Structural basis for transthiolation intermediates in the ubiquitin pathway.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins1-3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters4-6. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.

Nuclear pores dilate and constrict in cellulo.

In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryo­electron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.

Analysis and application of a suite of recombinant endo-ß(1,3)-D-glucanases for studying fungal cell walls.

BACKGROUND: The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. RESULTS: Here we examined the efficiency and reproducibility of 12 recombinant endo-ß(1,3)-D-glucanases for specifically degrading the cell wall ß(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, ß(1,3)-D-glucan and ß(1,6)-D-glucan. We found that the GH16 endo-ß(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade ß(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-ß(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant ß(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. CONCLUSIONS: The discovery of a reliable and efficient recombinant endo-ß(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall ß(1,3)-D-glucan. This new commercial endo-ß(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.

Characterization of the nanomechanical properties of the fission yeast (Schizosaccharomyces pombe) cell surface by atomic force microscopy.

Variations in cell wall composition and biomechanical properties can contribute to the cellular plasticity required during complex processes such as polarized growth and elongation in microbial cells. This study utilizes atomic force microscopy (AFM) to map the cell surface topography of fission yeast, Schizosaccharomyces pombe, at the pole regions and to characterize the biophysical properties within these regions under physiological, hydrated conditions. High-resolution images acquired from AFM topographic scanning reveal decreased surface roughness at the cell poles. Force extension curves acquired by nanoindentation probing with AFM cantilever tips under low applied force revealed increased cell wall deformation and decreased cellular stiffness (cellular spring constant) at cell poles (17 ± 4 mN/m) relative to the main body of the cell that is not undergoing growth and expansion (44 ± 10 mN/m). These findings suggest that the increased deformation and decreased stiffness at regions of polarized growth at fission yeast cell poles provide the plasticity necessary for cellular extension. This study provides a direct biophysical characterization of the S. pombe cell surface by AFM, and it provides a foundation for future investigation of how the surface topography and local nanomechanical properties vary during different cellular processes.

Closed mitosis requires local disassembly of the nuclear envelope.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Roles of Mso1 and the SM protein Sec1 in efficient vesicle fusion during fission yeast cytokinesis.

Membrane trafficking during cytokinesis is essential for the delivery of membrane lipids and cargoes to the division site. However, the molecular mechanisms are still incompletely understood. In this study, we demonstrate the importance of uncharacterized fission yeast proteins Mso1 and Sec1 in membrane trafficking during cytokinesis. Fission yeast Mso1 shares homology with budding yeast Mso1 and human Mint1, proteins that interact with Sec1/Munc18 family proteins during vesicle fusion. Sec1/Munc18 proteins and their interactors are important regulators of SNARE complex formation during vesicle fusion. The roles of these proteins in vesicle trafficking during cytokinesis have been barely studied. Here, we show that fission yeast Mso1 is also a Sec1-binding protein and Mso1 and Sec1 localize to the division site interdependently during cytokinesis. The loss of Sec1 localization in mso1Δ cells results in a decrease in vesicle fusion and cytokinesis defects such as slow ring constriction, defective ring disassembly, and delayed plasma membrane closure. We also find that Mso1 and Sec1 may have functions independent of the exocyst tethering complex on the plasma membrane at the division site. Together, Mso1 and Sec1 play essential roles in regulating vesicle fusion and cargo delivery at the division site during cytokinesis.

Precise correlative method of Cryo-SXT and Cryo-FM for organelle identification.

Cryogenic soft X-ray tomography (Cryo-SXT) is ideally suitable to image the 3D sub-cellular architecture and organization of cells with high resolution in the near-native preservation state. Cryogenic fluorescence microscopy (Cryo-FM) can determine the location of a molecule of interest that has been labeled with a fluorescent tag, thus revealing the function of the cells. To understand the relations between the sub-cellular architecture and the function of cells, correlative Cryo-SXT and Cryo-FM was applied. This method required the matching of images of different modalities, and the accuracy of the matching is important. Here, a precise correlative method of Cryo-SXT and Cryo-FM is introduced. The capability of matching images of different modalities with high resolution was verified by simulations and practical experiments, and the method was used to identify vacuoles and mitochondria.

Coordinating septum formation and the actomyosin ring during cytokinesis in Schizosaccharomyces pombe.

During cytokinesis, animal and fungal cells form a membrane furrow via actomyosin ring constriction. Our understanding of actomyosin ring-driven cytokinesis stems extensively from the fission yeast model system. However, unlike animal cells, actomyosin ring constriction occurs simultaneously with septum formation in fungi. While the formation of an actomyosin ring is essential for cytokinesis in fission yeast, proper furrow formation also requires septum deposition. The molecular mechanisms of spatiotemporal coordination of septum deposition with actomyosin ring constriction are poorly understood. Although the role of the actomyosin ring as a mechanical structure driving furrow formation is better understood, its role as a spatiotemporal landmark for septum deposition is not widely discussed. Here we review and discuss the recent advances describing how the actomyosin ring spatiotemporally regulates membrane traffic to promote septum-driven cytokinesis in fission yeast. Finally, we explore emerging questions in cytokinesis, and discuss the role of extracellular matrix during cytokinesis in other organisms.

Functional Analysis of Conserved Transmembrane Charged Residues and a Yeast Specific Extracellular Loop of the Plasma Membrane Na+/H+ Antiporter of Schizosaccharomyces pombe.

The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu74 and Arg77 of transmembrane segment 3. Glu74 and Arg77 are conserved in yeast species while Glu74 is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg341 and Arg342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp389, Glu390, Glu392, Glu397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na+/H+ exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function.

Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system.

Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12-ATG5 complex retains the ability to facilitate ATG8-phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.

Set7 Is a H3K37 Methyltransferase in Schizosaccharomyces pombe and Is Required for Proper Gametogenesis.

Histone methylation by histone methyltransferases (HMTases) has a key role in transcriptional regulation. Discrepancies between the known HMTases and the histone lysine methylome suggest that HMTases remain to be identified. Here we report the discovery, characterization, and crystal structure of Schizosaccharomyces pombe Set7, an HMTase methylating the uncharted histone H3 lysine 37 (H3K37) mark. Set7 forms a dimer with its substrate-binding site structurally specific to K37, not the neighboring well-studied K36 mark. We also discovered that H3K37 methylation levels dramatically increase during gametogenesis. Set7 deletion mutant cells show defects in gametogenesis and produce the abnormal number of spores with aberrant morphology. S. pombe gametogenesis shares similarities with mammalian spermatogenesis. These findings extend our understanding of epigenetic regulation during gametogenesis and support a link between Set7, the epigenetic H3K37 methyl mark, and proper gametogenesis.

Fission yeast Adf1 is necessary for reassembly of actin filaments into the contractile ring during cytokinesis.

ADF/cofilin family proteins quickly disassemble actin in vitro, and are thought to be involved in various actin dynamics in the cell. Adf1 is a member of this family proteins expressed in fission yeast, and is thought to play roles in actin patch dynamics and also contractile ring formation during cytokinesis. We aimed to understand the function of this protein in cytokinesis in detail using the temperature-sensitive mutant adf1-1. Adf1 inactivation at a restrictive temperature during late G2 phase led to a clustering of actin patches at the cell ends. It was apparent that the inactivation occurred only in a few minutes. Furthermore, we found that the actin clusters migrated to the division site during anaphase possibly by the function of both myosin 5-1 and a myosin II. The migrated actin clusters, however, were not organized into the contractile ring. When Adf1 was inactivated at mid-anaphase B before contractile ring assembly, the ring was not formed, but it was formed when Adf1 was inactivated after this point. We conclude that Adf1 functions in the interphase actin dynamics and formation of the contractile ring during mitosis.

Fission yeast telosomes: non-canonical histone-containing chromatin structures dependent on shelterin and RNA.

Despite the prime importance of telomeres in chromosome stability, significant mysteries surround the architecture of telomeric chromatin. Through micrococcal nuclease mapping, we show that fission yeast chromosome ends are assembled into distinct protected structures ('telosomes') encompassing the telomeric DNA repeats and over half a kilobase of subtelomeric DNA. Telosome formation depends on the conserved telomeric proteins Taz1 and Rap1, and surprisingly, RNA. Although yeast telomeres have long been thought to be free of histones, we show that this is not the case; telomere repeats contain histones. While telomeric histone H3 bears the heterochromatic lys9-methyl mark, we show that this mark is dispensable for telosome formation. Therefore, telomeric chromatin is organized at an architectural level, in which telomere-binding proteins and RNAs impose a unique nucleosome arrangement, and a second level, in which histone modifications are superimposed upon the higher order architecture.

Dependency relationships within the fission yeast polarity network.

The ability to regulate polarised cell growth is crucial to maintain the viability of cells. Growth is modulated to facilitate essential cell functions and respond to the external environment. Failure to do so can lead to numerous developmental and disease states, including cancer. We have undertaken a detailed analysis of the regulatory interplay between molecules involved in the regulation and maintenance of polarised cell growth within fission yeast. Internally controlled live cell imaging was used to examine interactions between 10 key polarity proteins. Analysis reveals interplay between the microtubule and actin cytoskeletons, as well as multiple novel dependency pathways and feedback networks between groups of proteins. This study provides important insights into the conserved regulation of polarised cell growth within eukaryotes.

Role of nucleocytoplasmic transport in interphase microtubule organization in fission yeast.

The proper organization of microtubules is essential for many cellular functions. Microtubule organization and reorganization are highly regulated during the cell cycle, but the underlying mechanisms remain elusive. Here we characterized unusual interphase microtubule organization in fission yeast nuclear export mutant crm1-124. The mutant cells have an intranuclear microtubule bundle during interphase that pushes the nuclear envelope to assume a protruding morphology. We showed that the formation of this protruding microtubule bundle requires the nuclear accumulation of two microtubule-associated proteins (MAPs), Alp14/TOG and Mal3/EB1. Interestingly, the forced accumulation of Alp14 in the nucleus of wild type cells is sufficient to form the intranuclear microtubule bundle. Furthermore, the frequency of the intranuclear microtubule formation by Alp14 accumulated in the nucleus is prominently increased by a reduction in the nucleation activity of interphase cytoplasmic microtubules. We propose that properly regulated nucleocytoplasmic transport and maintained activity of cytoplasmic microtubule nucleation during interphase are important for the proper organization of interphase cytoplasmic microtubules.

Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments.

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

Specific detection of fission yeast primary septum reveals septum and cleavage furrow ingression during early anaphase independent of mitosis completion.

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival.

Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin-tropomyosin copolymer.

The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine-tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein.

Conserved and unique features of the fission yeast core Atg1 complex.

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.