Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori

The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species.     

Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl(2), and site-directed mutagenesis of the cysteine residue. HgCl(2) strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl(2) inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl(2) perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl(2) altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.     

Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly

The functions of Beclin‐1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K‐III complex. Here, we describe a new role for Beclin‐1 in mitotic chromosome congression that is independent of the PI3K‐III complex and its role in autophagy. Beclin‐1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP‐E, CENP‐F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin‐1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin‐1 interacts directly with Zwint‐1—a component of the KMN (KNL‐1/Mis12/Ndc80) complex—which is essential for kinetochore–microtubule interactions. This suggests that Beclin‐1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.     

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.     

Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells

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Roles and mechanisms of Kinesin-6 KIF20A in spindle organization during cell division

Mitotic kinesin is crucial for spindle assembly and chromosome segregation in cell division. KIF20A/MKlp2, a member of kinesin-6 subfamily, plays important roles in the central spindle organization at anaphase and cytokinesis. In this review, we briefly introduce the discovery and classification of kinesin-6 motors in model organisms, and summarize the biochemical features and mechanics of KIF20A proteins. We emphasize the complicated interactions of KIF20A with partner proteins, including MKlp1, Plk1 and Rab6. Particularly, we highlight the regulation of Cdk1 and chromosomal passenger complex on kinesin-6 KIF20A at late stage of mitosis. We summarized the multiple functions of KIF20A in central spindle assembly and the formation of cleavage furrow in both mitosis and meiosis. In addition, we conclude the expression patterns of KIF20A in tumorigenesis and its applications in tumor therapy.     

Dissecting the role of Aurora A during spindle assembly

The centrosomal kinase Aurora A (AurA) is required for cell cycle progression, centrosome maturation and spindle assembly. However, the way it participates in spindle assembly is still quite unclear. Using the Xenopus egg extract system, we have dissected the role of AurA in the different microtubule (MT) assembly pathways involved in spindle formation. We developed a new tool based on the activation of AurA by TPX2 to clearly define the requirements for localization and activation of the kinase during spindle assembly. We show that localized AurA kinase activity is required to target factors involved in MT nucleation and stabilization to the centrosome, therefore promoting the formation of a MT aster. In addition, AurA strongly enhances MT nucleation mediated by the Ran pathway through cytoplasmic phosphorylation. Altogether, our data show that AurA exerts an effect as a key regulator of MT assembly during M phase and therefore of bipolar spindle formation.     

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

癌症高表达蛋白--Hec1在纺锤体组装检查点中的作用

细胞增殖依赖于细胞分裂前染色体的复制及随后的姐妹染色单体分离到达两极。纺锤体组装检查点具有确保染色体信息传递保真性的作用,检查点的缺失可能导致染色体的分离异常和肿瘤形成。癌症高表达蛋白(Hec1)通过与调控G2/M期的蛋白间的相互作用而在染色体的分离中发挥重要作用。Hec1与Nuf2的复合物,在G2/M期与动粒相结合,Hec1的缺失将导致严重的染色体分离错误。Hec1具有召集Mps1和Mad1/Mad2复合物结合到动粒上的作用,这种结合可以激活纺锤体组装检查点途径中非常重要的APCCdc20途径。但是Hec1、Mps1、Mad1三者之间的相互作用仍未明了。Hec1还可以通过与26S蛋白酶复合物的不同亚基结合调控其功能。Hec1是一种丝氨酸磷酸化蛋白,其磷酸化是由Nek2在G2/M期完成的。     

Epistasis plays an important role as genetic basis of heterosis in rice

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Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase

Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, cassette mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The mutagenesis at His454 has no effects on expression or stability of the protein. The K_M values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The V_max values were similar for wild-type and mutant GDH. The k_cat/K_M value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in K_M values for NADH and 2-oxoglutarate. The results with cassette mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.     

High-quality genome assembly of an important biodiesel plant,Euphorbia lathyris L

Caper spurge, Euphorbia lathyris L., is an important energy crop and medicinal crop. Here, we generated a high-quality, chromosome-level genome assembly of caper spurge using Oxford Nanopore sequencing, Illumina sequencing, and Hi-C technology. The final genome assembly was ∼988.9 Mb in size, 99.8% of which could be grouped into 10 pseudochromosomes, with contig and scaffold N50 values of 32.6 and 95.7 Mb, respectively. A total of 651.4 Mb repetitive sequences and 36,342 protein-coding genes were predicted in the genome assembly. Comparative genomic analysis showed that caper spurge and castor bean clustered together. We found that no independent whole-genome duplication event had occurred in caper spurge after its split from the castor bean, and recent substantial amplification of long terminal repeat retrotransposons has contributed significantly to its genome expansion. Furthermore, based on gene homology searching, we identified a number of candidate genes involved in the biosynthesis of fatty acids and triacylglycerols. The reference genome presented here will be highly useful for the further study of the genetics, genomics, and breeding of this high-value crop, as well as for evolutionary studies of spurge family and angiosperms.     

Mechanism and regulation of kinesin‐5, an essential motor for the mitotic spindle

Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity. A core element of this machinery is the kinesin‐5 motor protein, which plays essential roles in spindle formation and maintenance. In this review, we discuss how the structural and mechanical properties of kinesin‐5 motors uniquely suit them to their mitotic role. We describe some of the small molecule inhibitors and regulatory proteins that act on kinesin‐5, and discuss how these regulators may influence the process of cell division. Finally, we touch on some more recently described functions of kinesin‐5 motors in non‐dividing cells. Throughout, we highlight a number of open questions that impede our understanding of both this motor's function and the potential utility of kinesin‐5 inhibitors.     

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird‐of‐paradise

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat‐rich and GC‐rich regions (genomic “dark matter”) limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long‐read, linked‐read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC‐rich microchromosomes and the repeat‐rich W chromosome. Telomere‐to‐telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.     

Glu434 is an important amino acid residue for the activity,structure and stability of tyrosine hydroxylase of the silkworm,Bombyx mori

In this study, silkworm (Bombyx mori) tyrosine hydroxylase was expressed in Escherichia coli. The enzymatic characteristics of the recombinant wild silkworm tyrosine hydroxylase were similar to that of native silkworm tyrosine hydroxylase (BmTH). We investigated the role of the amino acid residue Glu434 of BmTH using site-directed mutagenesis. The activity of the E434A mutant was approximately 35.6 percent of that exhibited by BmTH. Furthermore, the mutation dramatically reduced its substrate affinity for tetrahydrobiopterin and decreased its activation by Fe²⁺. The E434A mutation impaired the conformational structure of BmTH, resulting in a partially unfolded state with more hydrophobic exposure, a tendency to aggregate and structural instability during environmental stresses. This mutation did not significantly affect a three-step transitional folding process involving two intermediate states in GdnHCl. However, it did affect the structural compactness of the folding intermediates. The results suggest that the Glu434 residue is an important determinant of the activity, stability and conformational structure of BmTH.     

CD142 plays an important role in the mobility of colorectal cancer cells

ABSTRACT

CD142 promotes cell mobility, which contributes to carcinogenesis. However, the role of CD142 on colorectal cancer (CRC) mobility is unclear. This study showed that CD142 expression increased in CRC tissues, especially in those with invasion or metastasis. The positive sorting or overexpression of CD142 promoted the invasion and migration of CRC cells. Overall, CD142 may be responsible for CRC mobility.