ABSTRACT High-temperature stress is a major constraint on yeast growth and fermentation, and has traditionally been interpreted primarily in terms of intracellular molecular damage such as protein denaturation and aggregation. Despite this extensive focus on intracellular mechanisms, how physical factors within the extracellular environment influence yeast thermotolerance remains poorly understood. Here we demonstrate that increases in extracellular osmolarity markedly attenuate growth inhibition under high-temperature conditions in yeast. This protective effect was consistently observed in multiple laboratory and industrial strains of Saccharomyces cerevisiae , as well as in ascomycetous and basidiomycetous yeasts, indicating that osmotic pressure-dependent thermotolerance is a broadly conserved phenomenon. We also found that extracellular osmolarity dynamically increases during growth and then decreases in a diauxic shift-like pattern after growth arrest. At high temperature, the secretion of glucose-derived metabolites decreased, but that of other solutes increased, suggesting that heat stress alters the composition of extracellular solutes contributing to osmolarity. In addition, intracellular glycerol levels increased at high temperature, and this increase was further enhanced under high-osmolarity conditions. Notably, expression of a constitutively active Hog1 mutant exhibited raised intracellular glycerol levels, enhanced nuclear localization of Hog1, and improved growth under high-temperature conditions. Collectively, these findings support a model in which extracellular osmolarity is modulated to avoid excessive intracellular osmolarity under high-temperature conditions, while the intracellular accumulation of glycerol contributes to yeast adaptation at high-temperature. Our results highlight extracellular–intracellular osmotic coordination as an additional physiological layer of high-temperature stress adaptation in yeast.

Gene amplification plays a critical role in evolution and disease and is widely utilized to overexpress valuable gene products in biotechnology. To broaden these applications, we previously developed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx), a method utilizing Cas9 nickase (nCas9) to amplify genetic sequences by driving tandem array expansion through ectopic BIR. Since BITREx efficiency depends on the guide RNA (gRNA) recruiting nCas9 to the array's flanking regions, here we develop a plasmid-based reporter system in budding yeast for the rapid identification of high-performing gRNAs. Furthermore, we introduce BITREx 2.0, a dual-nicking strategy that targets both sides of the gene array. We demonstrate that BITREx 2.0 is effective for both natural and synthetic arrays, enhancing expansion efficiency by up to an order of magnitude compared to the original single-nicking format. These advancements significantly broaden the applicability and efficiency of nCas9-mediated gene amplification across diverse biological and biotechnological contexts.

Genetic diversity underlies adaptive evolution. Because genes often act in concert to execute biological processes, the dosage stoichiometry among cooperating genes represents an additional layer of diversity beyond sequence variation. We therefore hypothesized that combinatorial randomization of gene copy numbers could generate cell populations enriched for functional heterogeneity and evolutionary potential. To test this idea, we extended our previously developed Cas9 nickase-based gene amplification method, break-induced replication-mediated tandem repeat expansion (BITREx), to simultaneously target multiple genes. Applying parallel BITREx to three carotenogenic genes introduced into the budding yeast Saccharomyces cerevisiae, we generated a cell population exhibiting broad variation in both absolute copy numbers and their stoichiometric ratios. This population enabled the identification of elite genotypes—specific copy number combinations that conferred enhanced β-carotene production. These results suggest that parallel BITREx is a versatile strategy for increasing functional heterogeneity in cell populations, with potential applications in both basic and applied research.

Copper resistance in the budding yeast Saccharomyces cerevisiae is primarily mediated by the tandemly arrayed metallothionein gene CUP1 . We analyzed eight isogenic strains harboring CUP1 arrays of varying lengths, including those artificially expanded beyond their natural ranges. CUP1 mRNA levels and copper resistance increased with copy number before reaching a plateau. Increased dosage of the transcriptional activator Cup2 partially mitigated the plateaued resistance in strains with intermediate, but not high, copy numbers. These findings indicate that CUP1 confers resistance dose-dependently until transcriptional capacity becomes limiting, suggesting a possible strategy for engineering extreme copper resistance.

Tandem gene arrays are inherently unstable, particularly in recombination-prone organisms such as the budding yeast Saccharomyces cerevisiae . However, understanding the nature of this instability-how frequently and to what extent the target array contracts or expands within the genome-remains challenging. As a surrogate approach to this goal, we propose using nanopore long-read sequencing to directly determine the length distribution of a target gene array cloned onto a centromeric plasmid functioning as an artificial chromosome. This strategy will not only allow us to assess the intrinsic instability of the array but also help identify factors that may influence its stability.

The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants ( RME1, TAO3 , and MKT1 ) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae . By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance Statement ### Competing Interest Statement The authors have declared no competing interest. * Cas : CRISPR-associated protein; CRISPR : clustered regularly interspaced short palindromic repeats; crRNA : CRISPR RNA; DIC : differential interference contrast; LOBSTERS : League Of Backbone plasmid vector Series To Expand the Range of Selection markers; ORF : open reading frame; PAM : protospacer adjacent motif; PCR : polymerase chain reaction; QTV : quantitative trait variant; sgRNA : single guide RNA. Japan Science and Technology Agency, JPMJCR19S1 Japan Society for the Promotion of Science, 24H00505, 24K02015

Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.

Gene duplication followed by sequence diversification is a key driver of innovation in genome evolution. To mimic this process in genome engineering, a method for region-restricted mutagenesis is needed to selectively mutate one copy of a duplicated gene. Notably, regions flanking a double-strand break (DSB) become hypersensitive to mutagens due to end resection, which converts them into single-stranded DNA (ssDNA). Blocking end resection could, therefore, confine hypermutation to a limited region. To achieve this, we investigated a catalytically inactive variant of Streptococcus pyogenes Cas9 (dSpCas9) and demonstrated its ability to attenuate end resection in the budding yeast Saccharomyces cerevisiae using ssDNA-specific quantitative PCR, live-cell imaging, and Southern blot analysis. By leveraging the bisulfite sensitivity of ssDNA, we further validated the concept of DSB-coupled, dSpCas9-mediated region-restricted mutagenesis. We anticipate that dSpCas9-mediated modulation of end resection at induced DSB sites will have valuable applications in both genome engineering and mechanistic studies.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER.

AbstractCatalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Because each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here, we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

Localized extracellular matrix (ECM) remodeling is thought to stabilize the cleavage furrow and maintain cell shape during cytokinesis [1-14]. This remodeling is spatiotemporally coordinated with a cytoskeletal structure pertaining to a kingdom of life, for example the FtsZ ring in bacteria [15], the phragmoplast in plants [16], and the actomyosin ring in fungi and animals [17, 18]. Although the cytoskeletal structures have been analyzed extensively, the mechanisms of ECM remodeling remain poorly understood. In the budding yeast Saccharomyces cerevisiae, ECM remodeling refers to sequential formations of the primary and secondary septa that are catalyzed by chitin synthase-II (Chs2) and chitin synthase-III (the catalytic subunit Chs3 and its activator Chs4), respectively [18, 19]. Surprisingly, both Chs2 and Chs3 are delivered to the division site at the onset of cytokinesis [6, 20]. What keeps Chs3 inactive until secondary septum formation remains unknown. Here, we show that Hof1 binds to the Sel1-like repeats (SLRs) of Chs4 via its F-BAR domain and inhibits Chs3-mediated chitin synthesis during cytokinesis. In addition, Hof1 is required for rapid accumulation as well as efficient removal of Chs4 at the division site. This study uncovers a mechanism by which Hof1 controls timely activation of Chs3 during cytokinesis and defines a novel interaction and function for the conserved F-BAR domain and SLR that are otherwise known for their abilities to bind membrane lipids [21, 22] and scaffold protein complex formation [23].

Cytokinesis is essential for development and survival of all organisms by increasing cell number and diversity. It is a highly regulated process that requires spatiotemporal coordination of hundreds of proteins functioning in the assembly, constriction, and disassembly of a contractile actomyosin ring, targeted vesicle fusion, and localized extracellular matrix remodeling. Cytokinesis has been studied in multiple systems with a wide range of technologies to learn the common principles. In this chapter, we describe the analysis of protein dynamics during cytokinesis in the budding yeast Saccharomyces cerevisiae by several live-cell imaging methods. This, in combination with the power of yeast genetics, has yielded novel insights into the mechanism of cytokinesis. Similar approaches are increasingly used to study this fundamental process in more complex systems.

Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.

The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope‐labeled standards. In this study, we developed a strategy of hierarchical use of peptide‐concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique “ID‐tag peptides” were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a “secondary PCS” in which all “ID‐tag peptides” were concatenated. Furthermore, we varied the copy number of the “ID‐tag peptide” in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post‐transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance.

MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species.

Septins perform diverse functions through the formation of filaments and higher-order structures. However, the exact architecture of septin structures remains unclear. In the budding yeast Saccharomyces cerevisiae, septins form an 'hourglass' at the mother-bud neck before cytokinesis, which is converted into a 'double ring' during cytokinesis. Here, using platinum-replica electron microscopy, we find that the early hourglass consists of septin double filaments oriented along the mother-bud axis. In the late hourglass, these double filaments are connected by periodic circumferential single filaments on the membrane-proximal side and are associated with centrally located, circumferential, myosin-II thick filaments on the membrane-distal side. The double ring consists of exclusively circumferential septin filaments. Live-cell imaging studies indicate that the hourglass-to-double ring transition is accompanied by loss of septin subunits from the hourglass and reorganization of the remaining subunits into the double ring. This work provides an unparalleled view of septin structures within cells and defines their remodelling dynamics during the cell cycle.

Asymmetric cell division plays a crucial role in cell differentiation, unequal replicative senescence, and stem cell maintenance. In budding yeast, the identities of mother and daughter cells begin to diverge at bud emergence when distinct plasma-membrane domains are formed and separated by a septin ring. However, the mechanisms underlying this transformation remain unknown. Here, we show that septins recruited to the site of polarization by Cdc42-GTP inhibit Cdc42 activity in a negative feedback loop, and this inhibition depends on Cdc42 GTPase-activating proteins. Combining live-cell imaging and computational modeling, we demonstrate that the septin ring is sculpted by polarized exocytosis, which creates a hole in the accumulating septin density and relieves the inhibition of Cdc42. The nascent ring generates a sharp boundary that confines the Cdc42 activity and exocytosis strictly to its enclosure and thus clearly delineates the daughter cell identity. Our findings define a fundamental mechanism underlying eukaryotic cell fate differentiation.