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.

Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy.

Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage.

The tetraspan protein Dni1p is required for correct membrane organization and cell wall remodelling during mating in Schizosaccharomyces pombe.

In fungi, success of mating requires that both cells agglutinate, modify their extracellular envelopes, and fuse their plasma membranes and nuclei to produce a zygote. Here we studied the role of the Schizosaccharomyces pombe Dni1 protein in the cell fusion step of mating. Dni1p is a tetraspan protein bearing a conserved cystein motif similar to that present in fungal claudin-related proteins. Dni1p expression is induced during mating and Dni1p concentrates as discrete patches at the cell-cell contact area and along the mating bridge. Proper Dni1p localization depends on Fus1p, actin and integrity of lipid rafts. In dni1Delta mutants, cell differentiation and agglutination are as efficient as in the wild-type strain, but cell fusion is significantly reduced at temperatures above 25 degrees C. We found that the defect in cell fusion was not associated with an altered cytoskeleton, with an abnormal distribution of Fus1p, or with a defect in calcium accumulation, but with a severe disorganization of the plasma membrane and cell wall at the area of cell-cell contact. These results show that Dni1p plays a relevant role in co-ordinating membrane organization and cell wall remodelling during mating, a function that has not been described for other proteins in the fission yeast.

Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation.

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.

Deciphering the relationship among phosphate dynamics, electron-dense body and lipid accumulation in the green alga Parachlorella kessleri.

Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.

Fission yeast autophagy induced by nitrogen starvation generates a nitrogen source that drives adaptation processes.

Autophagy is a conserved bulk protein degradation process that is proposed to play a role in events that arise when organisms are forced to radically change their fate, including nutritional starvation, differentiation and development. In our present study, we have identified fission yeast autophagy as a bulk protein degradation process induced by the deprivation of environmental nitrogen, the effects of which are known to trigger sexual differentiation as an adaptive response. Autophagy-defective mutants were found to be sterile in the absence of environmental nitrogen, but could complete sexual differentiation when nitrogen was supplied, suggesting that the major function of autophagy is to provide a nitrogen source. In addition, the environmental nitrogen levels act as an autophagy "on/off" switch, whereas components essential for sexual differentiation were dispensable for this regulation. We propose that fission yeast autophagy functions to supply nitrogen and is activated when cells cannot access exogenous nitrogen, thus ensuring that they can adapt and subsequently propagate.

The Arabidopsis D-type cyclin CYCD4 controls cell division in the stomatal lineage of the hypocotyl epidermis.

Cyclin D (CYCD) plays an important role in cell cycle progression and reentry in response to external signals. Here, we demonstrate that Arabidopsis thaliana CYCD4 is associated with specific cell divisions in the hypocotyl. We observed that cycd4 T-DNA insertion mutants had a reduced number of nonprotruding cells and stomata in the hypocotyl epidermis. Conversely, CYCD4 overexpression enhanced cell division in nonprotruding cell files in the upper region of the hypocotyls, where stomata are usually formed in wild-type plants. The overproliferative cells were of stomatal lineage, which is marked by the expression of the TOO MANY MOUTHS gene, but unlike the meristemoids, most of them were not triangular. Although the phytohormone gibberellin promoted stomatal differentiation in the hypocotyl, inhibition of gibberellin biosynthesis did not prevent CYCD4 from inducing cell division. These results suggested that CYCD4 has a specialized function in the proliferation of stomatal lineage progenitors rather than in stomatal differentiation. We propose that CYCD4 controls cell division in the initial step of stomata formation in the hypocotyl.

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation.

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy.

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.

A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions.

The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.

In vitro activity of tebipenem, a new oral carbapenem antibiotic, against penicillin-nonsusceptible Streptococcus pneumoniae.

The in vitro activity of tebipenem (TBM), a new oral carbapenem antibiotic, against Streptococcus pneumoniae clinical isolates (n = 202) was compared with those of 15 reference agents. The isolates were classified into five genotypic classes after PCR identification of abnormal pbp1a, pbp2x, and pbp2b genes: (i) penicillin-susceptible S. pneumoniae (PSSP) isolates with no abnormal pbp genes (n = 34; 16.8%), (ii) genotypic penicillin-intermediate S. pneumoniae (gPISP) isolates with only an abnormal pbp2x gene [gPISP (2x)] (n = 48; 23.8%), (iii) gPISP isolates with abnormal pbp1a and pbp2x genes (n = 32; 15.8%), (iv) gPISP isolates with abnormal pbp2x and pbp2b genes (n = 16; 7.9%), and (v) genotypic penicillin-resistant S. pneumoniae (gPRSP) isolates with three abnormal pbp genes (n = 72; 35.6%). The majority of the strains tested had mefA (n = 59; 29.2%) or ermB (n = 91; 45%) gene-mediating macrolide resistance. For these isolates the MIC at which 90% of isolates are inhibited was significantly lower for TBM than for the reference oral antibiotics, as follows: 0.002 microg/ml for PSSP, 0.004 mug/ml for gPISP (2x), 0.016 microg/ml for gPISP (isolates with abnormal pbp1a and pbp2x genes and isolates with abnormal pbp2x and pbp2b genes), and 0.063 microg/ml for gPRSP. In addition, TBM showed excellent bactericidal activity against gPRSP isolates, which exhibited a 3-log(10) decrease within 2 h when they were incubated with a concentration greater than or equal to the MIC. Inhibition of cell wall synthesis toward the long axis and subsequent cell lysis were observed by scanning electron microscopy after a short-term exposure to TBM, unlike the effects seen with cephalosporins. These data suggest that TBM has potent activity against multidrug-resistant S. pneumoniae, the causative pathogen of community-acquired respiratory tract infections.

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells.

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.

Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy.

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.