A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.

E3 Ligases Regulate Organelle Inheritance in Yeast.

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

Targeted Protein Degradation Systems: Controlling Protein Stability Using E3 Ubiquitin Ligases in Eukaryotic Species.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin-proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.

Development of AlissAID system targeting GFP or mCherry fusion protein.

Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells.

Proteolysis of adaptor protein Mmr1 during budding is necessary for mitochondrial homeostasis in Saccharomyces cerevisiae.

In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase.

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Various Vertebrate Cell Lines.

Cells with a loss-of-function mutation in a gene (knockout cells) are powerful tools for characterizing the function of such gene product. However, for essential genes, conditional knockout cell lines must be generated. The auxin-inducible degron (AID) technique enables us to conditionally and rapidly deplete a target protein from various eukaryotic cell lines. A combination of CRISPR-/Cas9-based gene editing and AID technique allows us to generate AID-based conditional knockout cell lines. Using these two techniques, we recently proposed a simple and quick way to generate conditional knockout cells for essential genes. In this chapter, we introduce the reader to the experimental procedures to generate these AID-based conditional knockout cell lines.

A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines.

Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.

Alterations in hormonal signals spatially coordinate distinct responses to DNA double-strand breaks in Arabidopsis roots.

Plants have a high ability to cope with changing environments and grow continuously throughout life. However, the mechanisms by which plants strike a balance between stress response and organ growth remain elusive. Here, we found that DNA double-strand breaks enhance the accumulation of cytokinin hormones through the DNA damage signaling pathway in the Arabidopsis root tip. Our data showed that activation of cytokinin signaling suppresses the expression of some of the PIN-FORMED genes that encode efflux carriers of another hormone, auxin, thereby decreasing the auxin signals in the root tip and causing cell cycle arrest at G2 phase and stem cell death. Elevated cytokinin signaling also promotes an early transition from cell division to endoreplication in the basal part of the root apex. We propose that plant hormones spatially coordinate differential DNA damage responses, thereby maintaining genome integrity and minimizing cell death to ensure continuous root growth.

CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

Complete Genome Sequence of Sphingomonas paucimobilis Strain Kira, Isolated from Human Neuroblastoma SH-SY5Y Cell Cultures Supplemented with Retinoic Acid.

Because of its small size, Gram-negative Sphingomonas paucimobilis can pose a risk of nosocomial infection. We report the complete circular genome sequence of S. paucimobilis strain Kira, which was isolated from retinoic acid-supplemented SH-SY5Y human cell cultures, to be 3,917,410 bp (G+C content, 65.7%; 3,672 protein-coding sequences), with two plasmids (79,575 bp and 44,333 bp).

Essentiality of CENP-A Depends on Its Binding Mode to HJURP.

CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-AA59Q. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.

A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

Rapid turnover of transcription factor Rim101 confirms a flexible adaptation mechanism against environmental stress in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells activate the Rim101 pathway to adapt to alkaline and salt stresses. On activation of this pathway, the transcription factor Rim101 undergoes proteolytic activation and regulates the expression of responsive genes. We found Rim101 to be a short-lived protein with a half-life of approximately 15 min. Its rapid turnover was supposedly mediated by the ubiquitin-proteasome system. Excess accumulation of the processed active Rim101 through its over-expression conferred tolerance to both alkaline and salt stresses in yeast cells; in contrast, it had detrimental effects under cadmium stress condition. Cadmium ion inhibited proteolytic activation of Rim101, implying reciprocal interaction between the Rim101 pathway and cadmium stress. Our results showed yeast cells to be equipped with two protective systems to prevent overaccumulation of the processed active Rim101; Rim101 processing is inhibited when Rim101 level is high, and turnover of processed Rim101 is accelerated when it is abundant. Collectively, the results confirmed the flexible aspect of stress response in yeast cell; the cells not only prevent excess activation of one stress-responsive pathway but also facilitate its attenuation to cope with other environmental stresses.

Relationship between Lattice Strain and Efficiency for Sn-Perovskite Solar Cells.

In the composition of Q0.1(FA0.75MA0.25)0.9SnI3, Q is replaced with Na+, K+, Cs+, ethylammonium+ (EA+), and butylammonium+ (BA+), respectively, and the relationship between actually measured lattice strain and photovoltaic performances is discussed. The lattice strain evaluated by the Williamson-hall plot of X-ray diffraction data decreased as the tolerance factor was close to one. The efficiency of the Sn-perovskite solar cell was enhanced as the lattice strain decreased. Among them, EA0.1(FA0.75MA0.25)0.9SnI3 having lowest lattice strain gave the best result of 5.41%. Because the carrier mobility increased with a decrease in the lattice strain, these lattice strains would disturb carrier mobility and decrease the solar cell efficiency. Finally, the results that the efficiency of the SnGe-perovskite solar cells was gradually enhanced from 6.42 to 7.60% during storage, was explained by the lattice strain relaxation during the storage.

Suppression of Charge Carrier Recombination in Lead-Free Tin Halide Perovskite via Lewis Base Post-treatment.

Lead-free tin perovskite solar cells (PSCs) show the most promise to replace the more toxic lead-based perovskite solar cells. However, the efficiency is significantly less than that of lead-based PSCs as a result of low open-circuit voltage. This is due to the tendency of Sn2+ to oxidize into Sn4+ in the presence of air together with the formation of defects and traps caused by the fast crystallization of tin perovskite materials. Here, post-treatment of the tin perovskite layer with edamine Lewis base to suppress the recombination reaction in tin halide PSCs results in efficiencies higher than 10%, which is the highest reported efficiency to date for pure tin halide PSCs. The X-ray photoelectron spectroscopy data suggest that the recombination reaction originates from the nonstoichiometric Sn:I ratio rather than the Sn4+:Sn2+ ratio. The amine group in edamine bonded the undercoordinated tin, passivating the dangling bonds and defects, resulting in suppressed charge carrier recombination.

3D genomic architecture reveals that neocentromeres associate with heterochromatin regions.

The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere-heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.

Netrin Signaling Defines the Regional Border in the Drosophila Visual Center.

The brain consists of distinct domains defined by sharp borders. So far, the mechanisms of compartmentalization of developing tissues include cell adhesion, cell repulsion, and cortical tension. These mechanisms are tightly related to molecular machineries at the cell membrane. However, we and others demonstrated that Slit, a chemorepellent, is required to establish the borders in the fly brain. Here, we demonstrate that Netrin, a classic guidance molecule, is also involved in the compartmental subdivision in the fly brain. In Netrin mutants, many cells are intermingled with cells from the adjacent ganglia penetrating the ganglion borders, resulting in disorganized compartmental subdivisions. How do these guidance molecules regulate the compartmentalization? Our mathematical model demonstrates that a simple combination of known guidance properties of Slit and Netrin is sufficient to explain their roles in boundary formation. Our results suggest that Netrin indeed regulates boundary formation in combination with Slit in vivo.

An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9.

Generation of cells with a loss-of-function mutation in a gene (knockout cells) is a valuable technique for studying the function of a given gene product. However, if the product of the target gene is essential for cell viability, conditional knockout cell lines must be generated. Recently, as gene editing technology using CRISPR/Cas9 has developed, it has become possible to produce conditional knockout cell lines using this technique. However, to obtain final conditional knockout cell lines, it is necessary to perform several experiments with multiple complicated steps. In this paper, we introduce an easy and efficient method to generate conditional knockout cell lines based on combining auxin-inducible degron (AID) technology with CRISPR/Cas9 gene editing. Our method only requires performing a single transfection and is therefore an easy and rapid method to obtain a conditional knockout cell line.

Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis.

DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase.