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In this chapter we describe protocols for immunolabeling and FISH of pollen grains undergoing postmeiotic mitosis using Aegilops speltoides, Secale cereale, and Hordeum vulgare as models. Tissue sectioning of pollen overcomes the problem... more
In this chapter we describe protocols for immunolabeling and FISH of pollen grains undergoing postmeiotic mitosis using Aegilops speltoides, Secale cereale, and Hordeum vulgare as models. Tissue sectioning of pollen overcomes the problem of the pollen grain wall impermeability that interferes with immunolocalization and in situ hybridization. The crucial element of the protocol is the generation and immobilization of tissue sections. Our method facilitates the investigation of the microspore cell divisions and pollen grain maturation.
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Research Interests: Genetics, Biology, Cell Cycle, Medicine, Cell Division, and 15 morePlant evolution, Plant Genome Project, Cell Differentiation, Mutation, Gametophyte, Life Cycle, Gene Dosage, Arabidopsis, Retinoblastoma, Cell Cycle regulation, PLoS Genetics, Feedback loop, Cell Proliferation, Cell Fate, and Genome Duplication
A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched... more
A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.
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Research Interests: Chemistry, Electron Microscopy, Medicine, Cartilage, Collagen, and 15 moreFemale, Animals, Connective tissue, Periodontal Ligament, Glycosaminoglycan, Clinical Sciences, Cattle, Proteoglycans, Electrolytes, Molecular weight, Organometallic Compounds, Collagen Fibril, Biochemistry and cell biology, Cardiovascular medicine and haematology, and coloring agents
Research Interests: Chemistry, Electron Microscopy, Molecular Biology, Biology, Medicine, and 15 moreGene expression, Biological Sciences, Cell line, Humans, Animals, Polymerase Chain Reaction, Capsid, Recombinant DNA, Viral Infection, Base Sequence, Full Length Movies, Infectivity, binding sites, Cricetinae, and Medical and Health Sciences
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Rutten, Twan, Karsholt, Ole (2004): (Lepidoptera: Gelechiidae). Zootaxa 740: 1-42, DOI: 10.5281/zenodo.158360
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Research Interests: Developmental Biology, Biology, Medicine, Seed dormancy, Arabidopsis thaliana, and 15 moreBiological Sciences, Seed Development, Arabidopsis, Phenotype, Developmental, Anthocyanins, Amino Acids, Carbohydrate metabolism, DNA binding, Amino Acid Sequence, Base Sequence, Dormancy, Embryos, Molecular Sequence Data, and Medical and Health Sciences
Research Interests: Genetics, Plant Biology, Biology, Plant Molecular Biology, Medicine, and 15 moreProtein synthesis, Arabidopsis thaliana, Plant Genome Project, Seed Development, Gene, Arabidopsis, Protein Domain, Brassica Napus, Seeds, Gene Function, Seed storage, PLANT PROTEINS, Fatty Acid Composition, Biochemistry and cell biology, and Protein content
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Research Interests: Biophysics, Biology, Actin, Callose, Nicotiana Tabacum, and 3 moreCell Wall, Cytoplasm, and Pollen tube
Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is... more
Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.
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Research Interests: Engineering, Genetics, Physics, Chemistry, Biology, and 15 moreCell Biology, Medicine, Multidisciplinary, Biological Sciences, Sequencing, Cell Differentiation, Expressed Sequence Tags, Cellular differentiation, Endosperm, Barley, Differentiation, PLoS one, Cells, PLANT PROTEINS, and Gene expression profiling
Research Interests: Evolutionary Biology, Genetics, Zoology, Biology, Scanning Electron Microscopy, and 14 moreEvolution, Medicine, Phylogeny, Sequence alignment, Molecular Phylogenetics and Evolution, Molecular cloning, Crocus, Seeds, Monophyly, Phylogenetic Tree, Species Specificity, Base Sequence, Bayes Theorem, and Molecular Sequence Data
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The trade-off between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin, cruciferin). The phenotypic... more
The trade-off between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin, cruciferin). The phenotypic outcomes were assessed using magnetic resonance and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics and immunological assays. Surprisingly, the storage product profile of the seeds of RNAi transgenics was rather moderately changed, but embryonic cells underwent remarkable architectural rearrangements. The most conspicuous structural changes featured the elaboration of membrane stacks enriched with oleosin (6-fold higher protein abundance). Protein rebalancing, amino acid and sulfur metabolism were focal points of the metabolic adjustments to maintain the carbon/nitrogen homeostasis of embryo. Flux balance analysis was evident for a rather minor additional demand for cofactors (ATP, NADPH). The conclusion was hat cellular plasticity in the seed provides a buffer that protects against perturbations to its storage activity, and hence contributes materially to the homeostasis required to assure successful sexual reproduction. The study provides novel mechanistic insights into the intriguing linkage between lipid and protein storage, which have implications for biotechnological strategies directed at the improvement of oilseed crops.
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PRD1 is a ds-DNA bacteriophage from the Tectiviridae family with an unusual structural feature: the viral genome is enclosed by a protein-rich membrane, which is in turn enclosed by an external icosahedral protein shell (capsid).... more
PRD1 is a ds-DNA bacteriophage from the Tectiviridae family with an unusual structural feature: the viral genome is enclosed by a protein-rich membrane, which is in turn enclosed by an external icosahedral protein shell (capsid). Three-dimensional reconstructions from cryo-electron microscopy (cryo-EM) images have revealed the structure of the PRD1 capsid at moderate resolution (28 Å), while X-ray crystallographic studies have recently provided a high resolution (1.85 Å) picture of the major coat protein, P3. We have now combined these results from different imaging methods to obtain a more detailed understanding of the virion organization. The combination has been made in a cyclic process: a preliminary fitting of the atomic structure of P3 to each one of its independent positions in the cryo-EM maps of the capsids provided initial models that could be used to improve the reconstructions; the refined maps then served as a base frame for an optimized fit. This process allows us to s...
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By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in... more
By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in others they are very scanty. The filaments are seen: around bundles of collagen fibrils, at places which seem electron microscopically almost empty, associated with basement membranes around elastin, and sometimes associated with individual collagen fibrils. After poststaining tiny threads--connecting the filaments--could sometimes be observed. The filaments are resistant to treatment with nitrous acid, heparitinase or pronase after prefixation. After digestion with chondroitinase ABC, chondroitinase AC or pronase without prefixation, the filaments are no longer detectable. The tiny threads are chondroitinase ABC resistant. It is concluded that the Cuprolinic Blue-positive filaments represent proteoglycans which contain chondroitin sulfate and/or glucuro...
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Flowering plants with indeterminate inflorescences often produce more floral structures than they require. We found that floral primordia initiations in barley ( Hordeum vulgare L.) are molecularly decoupled from their maturation into... more
Flowering plants with indeterminate inflorescences often produce more floral structures than they require. We found that floral primordia initiations in barley ( Hordeum vulgare L.) are molecularly decoupled from their maturation into grains. While initiation is dominated by flowering-time genes, floral growth is specified by light signaling, chloroplast, and vascular developmental programs orchestrated by barley CCT MOTIF FAMILY 4 ( HvCMF4 ), which is expressed in the inflorescence vasculature. Consequently, mutations in HvCMF4 increase primordia death and pollination failure, mainly through reducing rachis greening and limiting plastidial energy supply to developing heterotrophic floral tissues. We propose that HvCMF4 is a sensory factor for light that acts in connection with the vascular-localized circadian clock to coordinate floral initiation and survival. Notably, stacking beneficial alleles for both primordia number and survival provides positive implications on grain product...
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MADS-box transcription factors are crucial regulators of inflorescence and flower development in plants. Therefore, the recent interest in this family has received much attention in plant breeding programs due to their impact on plant... more
MADS-box transcription factors are crucial regulators of inflorescence and flower development in plants. Therefore, the recent interest in this family has received much attention in plant breeding programs due to their impact on plant development and inflorescence architecture. The aim of this study was to investigate the role of HvMADS-box genes in lateral spikelet development in barley (Hordeum vulgare L.). A set of 30 spike-contrasting barley lines were phenotypically and genotypically investigated under controlled conditions. We detected clear variations in the spike and spikelet development during the developmental stages among the tested lines. The lateral florets in the deficiens and semi-deficiens lines were more reduced than in two-rowed cultivars except cv. Kristina. Interestingly, cv. Kristina, int-h.43 and int-i.39 exhibited the same behavior as def.5, def.6, semi-def.1, semi-def.8 regarding development and showed reduced lateral florets size. In HOR1555, HOR7191 and HOR...
Research Interests: Biology, Medicine, Inflorescence, Plants, Gene, and 3 moreHordeum vulgare, MADS Box Genes, and TASSEL
The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox,... more
The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana. We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the unipa...
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Illuminating the mechanisms of inflorescence architecture of grain crops that feed our world may strengthen the goal towards sustainable agriculture. Lateral spikelet development of barley (Hordeum vulgare L.) is such an example of a... more
Illuminating the mechanisms of inflorescence architecture of grain crops that feed our world may strengthen the goal towards sustainable agriculture. Lateral spikelet development of barley (Hordeum vulgare L.) is such an example of a floral architectural trait regulated by VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1, syn. HvHOX1). Its lateral spikelet-specific expression and the quantitative nature of suppressing spikelet development were previously shown in barley. However, the mechanistic function of this gene and its paralog HvHOX2 on spikelet development is still fragmentary.Here, we show that these duplicated transcription factors (TFs) have contrasting nucleotide diversity in various barley genotypes and several Hordeum species. Despite this difference, both proteins retain their basic properties of the homeodomain leucine zipper class I family of TFs. During spikelet development, these genes exhibit similar spatiotemporal expression patterns yet with anticyclic expres...
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Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed... more
Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but ...
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With the notable exception of angiosperms, all phototrophs contain different sets of flavodiiron proteins that help to relieve the excess of excitation energy on the photosynthetic electron transport chain during adverse environmental... more
With the notable exception of angiosperms, all phototrophs contain different sets of flavodiiron proteins that help to relieve the excess of excitation energy on the photosynthetic electron transport chain during adverse environmental conditions, presumably by reducing oxygen directly to water. Among them, the Flv2-Flv4 dimer is only found in β-cyanobacteria and induced by high light, supporting a role in stress protection. The possibility of a similar protective function in plants was assayed by expressing Synechocystis Flv2-Flv4 in chloroplasts of tobacco and Arabidopsis. Flv-expressing plants exhibited increased tolerance toward high irradiation, salinity, oxidants, and drought. Stress tolerance was reflected by better growth, preservation of photosynthetic activity, and membrane integrity. Metabolic profiling under drought showed enhanced accumulation of soluble sugars and amino acids in transgenic Arabidopsis and a remarkable shift of sucrose into starch, in line with metabolic...
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High-resolution spatial transcriptomics identifies previously unknown regulators for barley floral meristem development.
Research Interests: Botany, Science, Biology, Morphogenesis, Medicine, and 4 moreInflorescence, Hordeum vulgare, Poaceae, and Meristem
Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. Here, we identify the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressing in... more
Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. Here, we identify the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressing in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in barley (Triticeae) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts reveal barley COM1 regulates cell growth, thereby affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems. COM1 acts upstream of the boundary gene Liguleless1 and confers meristem identity partially independent of the COM2 pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding...
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Hexaploid wheat (Triticum aestivum L.) is a natural allopolyploid and provides a usable model system to better understand the genetic mechanisms that underlie allopolyploid speciation through the hybrid genome doubling. Here we aimed to... more
Hexaploid wheat (Triticum aestivum L.) is a natural allopolyploid and provides a usable model system to better understand the genetic mechanisms that underlie allopolyploid speciation through the hybrid genome doubling. Here we aimed to identify the contribution of chromosome 1D in the development and evolution of hexaploid wheat. We identified and mapped a novel DEFECTIVE ENDOSPERM–D1 (Dee-D1) locus on 1DL that is involved in the genetic control of endosperm development. The absence of Dee-D1 leads to non-viable grains in distant crosses and alters grain shape, which negatively affects grain number and thousand-grain weight. Dee-D1 can be classified as speciation locus with a positive effect on the function of genes which are involved in endosperm development in hybrid genomes. The presence of Dee-D1 is necessary for the normal development of endosperm, and thus play an important role in the evolution and improvement of grain yield in hexaploid wheat.
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Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. We identified the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressed in inflorescence... more
Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. We identified the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressed in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in Triticeae (barley) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts revealed barley COM1 regulates cell growth, affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems.COM1acts upstream of the boundary geneLiguleless1and confers meristem identity partially independent of theCOM2pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.
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TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in... more
TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together wi...
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The ability of plants to maintain photosynthesis in a dynamically changing environment is of central importance for their growth. As their photosynthetic machinery typically cannot adapt rapidly to fluctuations in the intensity of... more
The ability of plants to maintain photosynthesis in a dynamically changing environment is of central importance for their growth. As their photosynthetic machinery typically cannot adapt rapidly to fluctuations in the intensity of radiation, the level of photosynthetic efficiency is not always optimal. Cyanobacteria, algae, non-vascular plants (mosses and liverworts) and gymnosperms all produce flavodiirons (Flvs), a class of proteins not represented in the angiosperms; these proteins act to mitigate the photoinhibition of photosystem I. Here, genes specifying two cyanobacterial Flvs have been expressed in the chloroplasts of Arabidopsis thaliana in an attempt to improve the robustness of Photosystem I (PSI). The expression of Flv1 and Flv3 together shown to enhance the efficiency of the utilization of light and to boost the plant’s capacity to accumulate biomass. Based on an assessment of the chlorophyll fluorescence in the transgenic plants, the implication was that photosynthetic...
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SummaryCentromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed... more
SummaryCentromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non‐nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co‐expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N‐terminal tail and the histone fold domain of non‐nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.