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
  • 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
  • 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
  • Asymmetric assembly of centromeres epigenetically regulates stem cell fate
  • 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
  • Spt6 is a maintenance factor for centromeric CENP-A
  • Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin
  • Kinetochore-independent mechanisms of sister chromosome separation
    Kinetochore in oocytes
  • Multiple pools of PP2A regulate spindle assembly, kinetochore attachments and cohesion in Drosophila oocytes
  • Lateral and end-on kinetochore attachments are coordinated to achieve bi-orientation in Drosophila oocytes
  • Asymmetric assembly of centromeres epigenetically regulates stem cell fate
  • The careful control of Polo kinase by APC/C-Ube2C ensures the intercellular transport of germline centrosomes during Drosophila oogenesis
    Kinetochore in spermatocytes
  • Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes


  • 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).

    Although there is no precedent for core kinetochore proteins functioning outside of chromosome mechanics, several lines of evidence argue that the observed Drosophila phenotypes in neurons are not secondary to chromosome segregation defects. First, errors of kinetochore assembly are lethal during cell division, arresting at the spindle assembly checkpoint, and this would have prevented motor neurons from forming; this study saw no loss of motor neurons in mis12 mutants. Second, were aneuploid or polyploid cells sometimes to escape that lethality, they would do so rarely, randomly, and with heterogeneity in the chromosomes lost. This study, however, detect synaptic phenotypes consistently at the NMJs of all mutant embryos examined. Third, the selective nature of the NMJ defect is difficult to reconcile with chromosomal aberrations: muscles are correctly patterned and have the correct complement; motor neurons are born and target consistently and appropriately to their muscles; and synaptic specializations form with appropriate components, despite the failure to form large boutons. Fourth, Ndc80 immunoreactivity is present at the embryonic NMJ and elsewhere in the nervous system; tagged kinetochore proteins, expressed under control of their normal promoters, were detected outside of nuclei in synaptic and axonal regions. In mammalian neurons, although only mis12 has been examined so far, the function of mis12 is clearly post-mitotic. It was detected by western blot in post-mitotic neurons, and the knockdown of mis12 in post-mitotic hippocampal neurons altered the morphology of hippocampal dendrites. A similar post-mitotic requirement for the proteins of the kinetochore has also been demonstrated in C. elegans, where the degradation of kinetochore proteins selectively in post-mitotic and differentiated sensory neurons disrupts their morphogenesis. Interestingly, there is precedent for the repurposing of other mitotic proteins, such as those of the anaphase-promoting complex, for post-mitotic functions in synaptogenesis (Zhao, 2019).

    This study was fortunate to find the synaptogenic role of mis12 in screen: had the maternal contribution been less, the motor neurons might not have formed, and had the contribution persisted into late-stage embryos, it might have been sufficient fully to support synaptogenesis. The observation that at least eight kinetochore components, including representatives of each of the kinetochore subcomplexes, give rise to the same phenotype at the NMJ suggests that they are functioning in a complex very much similar to that at the centromere. This supposition is further strengthened by the observations that, in several cases examined, the proper localization of one component was altered in a genetic background mutant for another component (Zhao, 2019).

    Although some alleles and RNAi lines did not have a phenotype, their role may have been obscured by the persistence of sufficient maternal contribution, as in the case of Cenp-C, or poor efficacy of the RNAi line. This is particularly true for Nuf2, which was detectable in larval axons although its RNAi line lacked a phenotype. It remains to be determined if the complete complement of kinetochore proteins or only mis12 function in the development of hippocampal dendrites (Zhao, 2019).

    In light of the knockdown phenotype of ndc80, the microtubule-binding subunit, at the embryonic NMJ and its presence in sparse puncta at that synapse, one parsimonious hypothesis is that the kinetochore proteins interact with neuronal microtubules akin to their function at the centromere. Microtubule dynamics are crucial to the formation of both synapses and dendrites, and this is consistent with the observation of significant overextension of both synaptic branches and sensory dendrites in Drosophila and alterations in dendritic morphology in hippocampal neurons. At the embryonic NMJ and in larval nerves, where individual puncta of kinetochore proteins could be resolved, the puncta were sparse, with just one or two per bouton. The axonal puncta in larval nerves were not as bright as those in nearby glial nuclei. Whereas multiple kinetochores and microtubule + ends are present at each centromere, the dim axonal puncta may represent individual kinetochore-like complexes at individual + ends, and this would account for the difficulty of imaging the proteins outside of the densely synaptic neuropil of the ventral nerve cord. In axons, the + ends of axonal microtubules are oriented toward growth cones and synapses, and while microtubules are splayed in growth cones, they are replaced during synaptogenesis by more stable bundles. When microtubules are not appropriately stabilized, synapse formation is perturbed, giving rise to abnormal extensions of axons and improper bouton formation. The phenotypes now reported suggest that this developmental transition requires a kinetochore-like complex. Hippocampal dendrites contain microtubules of both polarities and the increased filopodia-like protrusions that appear upon the knockdown of mis12 may arise from misorganization or overgrowth of dendritic microtubules. Future studies will need to clarify how the kinetochore influences microtubule organization. Analysis of kinetochore mutations has thus uncovered a previously unknown mechanism that appears to co-opt the fundamental mitotic functions of the ancient kinetochore complex for non-mitotic functions in both invertebrate and vertebrate neurodevelopment. A deeper understanding of these synaptogenic functions should therefore illuminate a process central to the accurate wiring of the brain (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).

    Asymmetric assembly of centromeres epigenetically regulates stem cell fate

    Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

    Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

    In this study, a detailed characterization was carried out of centromere dynamics throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model used in this study, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

    According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase]. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (1) a rapid pulse during G1 in mitotically dividing cells; and (2) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results of this study also support this model (Dattoli, 2020).

    Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals. Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase, further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

    According to the results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (1) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (2) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids. The current data confirm this model, as symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, this study observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2%-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported . This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

    These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In the favored scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be ruled out that the effects on cell fate observed in this functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

    Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

    Ultimately, these results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, the data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

    The careful control of Polo kinase by APC/C-Ube2C ensures the intercellular transport of germline centrosomes during Drosophila oogenesis

    A feature of metazoan reproduction is the elimination of maternal centrosomes from the oocyte. In animals that form syncytial cysts during oogenesis, including Drosophila and human, all centrosomes within the cyst migrate to the oocyte where they are subsequently degenerated. The importance and the underlying mechanism of this event remain unclear. This study shows that, during early Drosophila oogenesis, control of the Anaphase Promoting Complex/Cyclosome (APC/C), the ubiquitin ligase complex essential for cell cycle control, ensures proper transport of centrosomes into the oocyte through the regulation of Polo/Plk1 kinase, a critical regulator of the integrity and activity of the centrosome. This study shows that novel mutations in the APC/C-specific E2, vihar/Ube2c, that affect its inhibitory regulation on APC/C cause precocious Polo degradation and impedes centrosome transport, through destabilization of centrosomes. The failure of centrosome migration correlates with weakened microtubule polarization in the cyst and allows ectopic microtubule nucleation in nurse cells, leading to the loss of oocyte identity. These results suggest a role for centrosome migration in oocyte fate maintenance through the concentration and confinement of microtubule nucleation activity into the oocyte. Considering the conserved roles of APC/C and Polo throughout the animal kingdom, these findings may be translated into other animals (Braun, 2021).

    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).

    The kinetochore is a quintessential mitosisand meiosis-specific structure that attaches chromosomes to the mitotic spindle for segregation of genetic material to daughter cells. In many species, including Drosophila, it is built on centromeric DNA that is recognized by the histone Cid/Cenp-A that recruits inner kinetochore protein Cenp-C to serve as a binding site for the KMN network (Knl1, Mis12, and Ndc80 complexes). Of these, the Mis12 complex is most central, while the remainder make up more distal parts of the structure that is involved in connecting to microtubules. Many animals have other inner kinetochore proteins, including the constitutive centromere-associated network (CCAN), but these are absent in Drosophila (Drinnenberg, 2016). In cycling mammalian cells, inner centromere proteins including Cenp-A, Cenp-C, and CCAN are found on the centromere in interphase as well as mitosis. The Mis12 complex has some nuclear staining in interphase, but it is not as clearly punctate as CENP-A. In Drosophila, Cenp-A/Cid, Cenp-C, and Mis12 localize to centromeres in interphase and mitosis. In mammals and Drosophila, the other kinetochore components including the Knl1 and Ndc80 complexes are recruited to the kinetochore only in mitosis. Once the kinetochore is fully assembled, Nuf2 and Ndc80 in the outer kinetochore capture microtubule plus ends growing into the central spindle to allow chromosome segregation (Hertzler, 2020).

    In addition to the kinetochore proteins that bridge chromosomes to microtubules, several regulatory complexes are present at the kinetochore before final bioriented microtubule attachment. The chromosome passenger complex (CPC) targets the kinase Aurora B to the centromere, where it helps correct errors in chromosome attachment to microtubules by phosphorylation of substrates including Ndc80. The targeting subunits of the CPC are borealin (or in flies borealin-related, borr), survivin (or in flies Deterin, Det), and Incenp, which are tightly associated with one another through a three-helix bundle. Aurora B is also critical for recruiting spindle assembly checkpoint (SAC) proteins to unattached kinetochores. The major effector of the SAC is cdc20/fzy, which is a subunit of the anaphase-promoting complex or cyclosome (APC/C). When bound by SAC subunits at the kinetochore, cdc20/fzy is inactive. A third complex, Rod1-Zw10-Zwilch (RZZ) complex, interacts with both SAC and KMN proteins. Together these regulatory complexes ensure that anaphase does not begin until all sister chromatids are correctly attached to opposite spindle poles. Attachment causes a shift in kinase and phosphatase balance, such that Aurora B starts to lose, and 'stripping' of the SAC by dynein connected to the RZZ complex is initiated. As a result, cdc20/Fzy is freed to activate APC/C, the ubiquitin ligase that triggers degradation of Cyclin B and Securin to initiate sister chromatid separation. KMN network proteins remain at the kinetochore to mediate attachment of separating sister chromatids to spindle microtubules (Hertzler, 2020).

    Most of the proteins that make up the kinetochore and its associated regulatory complexes have not been linked to functions in interphase or postmitotic cells. However, two recent studies demonstrated neuronal defects after postmitotic reduction of kinetochore proteins, suggesting that they do function in noncycling cells (Cheerambathur, 2019; Zhao, 2019). In Caenorhabditis elegans, KMN network components localize to ciliated dendrites of amphid neurons and play a role in their extension (Cheerambathur, 2019). Although the microtubule-binding domains of Ndc80 are required to support normal amphid development, specific defects in microtubules were not detected (Cheerambathur, 2019). In Drosophila, mis12 mutants were identified in a genetic screen to identify modulators of synaptic bouton structure (Zhao, 2019). Reduction of other KMN network proteins had similar effects on neuromuscular junction structure, and intriguingly, so did targeting the centromeric histone cid/CENP-A (Zhao, 2019). Knockdown of Mis12 in primary rodent hippocampal neurons increased the number of protrusions from dendrites (Zhao, 2019), indicating that kinetochore proteins likely function in neurons broadly across evolution. Specific alterations in the neuronal cytoskeleton were not reported in this study either. It therefore remains to be determined how kinetochore proteins impact neuronal structure (Hertzler, 2020).

    One of the most intense structural challenges neurons face is rebuilding axons or dendrites after injury. Key regulators of axon regeneration have been identified, including sensors like the dual leucine zipper kinase (DLK), epigenetic regulators like HDAC5, transcription factors including fos, jun, and cebp-1, and a myriad of downstream regeneration-associated genes (RAGS). However, it was only recently shown that neurons survive removal of all dendrites and can regrow a new arbor. Only a handful of genes including Akt and Ror have been linked to dendrite regeneration. To identify dendritic RAGs, RNA was isolated from Drosophila neurons after in vivo dendrite removal. Surprisingly, many kinetochore gene transcripts were coordinately up-regulated 6 h after dendrite injury. These included not only transcripts encoding structural kinetochore proteins in the inner kinetochore and KMN network, but also transcripts encoding regulatory proteins in the CPC, SAC, and RZZ complexes. Postmitotic RNA interference (RNAi) was used to confirm a role of some of these proteins in dendrite regeneration. In the same genetic backgrounds, no effect was seen on axon regeneration. In uninjured neurons, kinetochore protein reduction specifically increased microtubule plus-end number (also referred to as microtubule dynamics) in dendrites without altering other metrics of microtubule behavior such as polarity, speed, or minus-end density. Unexpectedly, axonal microtubule dynamics was unaffected. Therefore, kinetochore proteins act in a compartment-specific manner to suppress microtubule dynamics in dendrites. The dendritic microtubule phenotype and dendrite regeneration defect were rescued by concurrent reduction of γ-tubulin, suggesting that kinetochore proteins normally function to temper microtubule nucleation in dendrites. Together the data suggest that the KMN network and CPC, SAC, and RZZ complexes function together to promote dendrite regeneration and modulate dendritic microtubule dynamics through control of nucleation in postmitotic neurons (Hertzler, 2020).

    On the basis of the surprising appearance of transcripts of structural and regulatory kinetochore proteins in RNASeq data from injured neurons, their role in dendrite regeneration was investigated. Regeneration after dendrite, but not axon, injury was impaired when they were reduced using postmitotic RNAi. Because kinetochore proteins function to attach, and monitor attachment, to microtubules in mitotic spindles, microtubule behavior was surveyed in neurons in which they were reduced. RNAi knockdowns of many kinetochore proteins caused an up-regulation of dendritic microtubule dynamics, an effect not seen in axons. Other parameters of microtubule behavior, including number of growing minus ends, were not affected. The increase in plus-end, but not minus-end, number suggested that nucleation rather than severing was responsible. The involvement of nucleation was confirmed by rescue of the plus end and regeneration phenotypes in neurons with reduced γ-tubulin. The increase in microtubule dynamics when kinetochore proteins were reduced suggests that they normally function to dampen nucleation of dendritic microtubules in postmitotic neurons (Hertzler, 2020).

    Two recent studies indicate that kinetochore proteins are likely to function broadly in different neuron types. In one study, the authors identified Mis12 mutants in a Drosophila forward genetic screen. Other components of the KMN network complexes (Ndc80, Knl1, and Nnf1a), as well as centromeric protein Cenp-A (Cid), had similar neuromuscular junction phenotypes and reduced neuropil in the CNS (Zhao, 2019). The authors went on to show that mammalian neurons with reduced Mis12 also had structural defects, this time in dendrites (Zhao, 2019). The group reported some localization of KMN network proteins to spots in peripheral nerves and neuropil in the CNS (Zhao, 2019), but the pattern did not help to suggest how the proteins might function in neurons (Hertzler, 2020).

    The other study demonstrated that Cenp-C, Ndc80, and Nuf2 localize postmitotically to the ciliated dendrites of amphid neuron bundles in C. elegans. When these proteins were knocked down with an elegant GFP-degrader system, amphid dendrite extension was impaired (Cheerambathur, 2019). Effects on egg laying and fertility were also seen. Notably, a deletion of only the microtubule-binding domains of Ndc80 was able to phenocopy the knockdowns, indicating that an interaction with microtubules likely mediates the phenotypes. Degradation of GIP2, an essential γTuRC subunit, showed the same neuron extension deficits, supporting the hypothesis that microtubules in these neurons are affected by KMN network protein knockdown, although the authors were unable to pinpoint any specific changes in the microtubule cytoskeleton (Hertzler, 2020).

    The two previous studies on neuronal roles for kinetochore proteins focused on the structural components of the kinetochore, including inner centromere components Cenp-A and C and KMN network proteins (Cheerambathur, 2019; Zhao, 2019). The three regulatory complexes-CPC, RZZ, and SAC- are added to the list of kinetochore proteins with neuronal function. Moreover, it was found that players in all structural and regulatory complexes have similar roles in controlling microtubule dynamics. This suggests that, as in mitosis, these complexes function together in a single pathway in neurons. It is therefore likely that they are not only involved in attaching microtubules to something, but are also acting as sensors of microtubule behavior. If they have a similar role in neurons as in mitosis, they may recognize plus-end arrival at a specific cellular location. Again, in analogy with mitosis, plus-end arrival could trigger release of regulatory complexes and free them to send signals to other cellular locations about the status of the microtubule cytoskeleton. These signals could involve ubiquitination mediated by association of Cdc20/Fzy with an E3 ligase complex, or phosphorylation by Aurora B. One target of either output signal could be suppression of γTuRC activity (Hertzler, 2020).

    One intriguing aspect of these findings is that changes were only observed in the microtubule cytoskeleton in dendrites, and dynamics in the axon was unaffected. While phenotypes in C. elegans and mammalian neurons occurred in dendrites, changes in motor axon terminals were seen in Drosophila. One possible way to reconcile the axonal and dendritic phenotypes would be if kinetochore proteins are important throughout dendrites but function specifically at synaptic regions of axons. There are several hints that microtubule nucleation might be particularly important near presynaptic sites. In Drosophila motor neurons, concentrations of γ-tubulin were seen in large synaptic boutons at the neuromuscular junction. In cultured mammalian neurons, γ-tubulin also concentrates at presynaptic sites, and increases in neuronal activity lead to increases in EB comet formation at these sites, suggesting that regulation of nucleation is important in axons. It is therefore possible that kinetochore proteins regulate presynaptic nucleation in addition to dendritic nucleation (Hertzler, 2020).

    In mitotic cells, kinetochore proteins localize to tight spots around centromeric DNA, and their localization has provided invaluable clues to their function. So far, localization patterns in neurons have not helped pin down specific sites of action. In C. elegans, expression levels in amphid neurons were high enough to detect tagged proteins expressed at endogenous levels, and they were seen throughout the linear ciliated dendrite region (Cheerambathur, 2019). In contrast, expression seems low in Drosophila neurons, and beyond being present in neuropil, a pattern has not been discernible (Zhao, 2019). Attempts of this study to visualize meaningful localization in sensory neurons also did not provide any insights. Use of amplification systems like SunTag may be required to acquire meaningful information about where structural and regulatory kinetochore proteins function in neurons (Hertzler, 2020).

    One possible model for neuronal kinetochore protein function is that, in analogy with mitosis, the structural and regulatory complexes colocalize at a specific cellular site, perhaps in the cell body, when microtubule plus ends are absent or in low abundance. Upon plus-end arrival, the RZZ complex in conjunction with dynein could transport regulatory proteins toward microtubule minus ends. Dendrites contain minus-end-out microtubules, so this would mean that regulatory proteins could be transported outward into dendrites to suppress microtubule dynamics distally after plus-end arrival. This type of mechanism could allow for global homeostatic control of microtubule dynamics in dendrites (Hertzler, 2020).

    Asymmetric assembly of centromeres epigenetically regulates stem cell fate

    Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

    Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

    A key player in centromere assembly in vertebrates is HJURP (holliday junction recognition protein), which localizes at centromeres during the cell cycle window of CENP-A deposition. Furthermore, centromere assembly is regulated by the cell cycle machinery. In flies, deposition of CID (the homologue of CENP-A) requires activation of the anaphase promoting complex/cyclosome (APC/C) and degradation of CYCLIN A (CYCA). In humans, centromere assembly is antagonized by Cdk1 activity, while the kinase Plk1 promotes assembly. Additionally, the CYCLIN B (CYCB)/Cdk1 complex inhibits the binding of CENP-A to HJURP, preventing CENP-A loading at centromeres. To date, little is known about centromere assembly dynamics and functions in stem cell asymmetric divisions. Drosophila melanogaster ovaries provide an excellent model to study stem cells in their native niche. In this tissue, germline stem cells (GSCs) are easily accessible and can be manipulated genetically. Moreover, centromere assembly mechanisms in GSCs and their differentiated cells, cystoblasts (CBs), could be used to epigenetically discriminate between these two cell types. In Drosophila, CID binds to CAL1 (fly functional homologue of HJURP) in a prenucleosomal complex, and its localization to centromeres requires CAL1 and CENP-C (Dattoli, 2020).

    This study investigated the dynamics of CENP-A deposition in Drosophila GSCs. GSC centromeres are assembled after replication, but before chromosome segregation, with neural stem cells following the same trend. Centromere assembly in GSCs is tightly linked to the G2/M transition. Indeed, CYCA localizes at centromeres, and its knockdown is responsible for a marked reduction of centromeric CID and CENP-C, but not CAL1. Surprisingly, excessive CID deposition is prevented by CYCB, through the kinase HASPIN. Superresolution microscopy analysis of GSCs at prometaphase and metaphase shows that CID incorporation on sister chromatids occurs asymmetrically, and chromosomes that will be inherited by the stem cell are loaded with more CID. Moreover, GSC chromosomes make stronger kinetochores, which anchor more spindle fibers. This asymmetric distribution of CID between GSC and CB is maintained also at later stages of the cell cycle, while it is not observed in differentiated cells outside of the niche. This study also found that the depletion of CAL1 at centromeres blocks GSC proliferation and differentiation. Notably, overexpression of both CID and CAL1, as well as HASPIN knockdown, promotes stem cell self-renewal and disrupts the asymmetric inheritance of CID. Conversely, overexpression of CAL1 causes GSC-like tumors. Finally, CAL1 and CID knockdown at later stages of egg development have no obvious effect on cell division, suggesting that these cells inherit CID from GSCs. Taken together, these findings establish centromere assembly as a new epigenetic pathway that regulates stem cell fate (Dattoli, 2020).

    In this study a detailed characterization of centromere dynamics was performed throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, this study shows that overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

    According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (a) a rapid pulse during G1 in mitotically dividing cells; and (b) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results also support this model (Dattoli, 2020).

    Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with the current functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals (Stankovic, 2017). Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase (Ranjan, 2019), further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

    According to the current results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (a) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (b) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids. The current data confirm this model, symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, it was observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported. This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

    These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In a favorite scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be rule out that the effects on cell fate observed with the functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

    Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

    Ultimately, the results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, these data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

    Spt6 is a maintenance factor for centromeric CENP-A

    Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. This study demonstrates that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. It is concluded that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling (Bobkov, 2020).

    The CENP-A nucleosome is considered to be the key epigenetic mark for centromere identity in most organisms. Accordingly, CENP-A and epigenetic marks in general should meet three requirements: (1) Template its own deposition, (2) be replenished in a cell cycle-controlled manner to counteract dilution by half in each S-phase and (3) be stably transmitted to the next cell generation (Bobkov, 2020).

    New dCENP-A can be targeted to sites of previous CENP-A deposition by its chaperone CAL1, which is recruited to centromeres by dCENP-C. Loading of new CENP-A is restricted to mitosis and G1 and serves primarily to replenish CENP-A containing nucleosomes that became diluted by half during the preceding S-phase. During DNA replication the MCM2-7 replicative helicase along with other histone chaperones like HJURP, are instrumental for the stable transmission of parental CENP-A during S-phase (Bobkov, 2020).

    Recent work has shown in Drosophila S2 cells that transcription at the centromere is required for stable nucleosome incorporation of new dCENP-A9. This finding could be explained by a model in which transcription-mediated chromatin remodeling is re-purposed to evict placeholder H3 nucleosomes to make room for deposition of new dCENP-A. However, the induction of nucleosome eviction during CENP-A loading also bears the danger of losing previously incorporated CENP-A. This study reports the identification of the transcription elongation factor and histone chaperone Spt6 as a new CENP-A maintenance factor, which safeguards previously deposited CENP-A during centromeric transcription (see Model showing histone dynamics during dCENP-A loading) (Bobkov, 2020).

    Drosophila Spt6 localizes to centromeres during mitosis and G1, coinciding with the time window when transcription and dCENP-A loading occurs. The SH2 domain enables Spt6 to interact directly with RNAPII and it is therefore likely that recruitment of Spt6 to centromeres is a direct consequence of centromeric transcription. Because Spt6 prevents transcription-coupled loss of posttranslationally modified nucleosomes in gene bodies, whether Spt6 might act to maintain dCENP-A at the centromere was tested. Indeed, when Spt6 was depleted in Drosophila or human cells, the specific loss of old CENP-A was observed after passage through mitosis into G1 phase. This observation suggests that ongoing transcription evicts nucleosomes at centromeres and that Spt6 serves a conserved role to recycle CENP-A/H4 tetramers expelled by closely spaced polymerase complexes. A key point of this model is the transcription-mediated creation of nucleosomal gaps as a prerequisite for full incorporation of new dCENP-A. Consequently, the additional loss of nucleosomes in Spt6-depleted cells should create more opportunities to load new dCENP-A. Indeed, when an experimental system was used that provides elevated levels of transgenic, ready-made dCENP-A (TI-dCENP-AHA), a clear increase was observed in loading. This is further supported by the fact that the loss of total centromeric dCENP-A in Spt6-depleted cells is completely compensated under these conditions (Bobkov, 2020).

    It is currently unknown if the mitotic defects observed upon Spt6 depletion by RNAi are a direct or indirect consequence of Spt6 removal. As cells can tolerate very low CENP-A levels at the centromere down to 10%, the 2-day depletion of Spt6 likely leaves sufficient dCENP-A for centromere function. Despite this, chromosome segregation might be compromised due to the specific loss of old nucleosomes with specific PTMs. PTMs relevant for centromere function have been identified on CENP-A and shown to affect CENP-A stability and correct mitotic progression. Moreover, methylation of lysine 20 on the associated H4 plays an essential role for kinetochore formation. Likewise, in addition to CENP-A nucleosomes, centromeres contain canonical H3 nucleosomes with a specific set of posttranslational modifications that might need to be retained. It is therefore postulated that Spt6 should be able to distinguish between placeholder nucleosomes that need to be removed and epigenetically marked nucleosomes that should be kept. As previously demonstrated for H3/H4 in budding yeast39, direct binding of a bacterially expressed N-terminal fragment of Spt6 (199-338) was observed with both H3/H4 and dCENP-AΔNT/H4 tetramers. In addition, full length dCENP-AFLAG and H3 co-IP with endogenous Spt6 from S2 cell extracts with comparable efficiency (Bobkov, 2020).

    Interestingly, CENP-A is phosphorylated in various organisms including flies and humans and phosphorylation events have been linked to transcription-induced loss of centromeric CENP-A nucleosomes in mouse cells. To test whether phosphorylation of dCENP-A affects its maintenance, three previously identified phosphorylation sites were mutated in the N-terminal tail of dCENP-A (S20, S75 and S7757). Indeed, it was found that dCENP-A mutants carrying the phosphomimetic residue aspartate showed significantly reduced binding to Spt6, while the opposite was observed for the respective non-phosphorylatable alanine mutants. Furthermore, wild-type or non-phosphorylatable mutants of dCENP-A bound robustly to Spt6 when exposed to high salt washes while canonical H3 binding was abolished. This difference hints toward a mechanism how Spt6 distinguishes between the two histone H3-variants and allows selective retention of CENP-A, while placeholder nucleosomes are exchanged. Consistent with the observations described above, a pulse-chase experiment to follow the decline of old dCENP-A during cell division showed higher loss rate for the phosphomimetic dCENP-A construct. Interestingly, dCENP-A wild-type displayed less than perfect inheritance after two cell cycles (<25% expected for replicative dilution). In contrast S77A was on average more and S77D less stable than wild type, likely accounting for the accumulation of the non-phosphorylatable dCENP-A mutant at centromeres over time (Bobkov, 2020).

    Taken together, it is proposed that the transcription-mediated eviction of centromeric nucleosomes affects both placeholder H3 and previously deposited CENP-A nucleosomes. However, loss of the centromeric mark is prevented by specific recycling of CENP-A through Spt6, potentially involving phospho-regulation of the CENP-A/Spt6 interaction. It is concluded that Spt6 acts as an important CENP-A maintenance factor and contributes to the long-term stability of the epigenetic centromere mark (Bobkov, 2020).

    Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

    Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. This study investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, this study reveals a hierarchical organization of the PCH domains into distinct 'territories.' Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. These findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin (Lee, 2020).

    An appreciable fraction of most eukaryotic genomes comprises constitutive heterochromatin, which is enriched for megabases of repetitive DNA localized predominantly around centromeres (PCH). However, because of technical difficulties associated with repetitive DNA, a global and in-depth understanding of the 3D organization of the PCH domain, which encompasses at least a fifth of the human and a third of the D. melanogaster genomes, is lacking. This study provides a comprehensive and detailed picture of the 3D organization of PCH domains in D. melanogaster by combining genome-wide Hi-C analyses and cytological FISH studies. A novel analysis approach was developed that overcomes the challenges posed by repeated DNAs when determining 3D contact frequencies from Hi-C reads. Specifically, the single-locus mapping restriction was relaxed to include reads originating from the abundant repetitive DNA in PCH and different combinations of PCH reads (single-locus mapping or not) were used depending on the question being addressed. These investigations reveal significant, new insights into the interactions between different PCH regions and their 3D contacts with the euchromatic genome (Lee, 2020).

    The coalescence of PCHs on different D. melanogaster chromosomes contributes to the formation of a large PCH domain in 3D nuclear space. However, DNA contacts within the PCH domain are far from homogeneous. Hi-C analysis reveals the strongest interactions (~98%) involve PCH regions on the same chromosome arm (e.g., 2L), suggesting PCH regions from each arm are organized into distinct 'territories'. This is similar to identified chromosome territories for the euchromatic genome. It is clear from both the fusion of multiple PCH domains from different chromosomes and from Hi-C and FISH analyses presented in this study that PCH regions from all the chromosomes do interact. However, some interactions occur more often than random, in particular the inter-arm (2L-2R, 3L-3R) and specific inter-chromosomal (3L/3R-4) 3D associations. Most strikingly, ~14% of identified H3K9me2-enriched regions in epigenomically defined euchromatin display preferential 3D contacts with the central PCH domains. Quantitative FISH analysis further provides cytogenetic support for the Hi-C results. The bimodal distributions of PCH-PCH or EU-PCH distances in nuclei demonstrate that these 3D contacts are dynamic and can vary among cells, similar to what has been previously shown for the euchromatic Hox loci in mouse. Importantly, polymorphic TE insertions in euchromatin allowed direct comparison of homologous sequences with and without H3K9me2 enrichment, which strongly supports the conclusion that H3K9me2 enrichment is required for EU-PCH 3D contacts (Lee, 2020).

    Overall, the Hi-C and FISH analyses reveal a previously unknown picture of the 3D architecture of the PCH domains: the spatial interactions within the domains, instead of being random, are hierarchical. In addition, despite the separation of euchromatic and PCH territories on the same chromosome arm, short stretches of H3K9me2/3 enrichment in the euchromatic genome (with and without TEs) also dynamically interact with the main PCH domains. Both PCH-PCH and EU-PCH interactions happen most often within chromosome arms, which is consistent with the predictions of polymer physics on chromosome folding. Importantly, the tendency of H3K9me2 islands to interact with PCH strongly depends on their distance to PCH on a linear chromosome. This suggests that euchromatic regions and PCH could be in spatial proximities transiently with a frequency that largely follows the polymer physics of chromosome folding. The enrichment of H3K9me2/3 and the reader protein HP1a at specific euchromatic loci would then inevitably lead to their liquid-like fusion with HP1a-enriched PCH, resulting in frequent and/or maintained EU-PCH 3D interactions. Alternatively, this association with PCH may be an active process, regulating gene expression in specific subsets of cells. Indeed, in mice, the spatial clustering of olfactory receptor genes into heterochromatin domains silences all except for one receptor gene that spatially loops out from the cluster (Lee, 2020).

    The observed specific spatial contacts between PCH regions located on different chromosomes are surprising, but nevertheless consistent with the coalescence of PCH of all chromosomes into chromocenters. The varying frequencies of inter-chromosomal interactions could result from non-random positioning of PCH regions upon mitotic exit or constraints imposed by other nuclear structures. For example, nucleoli, whose formation is driven by the transcription of rDNA arrays on the X chromosome, may impose structural constraints that lead to less frequent than expected spatial contacts involving X PCH. In addition, variation in biophysical properties (e.g., viscosity or varying protein compositions) among PCH domains arising from specific chromosomes could result in different frequencies of liquid-liquid fusion. Indeed, the 4th chromosome has a unique composition of histone modifications and chromatin proteins and depends on a specific suite of genes for its regulation (e.g., requirement of Egg for histone methylation), both of which could result in biophysical properties that promote frequent 3D contacts between 4th chromosome and specific PCH regions (Lee, 2020).

    Importantly, the population genetic analysis reveals that euchromatic TEs with PCH interactions have lower population frequencies than TEs lacking frequent PCH contacts, suggesting that EU-PCH 3D interactions may influence individual fitness. What are the potential functional consequences of TE-PCH interactions that could influence individual fitness? TE-PCH interactions could lead to increased TE-induced enrichment of repressive epigenetic marks on neighboring sequences/genes. However, this study found no difference in the extent or the magnitude of H3K9me2 spread around TEs with and without PCH interactions, suggesting that TE-PCH interactions influence other aspects of nuclear organization critical for gene regulation and/or other genome functions. For instance, 3D interactions between PCH and TEs could bring neighboring euchromatic genes into the PCH domains and result in aberrant or enhanced silencing. On the other hand, the enrichment of HP1a, and likely spatial localization in the PCH domains, can promote the expression of genes in both PCH and the euchromatic genome. Still another possibility is that the spatial contact with PCH on one chromosome may 'drag' its homolog to the same nuclear compartment due to somatic homolog pairing, resulting in trans-silencing. A preliminary analysis found that ~15% of heterozygous TEs induced H3K9me2 enrichment not only in cis, but also in trans on the homologous chromosome without the TE insertion (i.e., trans-epigenetic effects). Accordingly, the fitness consequences of TE-PCH spatial interactions could potentially result from their positive or negative impacts on the expression of genes in cis or in trans to TEs, or from influencing other genome functions, such as replication and repair. Further studies are needed to test these hypotheses (Lee, 2020).

    It is important to note that TEs comprise an appreciable fraction of the euchromatic genomes of virtually all eukaryotes. For instance, more than 50% of assembled human euchromatin contains TEs or TE-derived sequences, many of which are interspersed with actively transcribed genes and can influence gene expression through H3K9me2/3 spreading. Moreover, the presence of many TE insertions at specific locations are polymorphic between individuals in natural populations (e.g., human, Caenorhabditis, Drosophila, and Arabidopsis). Spatial interactions between euchromatic TEs and PCH can thus generate polymorphic 3D organization of the euchromatic genomes, leading to variation in critical biological functions that depend on chromosome conformations and even varying fitness between individuals. This investigation of the spatial architecture of PCH domains could thus have strong implications for how such 3D organizations could influence gene regulation, genome function, and even genome evolution of both heterochromatin and the gene-rich euchromatin (Lee, 2020).

    Multiple pools of PP2A regulate spindle assembly, kinetochore attachments and cohesion in Drosophila oocytes

    Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, this study examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. Both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. At least three populations of PP2A-B56 regulate meiosis are proposed, two of which depend on SPC105R and a third that is associated with the spindle (Zhang, 2021).

    Vicars, H., Karg, T., Warecki, B., Bast, I. and Sullivan, W. (2021). Kinetochore-independent mechanisms of sister chromosome separation. PLoS Genet 17(1): e1009304. PubMed ID: 33513180

    Kinetochore-independent mechanisms of sister chromosome separation

    Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, this study discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, 117 RNAi gene knockdowns were screened for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on this analysis of EB1 localization and knockdown phenotypes, it is proposed that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation (Vicars, 2021).


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