Disordered region of nuclear membrane protein Bqt4 recruits phosphatidic acid to the nuclear envelope to maintain its structural integrity.

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion.

Secretory vesicle clusters transported on actin filaments by myosin V motors for local secretion underlie various cellular processes, such as neurotransmitter release at neuronal synapses,1 hyphal steering in filamentous fungi,2,3 and local cell wall digestion preceding the fusion of yeast gametes.4 During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes.5,6,7 The position and coalescence of the vesicle focus are controlled by local signaling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis,6,8,9,10 but the mechanisms of focusing have been elusive. Here, we show that the regulatory N terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude ribosomes. Fus1 lacking its IDR fails to concentrate in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, the replacement of Fus1 IDR with a heterologous low-complexity region that forms molecular condensates fully restores Fus1 focusing and function. By contrast, the replacement of Fus1 IDR with a domain that forms more stable oligomers restores focusing but poorly supports cell fusion, suggesting that condensation is tuned to yield a selectively permeable structure. We propose that condensation of actin structures by an IDR may be a general mechanism for actin network organization and the selective local concentration of secretory vesicles.

Improving Drug Sensitivity of HIV-1 Protease Inhibitors by Restriction of Cellular Efflux System in a Fission Yeast Model.

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Perturbation of kinetochore function using GFP-binding protein in fission yeast.

Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems.

Single-Agent and Fixed-Dose Combination HIV-1 Protease Inhibitor Drugs in Fission Yeast (Schizosaccharomyces pombe).

Successful combination antiretroviral therapies (cART) eliminate active replicating HIV-1, slow down disease progression, and prolong lives. However, cART effectiveness could be compromised by the emergence of viral multidrug resistance, suggesting the need for new drug discoveries. The objective of this study was to further demonstrate the utility of the fission yeast cell-based systems that we developed previously for the discovery and testing of HIV protease (PR) inhibitors (PIs) against wild-type or multi-PI drug resistant M11PR that we isolated from an infected individual. All thirteen FDA-approved single-agent and fixed-dose combination HIV PI drugs were tested. The effect of these drugs on HIV PR activities was tested in pure compounds or formulation drugs. All FDA-approved PI drugs, except for a prodrug FPV, were able to suppress the wild-type PR-induced cellular and enzymatic activities. Relative drug potencies measured by EC50 in fission yeast were discussed in comparison with those measured in human cells. In contrast, none of the FDA-approved drugs suppressed the multi-PI drug resistant M11PR activities. Results of this study show that fission yeast is a reliable cell-based system for the discovery and testing of HIV PIs and further demonstrate the need for new PI drugs against viral multi-PI resistance.

Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

Tell the Difference Between Mitosis and Meiosis: Interplay Between Chromosomes, Cytoskeleton, and Cell Cycle Regulation.

Meiosis is a specialized style of cell division conserved in eukaryotes, particularly designed for the production of gametes. A huge number of studies to date have demonstrated how chromosomes behave and how meiotic events are controlled. Yeast substantially contributed to the understanding of the molecular mechanisms of meiosis in the past decades. Recently, evidence began to accumulate to draw a perspective landscape showing that chromosomes and microtubules are mutually influenced: microtubules regulate chromosomes, whereas chromosomes also regulate microtubule behaviors. Here we focus on lessons from recent advancement in genetical and cytological studies of the fission yeast Schizosaccharomyces pombe, revealing how chromosomes, cytoskeleton, and cell cycle progression are organized and particularly how these are differentiated in mitosis and meiosis. These studies illuminate that meiosis is strategically designed to fulfill two missions: faithful segregation of genetic materials and production of genetic diversity in descendants through elaboration by meiosis-specific factors in collaboration with general factors.

Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast.

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces pombe.

Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast Schizosaccharomyces pombe. We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent. Our proteome datasets revealed peptides corresponding to short open reading frames (sORFs) that have been previously identified by ribosome profiling as new translated regions. We verified expression of selected sORFs by Western blotting and analyzed the phenotype of deletion mutants. Our data provide a resource for studying meiosis that may help understand differences between meiosis I and meiosis II and how S phase is suppressed between the two meiotic divisions.

Development of A Fission Yeast Cell-Based Platform for High Throughput Screening of HIV-1 Protease Inhibitors.

BACKGROUND: HIV-1 protease inhibitor (PI) is one of the most potent classes of drugs in combinational antiretroviral therapies (cART). When a PI is used in combination with other anti- HIV drugs, cART can often suppress HIV-1 below detection thus prolonging the patient's lives. However, the challenge often faced by patients is the emergence of HIV-1 drug resistance. Thus, PIs with high genetic-barrier to drug-resistance are needed. OBJECTIVE: The objective of this study was to develop a novel and simple fission yeast (Schizosaccharomyces pombe) cell-based system that is suitable for high throughput screening (HTS) of small molecules against HIV-1 protease (PR). METHODS: A fission yeast RE294-GFP strain that stably expresses HIV-1 PR and green fluorescence protein (GFP) under the control of an inducible nmt1 promoter was used. Production of HIV-1 PR induces cellular growth arrest, which was used as the primary endpoint for the search of PIs and was quantified by an absorbance-based method. Levels of GFP production were used as a counter-screen control to eliminate potential transcriptional nmt1 inhibitors. RESULTS: Both the absorbance-based HIV-1 PR assay and the GFP-based fluorescence assay were miniaturized and optimized for HTS. A pilot study was performed using a small drug library mixed with known PI drugs and nmt1 inhibitors. With empirically adjusted and clearly defined double-selection criteria, we were able to correctly identify the PIs and to exclude all hidden nmt1 inhibitors. CONCLUSION: We have successfully developed and validated a fission yeast cell-based HTS platform for the future screening and testing of HIV-1 PR inhibitors.

Capping Protein Insulates Arp2/3-Assembled Actin Patches from Formins.

How actin structures of distinct identities and functions coexist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits, and the formins For3, Cdc12, and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin, and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in cells lacking CP. CP deletion also rescues the fusion defect of a mutation in the Fus1 knob region. However, myosin V and exocytic cargoes are reduced at the fusion focus and diverted to ectopic foci, which underlies the fusion defect. Remarkably, the ectopic foci coincide with Arp2/3-assembled actin patches, which now contain low levels of Fus1. We further show that CP localization to actin patches is required to prevent the formation of ectopic foci and promote efficient cell fusion. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12, normally absent from patches, and are co-decorated by the linear filament-binding protein tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast.

The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT.

Nuclear displacement and fluorescence recovery after photobleaching (FRAP) assays to study division site placement and cytokinesis in fission yeast.

Cytokinesis is an essential cellular event that completes the cell division cycle. It begins with the assembly of an actomyosin contractile ring that undergoes constriction concomitant with the septum formation to divide the cell in two. Placement of the septum at the right position is important to ensure fidelity of the division process. In fission yeast, the medially placed nucleus is a major spatial cue to position the site of division. In this chapter, we describe a simple synthetic biology-based approach to displace the nucleus and study the consequence on division site positioning. We also describe how to perform fluorescence recovery after photobleaching to follow the dynamics of cytokinetic proteins at defined time points by live-cell microscopy.

Are all repeats created equal? Understanding DNA repeats at an individual level.

Repetitive DNA sequences, comprising up to 50 % of the genome in all eukaryotes, play important roles in a wide range of cellular functions, such as transcriptional regulation, genome stability, and cellular differentiation. However, due to technical difficulties in differentiating their sequences, DNA repeats remain one of the most mysterious parts of eukaryotic genomes. Key questions, such as how repetitive entities behave at individual level and how the internal architecture of these repeats is organized, are still poorly understood. Recent advances from our group reveal unexpected position-dependent variation within tandem DNA repeats in fission yeast. Despite sharing identical DNA sequences, the peri-centromeric repeats are organized into diverse epigenetic states and chromatin structures. We demonstrate that this position-dependent variation requires key heterochromatin factors and condensin. Our works further suggest that the peri-centromeric repeats are organized into distinct higher order structures that ensure a proper positioning of CENP-A, the centromere-specific histone H3 variant, to centromeres. These most recent developments offer insights into the mechanisms underlying the position effect within tandem DNA arrays, and have broad implications in the field of epigenetics and chromatin biology.

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell-cell fusion.

Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone-GPCR (G-protein-coupled receptor)-MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell-cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception.

Pom1 gradient buffering through intermolecular auto-phosphorylation.

Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.