Biology subjects

Kiuchi, T.

Publications and source records attributed to Kiuchi, T..

14 recordsLinked to original sources

bioRxiv2025

Cis-regulatory evolution of Wnt-family genes contributes to a morphological difference between silkworm species.

Closely related species often exhibit distinct morphologies that can contribute to species-specific adaptations and reproductive isolation. One example are Lepidopteran caterpillar appendages, such as the "caudal horn" of Bombycoidea moths, which have evolved substantial morphological diversity among species in this group. Using interspecific crosses, we identify the genetic basis of the caudal horn size difference between Bombyx mori and its closest relative B. mandarina. The three largest of eight QTL account for one third the mean horn length difference between the species. The largest of these, on chromosome 4, encompasses a conserved Wnt family gene cluster, key upstream regulators that are well-known for their roles in morphological diversification in animals. Using allele-specific expression analysis and CRISPR/Cas9 knockouts, we show that tissue-specific cis-regulatory changes to Wnt1 and Wnt6 contribute to the species difference in caudal horn size. This kind of modularity enables highly pleiotropic genes, including key upstream growth regulators, to contribute to the evolution of morphological traits without causing widespread deleterious effects. SignificanceThis study explores the genetic basis of a distinct morphological trait that varies between two closely related moth species, providing insights into the evolution of morphological diversity. By identifying cis-regulatory changes in two Wnt family genes as major contributors, this work underscores the importance of developmental gene regulatory networks in shaping species-specific traits. The findings illustrate how even small modifications in major upstream regulator genes can drive significant phenotypic variation, revealing how genetic changes in key growth regulators fuel the diversification of form and function. These results advance our understanding of the mechanisms behind the evolution of complex morphological traits.

evolutionary biology↗

bioRxiv2025

The Masc-PSI complex directly induces male-type doublesex splicing in silkworms

The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori, a lepidopteran model insect, females carry the W chromosome with the feminizing gene Feminizer (Fem), which is the source of a female-specific PIWI-interacting RNA (piRNA). The Fem piRNA-PIWI complex cleaves an mRNA encoding the masculinizing protein Masculinizer (BmMasc), resulting in the production of the female-type splice variant of Bombyx mori doublesex (Bmdsx), which is the master genetic switch of B. mori sex differentiation. In contrast, in males, BmMasc induces the production of the male-type Bmdsx splice variant (BmdsxM). However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the male-specific Bmdsx splicing event remains unknown. In this study, we showed that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), that is a RNA binding protein required for BmdsxM expression. BmMasc overexpression resulted in the production of BmdsxM in B. mori ovary-derived BmN-4 cells, but this induction was severely inhibited in BmPSI-knocked down cells, indicating that BmPSI is essential for the masculinizing activity of BmMasc. We also identified that BmMasc-containing protein complex was associated with a region of Bmdsx pre-mRNA spanning introns 2 to 4, particularly around the junctions of intron 2 with exon 3 and intron 3 with exon 4. Exons 3 and 4 of Bmdsx are female-specific units and are skipped in BmdsxM. Together with a previous report of the binding of BmPSI to the CE1 sequence located in exon 4 of Bmdsx, the current results strongly suggest that the BmMasc-BmPSI complex is required for male-specific exon skipping in Bmdsx pre-mRNA through its binding to the female-specific Bmdsx introns and exons.

molecular biology↗

bioRxiv2024

Laminar endocytic machinery revealed by quantitative multiplexed super-resolution imaging

Super-resolution microscopy achieves a few nanometers resolution, but protein quantification and colocalization is limited by its labeling density. Here we present a method to map molecular complexes using the multiplexed data of IRIS super- resolution imaging. We developed antiserum-derived Fab IRIS probes for high- density labeling of endogenous targets, and image analysis, Protein Cluster coloring (PC-coloring), which employs pixel-based principal component analysis and clustering. PC-coloring was first used to map regions of distinct ratios of targets on the image. The molecular complex formation was then evaluated by correlation between all combinations of two proteins in each PC-colored region. We elucidate receptor recruitment and the complex formation in a clathrin-coated structure (CCS) which is pre-assembled prior to endocytosis. Upon EGF stimulation, EGFR-, EGFR-Grb2-complex- and Grb2-dominant regions lined up from the CCS rim. Along the interior of Grb2-dominant regions, Eps15, FCHo1/2 and intersectin-1 formed a complex with Grb2. The interior of Eps15-FCHo1/2 complexes was lined with Eps15-intersectin-1 complexes. The results reveal a laminar complex formation of EGFR, Grb2 and CCS components at the recruitment sites.

cell biology↗

bioRxiv2024

Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the precise molecular mechanisms in this context remain unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on another clock gene, cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to induce photoperiodic diapause during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction. HighlightsO_LIKnockouts of core clock genes disrupted the rhythmic expression of cryptochrome 1 (cry1). C_LIO_LIA cry1 knockout strain was established using CRISPR/Cas9. C_LIO_LIThe cry1 knockout strain lost its eclosion rhythm. C_LIO_LIKnockout of cry1 disrupted photoperiodic diapause induction. C_LIO_LIFemales of a cry1/tim double knockout strain produced only non-diapause eggs regardless of larval photoperiod. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/593801v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a2b363org.highwire.dtl.DTLVardef@af3adcorg.highwire.dtl.DTLVardef@41490corg.highwire.dtl.DTLVardef@22bc30_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Disruption of a BTB-ZF transcription factor causes female sterility and melanization in the larval body of the silkworm, Bombyx mori

The dilute black (bd) of the silkworm Bombyx mori is a recessive mutant that produces a grayish-black color in the larval integument, instead of the characteristic white color found in wild-type larvae. In addition, eggs produced by bd females are sterile due to a deficiency in the micropylar apparatus. We identified candidate genes responsible for the bd phenotype using publicly available RNA-seq data. One of these candidate genes was homologous to the maternal gene required for meiosis (mamo) of Drosophila melanogaster, which encodes a broad-complex, tramtrack, and bric-a-brac-zinc finger (BTB-ZF) transcription factor essential for female fertility. In three independent bd strains, the expression of the B. mori mamo (Bmmamo) was downregulated in the larval integument. Using a CRISPR/Cas9-mediated knockout strategy, we found that Bmmamo knockout mutants exhibit a grayish-black color in the larval integument and female infertility. Moreover, larvae obtained from the complementation cross between bd/+ mutants and heterozygous knockouts for the Bmmamo also exhibited a grayish-black color, indicating that Bmmamo is responsible for the bd phenotype. Gene expression analysis using Bmmamo knockout mutants suggested that the BmMamo protein suppresses the expression of melanin synthesis genes. Previous comparative genome analysis revealed that the Bmmamo was selected during silkworm domestication, and we found that Bmmamo expression in the larval integument is higher in B. mori than in the wild silkworm B. mandarina, suggesting that the Bmmamo is involved in domestication-associated pigmentation changes of the silkworm. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/535244v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1cd37a1org.highwire.dtl.DTLVardef@1ff5b23org.highwire.dtl.DTLVardef@1b87dcforg.highwire.dtl.DTLVardef@14bd742_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Deleterious mutations in a heterozygous chromosome result in recessive lethality in silkworm semiconsomic strains

In this study, we found two embryonic lethal mutations, t04 lethal (l-t04) and m04 lethal (l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkworm Bombyx mandarina, is substituted with chromosomes of the domesticated silkworm B. mori, and l-t04 and l-m04 mutations are located on B. mandarina-derived chromosome 4. The mutations responsible for the l-t04 and l-m04 were identified as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA Box Binding Protein-associated factor 5 (BmTaf5), respectively. These findings indicate that both mutations were independently introduced during or after the development of semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/521575v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1c59ea3org.highwire.dtl.DTLVardef@131fbf0org.highwire.dtl.DTLVardef@93515borg.highwire.dtl.DTLVardef@1c23afe_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage

The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.

biochemistry↗

bioRxiv2022

Knockouts of positive and negative elements of the circadian clock disrupt photoperiodic diapause induction in the silkworm, Bombyx mori

Diapause is one of the most important traits that have sustained insects to thrive. Insects can prospectively arrest their development and increase environmental stress-resistance by entering diapause. The photoperiod is the signal that indicates insects the proper timing to enter diapause. Circadian clock genes are shown to be involved in photoperiodic diapause induction in various insect species. The silkworm, Bombyx mori, enters diapause at the embryonic stage. In bivoltine strains, diapause determination is affected by embryonic temperature and photoperiodic conditions during embryonic and larval stages. Two independent studies showed that knocking out the core clock gene, period, perturb photoperiodic diapause induction. However, whether the circadian clock as whole or individual clock genes are responsible for the photoperiodic diapause induction remains unknown. In this study, using CRISPR/Cas9 we knocked out negative (period and timeless) and positive elements (Clock and cycle) in p50T, a bivoltine strain which exhibits photoperiodic diapause induction during both embryonic and larval stages. The temporal expression patterns of clock genes changed in each core clock gene knockout strain, suggesting disruption of normal feedback loops produced by circadian clock genes. Furthermore, photoperiodic diapause induction during both embryonic and larval stages was lost in all knockout strains. Our results indicate the involvement of circadian clock in photoperiodic diapause induction in B. mori. HighlightsO_LIWe established knockout Bombyx mori strains of four core clock genes C_LIO_LIThe temporal expression patterns of clock genes changed in knockout strains C_LIO_LIPhotoperiodic diapause induction was not observed in any knockout strains C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

genetics↗

bioRxiv2022

Transcriptome analysis in the silkworm Bombyx mori overexpressing piRNA-resistant Masculinizer gene

Dosage compensation is a process that produces a similar expression of sex-linked and autosomal genes. In the silkworm Bombyx mori with a WZ sex-determination system, the expression from the single Z in WZ females matches that of ZZ males due to the suppression of Z-linked genes in males. A primary maleness determinant gene, Masculinizer (Masc), is also required for dosage compensation. In females, P-element induced wimpy testis (PIWI) is complexed with the W chromosome-derived female-specific Feminizer (Fem) PIWI-interacting RNA (piRNA) and cleaves Masc mRNA. When Fem piRNA-resistant Masc cDNA (Masc-R) is overexpressed in both sexes, only female larvae are dead during the larval stage. In this study, transcriptome analysis was performed in neonate larvae to examine the effects of Masc-R overexpression on a global gene expression profile. Z-linked genes were globally repressed in Masc-R-overexpressing females due to force-driven dosage compensation. In contrast, Masc-R overexpression had little effect on the expression of Z-linked genes and the male-specific isoform of B. mori insulin-like growth factor II mRNA-binding protein in males, indicating that excessive Masc expression strengthens neither dosage compensation nor maleness in males. Fourteen genes were differentially expressed between Masc-R-overexpressing and control neonate larvae in both sexes, suggesting Masc functions other than dosage compensation and masculinization. HighlightsO_LITranscriptome analysis was performed in Masc-R-overexpressing neonate Bombyx mori larvae. C_LIO_LIZ-linked genes were globally suppressed in Masc-R-overexpressing females. C_LIO_LIMasc-R overexpression had little effect on BmImpM expression in males. C_LIO_LISeveral genes may be controlled by Masc-R regardless of sex. C_LI

genetics↗

bioRxiv2022

Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts

Wolbachia is an extremely widespread endocellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of O. furnacalis and O. scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint. SignificanceDespite the growing number of publicly available Wolbachia genome sequences, only a few high-quality male-killer genomes exist, particularly those found in lepidopteran hosts. The complete genomes of two male-killing Wolbachia of Ostrinia moth hosts were determined in this study. The genomic data obtained here will be used to elucidate the mechanism of reproductive manipulation and the origins of this endosymbionts extraordinary diversity. Additionally, phylogenetic analysis of mitochondria and Wolbachia revealed the evolutionary history of Ostrinia hosts and Wolbachia. The inferred dynamic pattern of infection adds to our understanding of evolution and ecology of Wolbachia endosymbiont, a promising agent for biological pest control.

microbiology↗

bioRxiv2022

Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in Bombyx mori

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori x B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication. Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene"s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori x B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication. Graphical TOC/Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/490996v2_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@10e5708org.highwire.dtl.DTLVardef@73ec82org.highwire.dtl.DTLVardef@ac6094org.highwire.dtl.DTLVardef@f8a54e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm, Bombyx mori

Dosage compensation balances the expression of sex-chromosome-linked genes with autosome-linked genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran model insect, uses the female heterogametic WZ sex determination system. In B. mori, a Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation. However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analyzed the allele-specific expression (ASE) to solve the problem. The ASE analysis revealed that the genes located on the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. Also, our results revealed that two Z chromosomes are transcriptionally downregulated in B. mori males as observed in Caenorhabditis elegans.

genetics↗

bioRxiv2021

Mutations in a β-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the {beta}-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes. HighlightO_LIResponsible region for the brown egg 4 (b-4) mutation was narrowed down by double digest restriction-site associated DNA sequencing (ddRAD-seq). C_LIO_LIThe gene structure was disrupted in one of the candidate genes, Bombyx mori mahogany (Bmmah), in the b-4 mutant strains. C_LIO_LICRISPR/Cas9-mediated gene knockout and complementation test confirmed that the Bmmah is responsible for the b-4 phenotypes. C_LIO_LIThe Bmmah encoded a putative amino acid transporter that belongs to the {beta}-group of solute carrier family. C_LIO_LIThe Bmmah gene is essential for normal colorization of eggs, compound eyes, and ganglions. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/436376v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10d1d56org.highwire.dtl.DTLVardef@12df5org.highwire.dtl.DTLVardef@1703701org.highwire.dtl.DTLVardef@2b5bcf_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

molecular biology↗