chromosome inheritance, but their evolutionary dynamics is poorly understood. This study generate high-quality reference genomes for multiple Drosophila obscura group species to reconstruct karyotype evolution. All chromosomes in this lineage were ancestrally telocentric and the creation of metacentric chromosomes in some species was driven by de novo seeding of new centromeres at ancestrally gene-rich regions, independently of chromosomal rearrangements. The emergence of centromeres resulted in a drastic size increase due to repeat accumulation, and dozens of genes previously located in euchromatin are now embedded in pericentromeric heterochromatin. Metacentric chromosomes secondarily became telocentric in the pseudoobscura subgroup through centromere repositioning and a pericentric inversion. The former (peri)centric sequences left behind shrunk dramatically in size after their inactivation, yet contain remnants of their evolutionary past, including increased repeat-content and heterochromatic environment. Centromere movements are accompanied by rapid turnover of the major satellite DNA detected in (peri)centromeric regions (Bracewell, 2019).

    Asymmetric centromeres differentially coordinate with mitotic machinery to ensure biased sister chromatid segregation in germline stem cells

    Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. How non-genic information could be inherited differentially to establish distinct cell fates is not well understood. This study reports a series of spatiotemporally regulated asymmetric components, which ensure biased sister chromatid attachment and segregation during ACD of Drosophila male germline stem cells (GSCs). First, sister centromeres are differentially enriched with proteins involved in centromere specification and kinetochore function. Second, temporally asymmetric microtubule activities and polarized nuclear envelope breakdown allow for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. Abolishment of either the asymmetric sister centromeres or the asymmetric microtubule activities results in randomized sister chromatid segregation. Together, these results provide the cellular basis for partitioning epigenetically distinct sister chromatids during stem cell ACDs, which opens new directions to study these mechanisms in other biological contexts (Ranjan, 2019).

    The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. Overexpression of CAL1 is associated with increased CENP-A levels at centromeres and uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration suggesting crosstalk of CENP-A loading with the regulatory machinery of mitosis. Mitosis length is influenced by the spindle assembly checkpoint (SAC), and this study found that CAL1 interacts with the SAC protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the SAC to ensure mitotic fidelity (Pauleau, 2019).

    The formation of two genetically identical daughter cells with a correct and stable genome is of utmost importance during mitosis. Condensed chromosomes are attached and segregated to the opposing poles of the dividing cell at anaphase by the mitotic spindle. At the interface between the chromosomes and the spindle microtubules lies the kinetochore. This multi-protein complex is formed by the components of the KMN network (formed by the Knl1 complex, the Mis12 complex, and the Ndc80 complex) (Joglekar, 2017). The Ndc80 complex is mainly responsible for connecting microtubules with kinetochores while the Knl1 complex primarily coordinates the Spindle Assembly Checkpoint (SAC) (Musacchio, 2017). The SAC delays entry into anaphase until all chromosomes are properly attached and aligned at the metaphase plate. The metaphase to anaphase transition is controlled by the activation of Cdc20 of the APC/C, a multisubunit ubiquitin ligase that triggers the degradation of cell cycle regulators by the proteasome. The SAC proteins Mad2, BubR1, and Bub3 sequester Cdc20 by forming the Mitotic Checkpoint Complex (MCC) thereby preventing the activation of the APC/C. Besides, other proteins have been implicated in SAC activity including Bub1, Mad1, the Mps1 and Aurora B kinases, and the RZZ complex (formed by the three proteins Rough Deal (ROD), Zw10 and Zwilch∂ (Musacchio, 2015). Finally, the Mis12 complex serves as a hub at the kinetochore interacting with all kinetochore complexes as well as with the centromere (Pauleau, 2019).

    The kinetochore assembles on the centromere during mitosis, a highly specialized chromatin region that is defined by an enrichment of nucleosomes containing the histone H3 variant CENP-A, also called CID in Drosophila. In contrast to canonical histones, CENP-A deposition at centromeres is independent of DNA replication and is temporally restricted to a specific cell cycle stage, which varies between organisms: late telophase/early G1 in mammalian cultured cells, G2 in S. pombe and plants, and mitosis to G1 in Drosophila. The timing of CENP-A is particularly intriguing in Drosophila cultured cells as centromeric CENP-A is replenished during prometaphase-metaphase thus coinciding with kinetochore assembly. CENP-A loading requires the action of its dedicated chaperones: HJURP in humans, Scm3 in fungi and CAL1 in Drosophila (Chen, 2014, Erhardt, 2008, Schittenhelm, 2010). Deregulation of CENP-A and its loading machinery can result in the misincorporation of CENP-A into regions along the chromosome arms. Misincorporated CENP-A is usually rapidly degraded. If, however, CENP-A-containing nucleosomes remain at non-centromeric sites ectopic formation of functional kinetochores can occur that may lead to chromosome segregation defects and aneuploidy (Pauleau, 2019).

    In Drosophila melanogaster, two other proteins are constitutively present at centromeres and essential for centromere function: the conserved protein CENP-C and the CENP-A chaperone CAL1. CENP-C has been shown to act as a linker between CENP-A nucleosomes and the Mis12 complex, therefore, providing a platform for kinetochore assembly (Przewloka, 2011). CENP-C is also implicated in CENP-A replenishment at centromeres during mitosis by recruiting CAL1 (Chen, 2009; Erhardt, 2008). CAL1 interacts with CENP-A in both pre-nucleosomal and nucleosomal complexes and is necessary for CENP-A protein stabilization via Roadkill-Cullin3-mediated mono-ubiquitination. Moreover, CAL1 has been previously shown to be the limiting factor for CENP-A centromeric incorporation in fly embryos. However, differences in centromere assembly have been reported between Drosophila cultured cells and embryos. Firstly, CENP-A loading has been shown to take place during mitotic exit in early embryos and prometaphase to early G1 in cultured cells. Second, CENP-C incorporates concomitantly to CENP-A in embryos while this time window seems to be larger in cultured cells. This study, therefore, set out to determine more precisely the function of CAL1 in CENP-A loading regulation in Drosophila cultured cells (Pauleau, 2019).

    During the course of these studies, overexpression of CAL1 was found not only to increase endogenous and exogenous CENP-A abundance at centromeres, but also was found to uncouple CENP-A loading from mitosis. Strikingly, it was discovered a co-dependence of mitotic duration and accurate CENP-A loading that may be coordinated by an interaction of the CENP-A loading machinery with the SAC protein and RZZ subunit Zw10. These data suggest an intricate coordination of the spindle assembly checkpoint, CENP-A loading, and mitotic duration in order to safeguard accurate mitotic progression (Pauleau, 2019).

    In Drosophila cells, CENP-A loading takes place primarily during prometaphase-metaphase. Additional turnover of CENP-A in G1 has been reported leading to the hypothesis that CENP-A could be further incorporated at this stage, which was not observe in FRAP experiments when centromeric CENP-A was bleached at the end of cytokinesis. However, the FRAP experiments and most importantly live staining of newly synthesized SNAP-CENP-A confirmed that the majority of CENP-A loading takes place during mitosis in Drosophila cultured cells (Pauleau, 2019).

    In flies, CENP-A incorporation is controlled by its chaperone CAL1. It has been shown previously that co-overexpression of exogenous CENP-A and CAL1 leads to an increase of centromeric CENP-A in embryos. This study now shows that overexpression of CAL1 alone leads to increased endogenous CENP-A protein levels in Drosophila cultured cells. Ectopic incorporation of CENP-A, however, was never observed suggesting that CAL1 loads CENP-A exclusively to centromeres and that ectopic CENP-A incorporation in flies depends on alternative loading mechanisms similar to what has been suggested in human cells. Importantly, increased centromeric CENP-A levels following CAL1 overexpression correlated with faster mitosis. A similar acceleration of mitotic timing was observed when CENP-A was only mildly overexpressed, revealing a possible link between CENP-A loading and mitotic timing. Indeed, shortening mitosis duration by depleting Mad2 or BubR1 was associated with decreased CENP-A loading. However, just elongating the mitotic time window during which CENP-A can get loaded (Spindly or Cdc27 depletion, or by drug treatment) did not increase the amount of CENP-A incorporated at centromeres, showing that the length of mitosis alone is insufficient to control CENP-A amounts at centromeres. Rather, these experiments showed that only a defined amount of CENP-A can be incorporated at each mitosis probably correlating with CAL1's availability. Indeed, live analysis of CAL1-overexpressing cells allowed visualization of newly synthesized CENP-A incorporation to centromeres in all stages of the cell cycle. This strongly suggests that CAL1 controls CENP-A incorporation into centromeric chromatin both quantitatively and temporally. How exactly CENP-A levels at centromeres are sensed is unclear but this study identified the RZZ-component Zw10 as a new CAL1 interacting partner, which directly connects CENP-A loading to the SAC (Pauleau, 2019).

    It has been proposed that SAC activation is a 2-steps process: at the end of G2-beginning of mitosis, before the kinetochores are assembled, cytosolic Mad1-Mad2 dimers initiate MCC formation inhibiting APC/CCdc20 and determining the timing of mitosis. After nuclear envelope disassembly, kinetochore-dependent MCC are generated and regulated by kinetochore-microtubules attachment. Therefore, the following model is suggested: efficient CENP-A loading by CAL1 during mitosis recruits Zw10 up to a threshold, which is sensed by the SAC. Low CENP-A levels at centromeres could lead to more cytosolic Mad2 thereby keeping the timer active longer. Higher CENP-A levels at centromeres during early mitosis would accelerate the recruitment of RZZ and consequently Mad2 to the kinetochores or capture microtubules more efficiently, therefore, releasing the timer and shortening mitosis duration in cells where kinetochores attach properly to the spindle microtubules. Interestingly, Nocodazole treatment did not affect CENP-A loading confirming previous observations that kinetochore attachment to the microtubule spindle does not play a role in CENP-A loading. These results are pointing further to an additional function of the SAC independent of the control of microtubule attachment. Interestingly, recent evidence shows that RZZ together with Spindly plays a central role in kinetochore expansion during early mitosis to form a fibrous corona that then compacts upon microtubule capture. Whether and -if so- how the kinetochore expansion by RZZ and spindly is involved in CENP-A loading needs to be investigated in the future (Pauleau, 2019).

    Many essential components of the SAC require outer kinetochore components for their localization to centromeric regions. However, several outer components are missing from the Drosophila kinetochore and even though Mad1/2 recruitment to the kinetochore depends on the RZZ complex, the factors necessary for the localization of the RZZ to kinetochores are unknown. This study has shown that RZZ localization to the kinetochores does not require KNL1Spc105R but depends on the centromeric proteins CAL1 and CENP-A. Therefore, it is proposed that the Drosophila outer kinetochore and components of the SAC assemble through two independent pathways: the CENP-C-KMN-Bub1-Bub3/BubR1 branch or the CAL1-RZZ-Mad1/2 branch. How those two pathways communicate for the formation of MCC complexes remains to be determined. One link may be the KMN complex since Mad2 is diminished in the absence of KMN proteins. Interestingly, Spc105R mutation does not affect SAC function in fly embryos suggesting that flies rely more on the RZZ-Mad1/2 branch to engage the SAC (Pauleau, 2019).

    CENP-A expression and its stability together with its dependence on the low abundant and highly specific loading factor CAL1 and the connection to mitotic events are likely interconnected cellular surveillance mechanisms to avoid misincorporation of CENP-A and, therefore, securing genome stability. How CAL1 itself is regulated to obtain such specificity is currently unknown. In conclusion, this study has shown that there is direct crosstalk between the SAC and the maintenance of centromeric chromatin, ensuring mitotic fidelity not only by controlling microtubule attachment but also by regulating the accurate composition of centromeres (Pauleau, 2019).

    Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function
    The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10, and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the SAC by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. To explore the importance of the Rod_C domain in RZZ function in Drosophila, a series of point mutations was generated in a stretch of 200 residues within this domain, and their phenotypes are reported in this study. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores (Menant, 2020).

    Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing

    During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. This study aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis (Hatkevich, 2019).

    Accurate segregation of homologous chromosomes during the first meiotic division is essential to reestablish the diploid genome upon sexual fertilization. To ensure faithful meiosis I chromosomal segregation, homologs must become physically connected in part through crossover formation. To enable homolog crossover events, a series of chromosomal and cellular events occur in early meiotic prophase I (Hatkevich, 2019).

    During or just prior to the onset of meiosis, homologous chromosomes pair along their entire lengths. Between paired homologs, synapsis, the formation of the synaptonemal complex (SC), ensues. The SC is a tripartite scaffold built between homologs extending the length of the chromosomes and consists of a central region that is nestled between two lateral elements, which are successors of cohesin-based chromosome axes formed between sister chromatids. Coincident with synapsis, DSBs are formed and repaired using a homologous template via homologous recombination (HR), resulting in crossover formation between homologs (Hatkevich, 2019).

    Perhaps the most enigmatic event within early meiosis is the mechanism by which a meiotic chromosome selectively pairs and synapses with its unique homologous partner. Initial homolog pairing is believed to be facilitated through early meiotic chromosome movement and telomere or the centromere clustering. However, how homologous pairing is maintained during synapsis to ensure the SC is formed exclusively between homologs is unknown (Hatkevich, 2019).

    The model organism Drosophila melanogaster has been used to uncover meiotic mechanisms for over a century. In Drosophila, prior to meiosis, chromosomes enter the germline unpaired; throughout the pre-meiotic region, homologous chromosomes gradually pair. In the nuclei at the last mitotic division prior to meiotic onset (in the 8-cell cyst), centromere-directed chromosomal movements occur, presumably ensuring complete homologous pairing. Also during pre-meiotic mitotic cycles, several proteins, including the cohesin SMC1, are enriched at the centromere. The onset of meiotic prophase I occurs in the 16-cell cyst. At zygotene, the first cytologically resolved stage of prophase, centromeres are clustered into 1 or 2 groups, and the SC nucleates in patches along chromosome arms. As zygotene proceeds into early pachytene, the SC extends between paired chromosomes, yielding full-length SC exclusively between homologs. How these early meiotic events, particularly SMC1 enrichment at the centromere and centromere clustering, contribute to meiotic homologous pairing and synapsis in Drosophila is largely unknown (Hatkevich, 2019).

    This study used the Drosophila early meiotic program and a unique genetic mutant to investigate how homolog pairing is maintained during meiotic synapsis. Meiotic homologs in a previously described Drosophila mutant, Mcm5A7, initially pair, but are unable to maintain pairing during synapsis, suggesting that initial meiotic pairing must be subsequently stabilized by an unknown mechanism to ensure proper synapsis. Using Mcm5A7 as a genetic tool to interrogate pairing stabilization mechanism(s), it was found that SMC1 localization at the centromere is compromised, correlating with a severe defect in meiotic centromere clustering and a decrease of crossover formation. However, arm cohesion and SC structure appear unperturbed in these mutants. By overexpressing SMC1, this study shows that the defects in centromere clustering, meiotic homolog pairing, homosynapsis, and crossing over in Mcm5A7 mutants are caused by a lack of centromeric SMC1 localization at meiotic onset. From these results, a model for proper synapsis is suggested in which initial meiotic pairing must be stabilized by centromere clustering, a meiotic event produced by SMC1-enrichment at the centromere and dynamic chromosome movements (Hatkevich, 2019).

    At the beginning of this study, it was hypothesized that the crossover defect in Mcm5A7 mutants was due to a homolog pairing deficiency. FISH results support this hypothesis and revealed that homolog pairing can be lost during synapsis, resulting in seemingly normal SC between heterologous sequences. Centromere-directed chromosome movements occur in Mcm5A7 mutants, presumably to promote initial chromosome arm pairing; however, centromere pairing and clustering are perturbed. SMC1 enrichment at the centromere is decreased in Mcm5A7 mutants, while arm cohesion appears normal. Overexpression of SMC1 rescues centromeric-SMC1 localization and downstream meiotic defects, including centromere clustering, pairing, crossover formation, and segregation. From these data, it is proposed that centromeric SMC1 stabilizes initial homolog pairing through centromere clustering, securing meiotic pairing, ensuring homosynapsis and promoting crossover formation (Hatkevich, 2019).

    Prior to the onset of meiosis, cohesins are loaded onto centromeres, and homologous chromosomes pair, with arm pairing preceding centromere pairing. Initial homolog pairing is achieved in part by centromere-directed movements in the division prior to meiotic onset. A model in which initial homologous chromosomal pairing is stabilized throughout early meiosis by SMC1-dependent centromere clustering (Hatkevich, 2019).

    According to this model, the enrichment of SMC1 at the centromere combined with chromosome movements in pre-meiotic stages yield centromere clustering at meiotic onset. While chromosome arms and centromeres enter meiosis paired, heterologous centromere clustering and/or centromere pairing are required to stabilize pairing during SC assembly. As initial euchromatic SC patches extend along the arms of paired homologs, DSBs are formed and subsequently repaired via HR to yield crossovers, which promote accurate disjunction at the end of meiosis (Hatkevich, 2019).

    In Mcm5A7 mutants, coordinated pre-meiotic centromere-directed movements occur, but a sufficient amount of SMC1 is not localized at the centromere to yield centromere clustering. Thus, at meiotic onset, arms are paired, but centromeres are not clustered. As euchromatic SC nucleation occurs, the stabilization provided by centromere clustering is absent, and homologous loci become unpaired. As synapsis extends, the SC is able to form between nearby chromosomes, regardless of homology, yielding heterosynapsis (intrachromosomal and/or interchromomosomal). DSBs made within regions of heterosynapsis are not repaired via HR due to the absence of an available homologous template. Therefore, crossovers are reduced, and nondisjunction occurs at high frequency in Mcm5A7 mutants (Hatkevich, 2019).

    The SMC1-dependent centromere clustering pairing model highlights the finding that initial meiotic pairing is not sufficient to yield complete homosynapsis. Rather, centromeric SMC1-dependent stabilization must occur after pairing and during synapsis. The inherent requirement of pairing stabilization for proper synapsis suggests that there is a force that opposes homolog alignment during synapsis. Perhaps the SC assembly process itself creates an opposing force that can push paired homologs away from one another in the absence of stabilization; a similar hypothesis was previously proposed in C. elegans. An alternative hypothesis is that recombination, which coincides temporally with synapsis assembly, creates a destabilizing force. However, when meiotic DSBs are eliminated in Mcm5A7 mutants (as shown through mei-P22 Mcm5A7 double mutants), homologs are unpaired at a frequency similar to Mcm5A7 mutants, indicating that the opposing force is independent of recombination. Regardless of the origin of the force, it is proposed that SMC1-dependent centromere clusters act as anchors at the nuclear envelope to maintain the proximity of homologous axes (Hatkevich, 2019).

    Although meiotic pairing programs vary among organisms, the SMC1-dependent centromere clustering pairing model may be broadly applicable. In Drosophila and C. elegans, meiotic pairing is independent of meiotic recombination. In contrast, meiotic pairing in organisms such as yeast, plants, and mice require DSB formation (although recombination-independent alignment is required for pairing in these organisms). In DSB-dependent pairing programs, homologs are considered paired at ~400 nm, where DSB-mediated interhomolog interactions can be visualized as bridges. However, contemporaneous with DSB formation, centromeres are coupled or clustered. It is speculated that these centromere interactions stabilize the DSB-dependent arm pairing to ensure synapsis exclusively between homologs in many sexually-reproducing organisms (Hatkevich, 2019).

    This study reveals the interesting phenomenon of stable heterosynapsis in Drosophila. Extensive heterosynapsis has been previously reported in C. elegans and yeast with variable SC integrity. Though SC aberrations in Mcm5A7 mutants cannot be ruled out, the data reveal no structural defects, supporting the notion that 'normal' synapsis is largely homology-independent in Drosophila, as has been observed in C. elegans (Hatkevich, 2019).

    In Drosophila, synapsis along the arms initiates as patches during zygotene. In Mcm5A7 mutants, synapsis initiation between paired homologs appears normal in zygotene but SC elongation fails to be limited to homologous regions. Thus, initiation of synapsis may require homology, but elongation may not. Because this study examined only specific loci and not whole chromosomes, future studies determining the degree of heterosynapsis in Mcm5A7 mutants may provide more insight into how synapsis and homology interact in flies (Hatkevich, 2019).

    While Mcm5A7 has proven to be a valuable genetic tool, how this particular mutation affects SMC1 localization at the meiotic centromere is unknown. Mcm5A7 mutants do not affect centromere clustering and pairing in a manner similar to that of mutants that disrupt centromere integrity, such as cal1 Cenp-C double heterozygotes. However, the results do not exclude a role for MCM5 in centromere function or integrity (Hatkevich, 2019).

    The canonical role of MCM5 is to function within the replicative helicase, MCM2-7, unwinding double-stranded DNA ahead of the replication fork during S-phase. Because of its important replication role, Mcm5 is an essential gene in every proliferating cell. Numerous studies have shown that replication is required for cohesion localization and establishment, but examining a direct role for any MCM protein in cohesin deposition is difficult since MCMs are essential for replication, which in turn is required for establishing cohesion (Hatkevich, 2019).

    Because MCM5 functions within the MCM2-7 replicative helicase, it is tempting to speculate that the Mcm5A7 mutation may directly perturb SMC1 localization, either through defects in replication or cohesin deposition. No replication defect in Mcm5A7 mutants has been detected, in either a mitotic or meiotic context. In the future, when individual pre-meiotic nuclei can be isolated from cysts, higher-resolution replication assays may determine whether replication is subtly disrupted in Mcm5A7 mutants. At this point, however, it seems more likely that the Mcm5A7 cleanly separates the replication role of MCM5 from a role in meiotic cohesin deposition (Hatkevich, 2019).

    Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration

    Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are post-mitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, post-mitotic RNAi was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, microtubule behavior was analyzed in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of DLK-mediated stress, but was rescued by concurrent reduction of γTubulin, the core microtubule nucleation protein. Reduction of γTubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. It is concluded that kinetochore proteins function post-mitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation (Hertzler, 2020).


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