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. We 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.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.The centromere is an important chromosomal region responsible for correct chromosome segregation during cell division. Centromeres are found in the primary constriction region on the chromosome, upon which the kinetochore complex assembles to produce a platform for spindle binding (1). Centromere function is conserved among different species, and several epigenetic markers of active centromeres have been found, including a histone H3 variant referred to as CENH3 in plants (2, 3) or CENP-A in animals (4–6) and phosphorylation of histone H2A at Thr133 in plants (7). Correct loading of CENH3 to the centromere region is a key component of kinetochore assembly (8). Centromeric DNA sequences have experienced rapid evolution (9, 10), and arrangements of DNA sequence in centromere regions differ in species and even in different chromosomes of an individual organism (11). Myriad repeat sequences exist in the centromeres of higher plants. In maize (Zea mays), there are two major types of centromere specific DNA sequences: the simple satellite repeat sequence CentC (12) and centromeric retrotransposon of maize (CRM) (3). Many epigenetic features have been identified in centromeric regions, including DNA methylation levels, histone variants, histone modifications, and RNA components (11). Both epigenetic elements and DNA sequences take part in centromere formation and maintenance, but it is still unknown how genetic and epigenetic factors work together in this process.The centromere is one of the most complex regions on the chromosome, and complete DNA sequencing through the centromeric region is difficult to obtain due to their highly repetitive nature. Centromere sizes, defined by CENP-A/CENH3 binding regions, range from 125 bp in Saccharomyces cerevisiae to 500–1,500 kb in humans and mice (11). In plants, centromere sizes can range to several megabases (Mb) with many repetitive transposable elements, which makes it difficult to study centromere structure and function. For example, the sizes of centromere 2 and 5 in maize are roughly 2 and 7 Mb, respectively.Previous work sought misdivision derivatives of the B chromosome centromere using a translocation between the supernumerary chromosome and the short arm of chromosome 9 (9S) to reduce the size of the centromere for functional studies (13–15). B chromosomes are extra chromosomes that have been found in many plants, animals, and fungi. In maize, a reciprocal translocation between a B chromosome and the short arm of chromosome 9 produced two chromosomes referred to as B-9 and 9-B (13), together referred to as TB-9Sb. Chromosome 9-B contains the long arm of the B chromosome and most of chromosome 9, including its centromere. Correspondingly, chromosome B-9 contains part of the short arm of chromosome 9 and the other part of the B chromosome with the active B centromere. The translocation breakpoint is near Wx1, which is located on 9-B (16). The B centromere of B-9 can undergo misdivision during meiosis, producing many derivatives (14). The first misdivision derivative was a pseudoisochromosome, and subsequently, many telocentric chromosomes and isochromosomes were derived by additional misdivisions (13, 17). Misdivision events can be recognized in crosses of TB-9Sb onto a tester via a fusion-breakage cycle recognized by the behavior of the C1 color marker on the B-9 chromosome. The cycle continues during endosperm development to produce a mosaic phenotype but is “healed” in the embryo, which when grown and analyzed cytologically will reveal the nature of the new chromosomes formed (13, 17). This type of screen was used to assemble a large collection of misdivisions to examine the structural features of the B centromere (18). Centromere sizes of these derivatives were changed and progressively reduced. In these previous studies, molecular analysis of centromere size relied on studying the B centromere-specific DNA repeat before the maize centromere elements, CentC and CRM, were known. The B-specific repeat allows this centromere to be studied against the background of the other centromeres; it surrounds and is interspersed within the active core of the B centromere (15, 19).New functional centromeres formed at ectopic locations rather than native centromeric regions on the chromosomes are called de novo centromeres. Many de novo centromeres have been found in human patients and other organisms (20, 21). There are reports of de novo centromeres in plants, such as barley (Hordeum vulgare) (22), oat (Avena sativa)-maize addition lines (23), and maize (24, 25). The conditions for de novo centromere formation remain unclear (26). Recent research revealed that many de novo centromeres prefer to form near native centromeric regions or in the heterochromatic regions, such as the pericentromere and telomere (27, 28). There are also de novo centromeres in human formed far from native centromeres (20). We have previously described two de novo centromeres in maize: one is near the position of the native centromere (25) and the other is distal to the site of the corresponding native centromere (24). Specific chromatin environments may be required for centromere formation, but the major elements are as yet unknown.DNA sequence alone is insufficient to direct centromere formation, and dicentric chromosomes containing two centromeres are good examples. To be stable, structurally dicentric chromosomes must have one inactive and one active centromere; otherwise, two active centromeres will lead to chromosome breakage during cell division. In maize, many dicentric chromosomes have been reported from B-A translocation chromosome derivatives (29). Dicentric chromosomes can be produced through the process of the chromosome type breakage-fusion-bridge (BFB) cycle, and the inactive centromeres can be reactivated by intrachromosomal recombination (30). The DNA sequences of the active and inactive centromeres of dicentric chromosomes are essentially identical, but the centromere activity states are completely different. We screened several misdivision derivatives using FISH probes specific to maize centromere sequences, CentC and CRM, as well as probes specific to the B centromere repeat sequence (B-repeat) to gain further insight into the nature of the centromeres in this collection. We discovered that one such chromosome, derivative 3-3, lacks detectable CentC and CRM signals, but still has a functional centromere that is not associated with the B-repeat sequence. The results of chromatin immunoprecipitation sequencing (ChIP-seq) using maize CENH3 antibody revealed that a 288-kb region on 9S is involved in the de novo centromere formation. The functional B centromere of progenitor telo2-2 is deleted from derivative 3-3. Further, new derivatives of derivative 3-3 had been selected (31) but there was no change in the B-specific repeat patterns. Here, we found that the de novo centromere of 3-3 has become inactive in all of its derivatives, and in each case a shift to a new de novo centromere position occurred; one of these contains only a 200-kb CENH3 binding region within 9S. The other two are apparently in B chromosome sequences. Thus, sequential de novo centromere formation and exchange of centromere activity occurred in chromosome 3-3 and its derivatives, providing new insight into centromere formation and maintenance.These results help formulate the nature of de novo centromere formation. In all of the examples now documented in maize, the size range is within a few hundred kilobases. In contrast, normal maize centromeres, as noted above, are typically several megabases. The regular occurrence of de novo centromeres found here and previously (24, 25) indicates that they are capable of being formed regularly on chromosomal fragments that are structurally acentric; however, they do not persist in normal chromosomes. The reason might reside in the previous observation in maize (30) and wheat (32) that in functional dicentrics the smaller centromere becomes inactive in a tug of war between large and small. However, in the absence of a normal centromere, the present work illustrates that de novo centromeres can persist. Thus, in normal chromosomes, if a de novo is initiated, it will be as quickly inactivated in opposition to the much larger preexisting centromere; the chromosome will not be affected, and will seldom change structure over evolutionary time despite such a high rate of de novo formation. This hypothesis also suggests that a selective pressure will be placed on the normal centromeres to expand to a size that can regularly inactivate de novo centromeres based on their initial size at formation.     

From the Cover: PNAS Plus: The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90–100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.Centromeres, the chromosomal domains that attach to spindle microtubules to segregate eukaryotic chromosomes in mitosis and meiosis, are DNA elements bound by special nucleosomes that contain a centromere-specific variant of histone H3 (cenH3). In most plants and animals, cenH3 nucleosomes are found on centromeric DNA that comprises megabases of tandemly repeated “satellite” sequences. Despite this apparent preference for repetitive DNA, a fully functional centromere, called a neocentromere, can occasionally form by assembling cenH3 nucleosomes on a single-copy DNA sequence that was not previously part of a centromere, indicating that centromere specification is epigenetic in plants and animals (for reviews, see refs. 1–4).The tandem arrays of highly repeated satellite sequences that compose most plant and animal centromeres can differ dramatically between closely related species (5), and even between different chromosomes (6–8), suggesting that satellite arrays undergo rapid evolution through expansions, contractions, gene conversions, and transpositions. Monomers of satellite repeats range in length from 5 bp in Drosophila to 1,419 bp in cattle although more than half of described monomers in 282 species have lengths between 100 and 200 bp, often regarded as approximately the length of nucleosomal DNA (6, 9). The cenH3 nucleosomes typically occupy only a portion of the satellite repeats, often in discontinuous blocks (7, 10–12), and the same or similar repeats often underlie flanking pericentromeric heterochromatin composed of conventional nucleosomes. Some of these repeats, for example African green monkey α-satellite DNA, have long been known to position conventional nucleosomes, resulting in arrays of regularly spaced nucleosomes, said to be translationally phased (13–15). Nucleosomes can occupy multiple alternative translational phases on the same satellite (16, 17). Translationally phased nucleosomal arrays have also been observed on satellites in cucumber and in several cereal species, where phasing varies among repeats and chromosomal regions (18, 19).Recently deep-sequencing technology has been applied to centromeres treated with micrococcal nuclease (MNase), which preferentially digests linker DNA between nucleosomes, to determine the positioning of cenH3 nucleosomes on satellite repeats. In human cultured cells, substantial translational phasing of CENP-A, the human cenH3, was reported on α-satellite (20). In maize, a similar approach mapped CENH3 (the name used for plant cenH3s) on the 156-bp maize centromeric satellite CentC and on two retrotransposon-derived centromeric sequences, CRM1 and CRM2 (21). Evidence for translational phasing of CENH3 on CentC and CRM1 was lacking, but 190-bp phasing was observed on CRM2. CentC was shown to have a strong periodicity of AA or TT dinucleotides about every 10 bp, which corresponds to one turn of the DNA double helix. This periodicity is thought to favor a particular orientation of the DNA toward the nucleosome core particle, based on DNA bendability, and is known as rotational phasing of nucleosomes (22–24).Rice has centromeres characterized by the 155-bp satellite sequence CentO, which is related to maize CentC (25, 26). Although some rice centromeres have megabases of CentO satellites, other evolutionarily new centromeres have little CentO, so CENH3 nucleosomes are found on both CentO and non-CentO sequences (12). For example, Cen8 is comprised of mostly non-CentO sequences and has a CentO array (CentO_8) that is spanned by a sequenced BAC (27). Centromeres like Cen8 are thought to represent an intermediate stage in centromere evolution between rare neocentromeres that form on unique sequences and mature centromeres populated by megabase-sized arrays of satellites (7, 12). Cen8 therefore presents an opportunity to compare the organization of CENH3 nucleosomes on CentO and non-CentO sequences. To that end, we used an antibody to rice CENH3 (27) to perform chromatin immunoprecipitation (ChIP) of CENH3 nucleosomes digested with MNase and sequenced the bound DNA (ChIP-Seq) to determine the positions of CENH3 nucleosomes on rice centromeres. We analyzed the sizes and positions of CENH3 nucleosomal DNA fragments on both CentO and non-CentO sequences to address the role of satellites in organizing centromeric chromatin and analyzed the sequence features of these fragments to look for evidence of nucleosome positioning signals.     

Inbreeding drives maize centromere evolution

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000–95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems.Centromere-specific tandemly arranged DNA repeats vary in length and nucleotide sequence between species. The puzzling observation that centromeres can consist of highly variable sequences despite being involved in an essential cellular function (i.e., chromosome segregation) has been coined the “centromere paradox” (1). “Centromere drive” has been proposed to preferentially segregate the “favored” centromere into the female gamete and thereby provide the selective force that acts on centromere DNA sequences and interacting proteins (2).Maize (Zea mays ssp. mays) was domesticated between 7.5 and 10 thousand years ago (ka) from wind-pollinated outcrossing wild teosinte (Z. mays ssp. parviglumis) (3, 4) in a process that dramatically changed its morphology. Several quantitative trait loci (QTLs) responsible for these morphological changes were identified in pioneering work (5–8), and a large number of additional genetic loci involved in maize domestication and improvement were subsequently identified in genome-wide scans (9). Gene (and centromere) flow between the fully interfertile maize and teosinte subspecies has been documented (10, 11). Functional centromeres of maize consist of 1–2 Mb of DNA enriched for the tandemly arranged CentC repeat and members of the centromeric retrotransposon (CR) family (12), which are widely distributed in seed plants and have been extensively characterized (13–18). Elements belonging to the maize CR1, CR2, and CR3 subfamilies have the remarkable ability to target their integration to centromeres and thus mark the historic centromere positions (12).FISH analysis has revealed that most centromeres of teosinte, and all centromeres of other Zea species and the more distantly related genus Tripsacum, contain large amounts of CentC (19, 20), suggesting that CentC-rich centromeres represent the ancestral state. In contrast, centromeres of domesticated maize display a remarkable variation of CentC content in different inbreds (19). Whole genome shotgun sequence of maize and teosinte revealed lower amounts of CentC, but a higher proportion of CR2, DNA in the former (9, 21), suggesting that CR2 is replacing CentC in domesticated corn.Here we detail the processes that result in turnover of centromere repeats at unprecedented temporal and spatial resolution and identify selection for key centromere-linked genes as the driving force. Our analyses strongly suggest that prolonged inbreeding for favorable centromere-linked alleles results in a net loss of the tandem CentC repeat that forces spreading of the cenH3 nucleosomes to an adjacent region, or repositioning to a nearby region. Subsequent invasion of these neocentromeres by the centromere-targeting CR2 element will ultimately result in nested insertions that can give rise to novel tandem centromeric repeats.We describe these events in detail for CEN5, where they are easily observed owing to a tightly linked domestication locus and the acquisition of several CEN5 variants by rare recombinations, but all other chromosomes exhibit the same trends, albeit to lesser degrees. In the case of maize, strong selection for favorable domestication or agronomic alleles drives the turnover of centromere repeats, but selection for any centromere-linked allele that increases fitness may replace centromere repeats in a similar manner in nature during speciation (e.g., Oryza brachyantha CentO-F) (22).     

Bacterial scaffold directs pole-specific centromere segregation

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.The bacterial cytoplasm is a complex mixture of dynamic macromolecules densely packed into a tiny compartment. Recent studies have revealed unexpected levels of organization of bacterial cytoplasmic components, including hundreds of proteins, specific lipids, mRNA molecules, and even the nucleoid itself (1). One strategy used by bacteria to generate subcellular organization of specific macromolecular complexes is active segregation by ParA-mediated molecular partitioning machines. ParA-based partitioning systems are found throughout bacteria and have been shown to spatially organize diverse macromolecular complexes to facilitate their equal distribution to progeny during cell division (2). An important question is how directionality is provided to ParA partitioning machines.One family of highly conserved ParA-based partitioning systems segregates plasmid or chromosomal centromeres to daughter cells during cell division. ParA-mediated DNA partitioning systems (Par systems) are composed of three core components: a centromeric DNA sequence parS, a site-specific DNA binding protein ParB that binds to the centromere parS sequence, and the ATPase ParA. Structural studies demonstrate that the activity of ParA is regulated by a molecular switch in which ATP-bound ParA forms dimers that bind tightly to DNA, and ParB stimulates ATP hydrolysis and release of ADP-bound ParA as monomers (3). During centromere partitioning in vivo, ATP-bound ParA assembles into a multimeric nucleoid-bound structure (4). At the centromere, ParB binds to the parS locus and nearby DNA to create a compact nucleoprotein complex (5). This ParB/parS complex binds to ParA subunits within the ParA/nucleoid structure, stimulating ATP hydrolysis and release of ParA-ADP (6–8). The multivalent ParB/parS complex has thus been proposed to bind to and shorten the ParA superstructure on the nucleoid, moving along a receding track via a dynamic disassembly mechanism (6, 8–10). The result of this process is the movement of the chromosomal centromere (parS) relative to the nucleoid bulk, and therefore to the cell itself.Whereas the fundamental operating principles of ParA-mediated movement seem conserved, how these machines target transfer to specific subcellular destinations is unknown. Many chromosomal Par systems maintain a single origin-proximal ParB/parS complex at the old cell pole and, after replication, move one newly replicated parS locus to the opposite pole (9, 11, 12). Polar protein complexes that interact with chromosome segregation factors have been identified in various bacteria, but the mechanistic consequences of these interactions have not been established (13–15). In Caulobacter, two distinct polar protein factors affect ParA-mediated centromere segregation: the new pole-specific protein TipN (16, 17) and the polar organizing protein PopZ (18, 19). TipN is a large, membrane-anchored, coiled-coil rich protein that localizes to the new pole throughout the cell cycle and, in addition to roles in localization of flagellar synthesis (16, 17), affects processive parS segregation via an unknown mechanism (6, 20).In contrast, PopZ is a small, acidic protein that forms a polymeric network at the cell pole (18, 19). In the prereplicative cell, PopZ localizes exclusively at the old cell pole, where it anchors the ParB-bound parS locus via direct interactions with ParB (18, 19). During chromosome replication initiation, PopZ releases ParB from the old pole and adopts a bipolar PopZ distribution that seems to capture ParB/parS complexes during the segregation process (18, 19). Whereas cells lacking tipN are only mildly elongated, popZ deletion causes severe filamentation (16–19), suggesting that PopZ plays a more important role in the regulation of segregation. However, the molecular mechanism by which PopZ affects segregation has remained elusive.Here we demonstrate that the multifunctional PopZ complex plays a crucial role in pole-directed movement of ParA-mediated chromosome segregation by interacting directly with ParA. We show that PopZ, but not TipN, is required for robust polar recruitment of ParA and demonstrate that a polar PopZ scaffold recruits and concentrates free ParA released during segregation. Recruitment of ParA within the PopZ matrix sequesters free ParA and locally regenerates ParA DNA binding activity. Active ParA complexes are released for recycling into nucleoid-bound structures near the cell pole, which we propose drives centromere segregation toward pole-localized PopZ. Thus, PopZ orchestrates a positive feedback mechanism that forces ParA-mediated centromere transfer to the cell pole. The polar PopZ scaffold complex creates a unique 3D microenvironment at the pole that spatially separates distinct centromere tethering and ParA-modulation activities, enabling coupling between chromosome segregation with the initiation of cell division.     

Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe

Kinetochores in multicellular eukaryotes are usually associated with heterochromatin. Whether this heterochromatin simply promotes the cohesion necessary for accurate chromosome segregation at cell division or whether it also has a role in kinetochore assembly is unclear. Schizosaccharomyces pombe is an important experimental system for investigating centromere function, but all of the previous work with this species has exploited a single strain or its derivatives. The laboratory strain and most other S. pombe strains contain three chromosomes, but one recently discovered strain, CBS 2777, contains four. We show that the genome of CBS 2777 is related to that of the laboratory strain by a complex chromosome rearrangement. As a result, two of the kinetochores in CBS 2777 contain the central core sequences present in the laboratory strain centromeres, but lack adjacent heterochromatin. The closest block of heterochromatin to these rearranged kinetochores is ∼100 kb away at new telomeres. Despite lacking large amounts of adjacent heterochromatin, the rearranged kinetochores bind CENP-A^Cnp1 and CENP-C^Cnp3 in similar quantities and with similar specificities as those of the laboratory strain. The simplest interpretation of this result is that constitutive kinetochore assembly and heterochromatin formation occur autonomously.The fission yeast Schizosaccharomyces pombe is a key system for the experimental study of centromere function. To date, all of this work has involved the strain 968h⁹⁰ and its derivatives 972h⁻ and 975h⁺, each of which contains three chromosomes. Each of these chromosomes contains a kinetochore embedded in heterochromatin (1) and resembles the centromeres of many metazoan organisms; consequently they have been intensively studied. These studies have demonstrated that heterochromatin is necessary for the formation of new kinetochores (2–4), but that once formed, kintetochores (5) can be maintained on a circular plasmid in the absence of heterochromatin. In evolution, novel centromeres form on regions of chromosomes lacking heterochromatin but subsequently acquire it (6, 7), an order of events at odds with the conclusions of published studies of S. pombe. Thus, the relationship between heterochromatin and kinetochore assembly in S. pombe requires investigation using new approaches.A study of 88 independent natural isolates of S. pombe showed that this fission yeast is karyotypically variable, and that one Japanese strain, CBS 2777, contains four chromosomes (8). In the present study, we determined the genome sequence and organization of CBS 2777 and found that it is related to the karyotype of the laboratory strains by a complex rearrangement. We show that two of the centromeres in CBS 2777 lack flanking heterochromatic repeated sequences. This DNA is not detectably associated with siRNA and does not show any detectable enrichment of Rad21 (cohesin) binding. Despite these differences in flanking DNA, the rearranged centromeres in CBS 2777 bind the constitutive centromere proteins Cnp1 (CENP-A) and Cnp3 (CENP-C) similarly to the unrearranged centromere. The simplest interpretation of these results is that constitutive heterochromatin and constitutive kinetochore assembly occur autonomously.Although the difference in genome organization between the laboratory strains and CBS 2777 is complex and resembles the rearrangements seen in fungal plant pathogens (9) and in some types of cancer cells (10), it can be explained by a sequence of simple events.     

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N− media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.Cyanobacteria are phototrophic microbes that bear a Gram-negative cell envelope and are capable of oxygenic photosynthesis. Some cyanobacteria, such as the filamentous Anabaena sp. strain PCC 7120 (hereafter called Anabaena), are capable of fixing atmospheric N₂ when grown in media lacking combined nitrogen. Nitrogen fixation occurs in heterocysts, specialized cells that differentiate from vegetative cells along the filaments and provide a micro-oxic environment for the process (1). One long-standing attraction of Anabaena is its beautiful pattern of differentiation: new heterocysts differentiate midway between two heterocysts as the distance between them doubles due to division of the vegetative cells. This organism, which belongs to one of the first prokaryotic groups on earth to have evolved multicellularity, had to develop structures for intercellular communication. Intercellular communication between heterocysts and vegetative cells comprises small molecules, such as sucrose moving from vegetative cells to heterocysts (2–5) and a dipeptide, β-aspartyl-arginine, moving from heterocysts to vegetative cells (6, 7). The mechanism of communication between heterocysts and vegetative cells has been debated for the last 50 y. Two pathways have been proposed for such exchanges (1, 8–10). One is through the periplasm, suggested by the continuity of the outer membrane surrounding the entire filament (9, 11, 12). The other proposed means of communication requires structures between adjacent cells in the filament. Several structures connecting vegetative cells and heterocysts and vegetative cells with each other have been observed using freeze-fracture, conventional electron microscopy and cryo fixation with electron tomography (13–17). Different names have been given to these structures: microplasmodesmata, septosomes, septal junctions, or nanopores (12, 13, 18, 19). Using cryopreservation combined with electron tomography, we observed structures we call “channels” traversing the peptidoglycan layer in Anabaena (20). These channels are 12 nm long with a diameter of 12 nm, in the septa between vegetative cells. Longer channels, 21 nm long with a similar diameter of 12 nm, were seen in the septa between vegetative cells and heterocysts (20).Several Anabaena gene products were proposed to be involved specifically in intercellular communication. Three were characterized initially from a large set of mutants selected on the basis of their inability to fix nitrogen (21). These mutants manifest a fragmentation phenotype, meaning that they fragment into short filaments upon transfer to liquid medium lacking combined nitrogen, after which they die (15, 22, 23). Further characterization of these mutants led to uncovering a role for several fra gene products in intercellular molecular transfer (23–25).fraC encodes a 179-aa protein with three predicted transmembrane segments; fraD encodes a 343-aa protein with five predicted transmembrane segments and a coiled-coil domain; and fraG (also called sepJ) encodes a 751-aa protein predicted to have an N-terminal coiled-coil domain, an internal linker domain, and a C-terminal permease-like domain with either 10 transmembrane segments (22) or 9 or 11 transmembrane segments (26). fraG deletion prevents heterocyst differentiation and glycolipid layer formation, whereas the deletion of either fraC or fraD allows heterocyst differentiation, but the heterocysts formed show an aberrant neck and do not fix nitrogen (23, 25). Using GFP tags, FraC, FraD, and FraG proteins were shown to be located in the septum between cells (23, 26). FraD was further localized to the septum by immunogold labeling using an antibody raised against the N-terminal coiled-coil part of FraD (25). Fluorescence recovery after photobleaching (FRAP) experiments showed impairment in cell-cell transfer of small molecules such as calcein (622 Da) and 5-carboxyfluorescein (374 Da) in fraC, fraD, and fraG mutants, further indicating a role of these gene products in intercellular communication (23–25).In the work reported here, cryopreservation combined with electron tomography was used to investigate the role of these three fra gene products in channel formation. We found that FraC and FraG are clearly required for channel formation, whereas FraD plays a minor role. Immunogold labeling with antibody to the N-terminal coiled-coil domain of FraG yielded an improved localization for FraG.     

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.Eukaryotes use microtubules for the segregation of replicated DNAs during the fundamental processes of mitosis and meiosis (1). Some prokaryotes use mitosis-like machineries based on different cytomotive filament systems to maintain and segregate plasmid DNA (2, 3). For example, the ParMRC actin-like system, composed of ATPase protein ParM, adaptor protein ParR, and centromeric DNA parC, pushes plasmids to the cell poles by insertional elongation of bipolar ParM spindles, leading to efficient DNA transport and segregation (4–6).Bacillus thuringiensis (7), Bacillus anthracis (8), and Bacillus cereus (9) maintain their large, low-copy-number virulence plasmids using tubZRC loci. TubZRC loci encode the tubulin-like GTPase protein TubZ, adaptor protein TubR, and tubC centromeric DNA repeats, which are located directly upstream of the tubZ and tubR genes on the plasmids.For B. thuringiensis pBtoxis plasmid, it has been shown that the TubZRC maintenance system (referred as “TubZRC”) functions via TubZ filaments whose formation and dynamics are both essential for plasmid stability (10). However, TubZRC alone maintains synthetic plasmids only under artificial selective pressure (7, 11), and replication of pBtoxis occurs without the dnaA-box but not without TubZRC, prompting others to propose a role for the TubZRC system in plasmid replication (12). In vitro, TubZ assembles into two- and four-stranded polymers (13–16), and structural studies suggested that the centromeric TubRC complex forms a ring-like structure (17), interacting with the long C-terminal TubZ extensions (18). Thus, it was proposed that the TubRC complex tracks growing ends of TubZ filaments in analogy to the centromeric complex that follows growing filament ends of the actin-like partitioning systems (5, 6, 19). In bulk assays, TubRC has been reported to enhance TubZ filament formation, possibly indicating a switch in dynamic behavior (17, 20).To uncover how TubR protein and tubC DNA harness TubZ filament dynamics for DNA transport, we probed the ability of the three TubZRC components encoded on plasmid pBtoxis of B. thuringiensis to self-organize in vitro. We used exclusively untagged, full-length proteins, and molecules were labeled with small chemical dyes for detection. Single-filament dynamics of TubZ have not been described before in vitro. Hence, we first characterized growth and shrinkage of individual filaments, because treadmilling has been described for TubZ filaments in cells (10), in contrast to the dynamic instability that is a hallmark of microtubules and ParM filaments (21, 22).     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.