Download Spore Full Thai Bit
May 16, 2016 Holy fuck 50k views:O Please like and subscribe for more:D Crack+Game: Winrar: http://www.rarlab.com. However, these stages are an essential part of Spore and it is worth the time and patience that it takes to get through these stages to the later parts of the game. Overall, Spore is a great game and is already receiving rave reviews from a wide range of satisfied gamers. The game is virtually free from bugs and provides a very smooth experience.
What exactly is the Thailand Edition of a PC Game? Is the game labeled as the Thailand Edition simply because it is released to that particular country within the Asian Market or is there more to it than that? What would be different about the game as compared to say the US or UK Edition? Would the games manual be in Thai? What about the games content? Would any dialogue within the game also be in Thai? Would I be allowed to choose english as a language option?
I assume that the packaging might be a little different but it's primarily the games content that I'm concerned about. Can someone help? I bought NeverWinter Nights expac (Shadows of Undrentide or something.) used on the internet and it was from Thailand.
It was full English except for the English and Thailand. Langauge text on the case. I am sure it would depend on the company making the game for if it would have English or ONLY their native language. I am sure a lot of non English speaking countries have the English option.
AgentKaliaver EA was the only publisher with fully-localized games available in Thailand. Since the closure of EA Thailand, all EA titles are being marketed by EA Singapore, who has ordered a stop to all localization work. If I remember correctly, the last localized EA game in Thailand was Burnout Paradise: The Ultimate Box. Luckily, I was able to play it in English thanks to a registry hack. The Thai versions of Crysis and Spore cannot be reverted to English though.
If you go into a PC games store like Zest or i27games, you'll see that all the PC games are labelled 'Thai edition: not for resale outside of Thailand.' The game itself will be in whatever the original language is, but the manual will be in Thai.
EA Games is the exception with the game itself in Thai as well as the manual. They put the 'not for resale' stuff on the boxes because the game publishers have had to sell very cheaply in Thailand in order to have any chance against the bootleggers who hawk pirated DVDs in places like Pantip Plaza. If a seller gets caught putting the stuff up on ebay or mail order, the customs agents in either Thailand or the receiving country are supposed to seize the game, but they rarely do unless one of the major publishers bribes the right official. QUOTE='AgentKaliaver' I bought NeverWinter Nights expac (Shadows of Undrentide or something.) used on the internet and it was from Thailand.
It was full English except for the English and Thailand. Langauge text on the case. I am sure it would depend on the company making the game for if it would have English or ONLY their native language. I am sure a lot of non English speaking countries have the English option. Cqdemal EA was the only publisher with fully-localized games available in Thailand. Since the closure of EA Thailand, all EA titles are being marketed by EA Singapore, who has ordered a stop to all localization work. If I remember correctly, the last localized EA game in Thailand was Burnout Paradise: The Ultimate Box.
Luckily, I was able to play it in English thanks to a registry hack. The Thai versions of Crysis and Spore cannot be reverted to English though.
Actually, you can get Crysis to work in English, but it's a ridiculously involved process. I was able to get it working by cracking open a.cab file, extracting the sounds and replacing them with ones I got from the English language demo. That only partially worked (up until the point the demo ended) but I just got somebody to upload their.cab file from an English version of the game on rapidshare and was able to get the game working. QUOTE='cqdemal'QUOTE='AgentKaliaver' I bought NeverWinter Nights expac (Shadows of Undrentide or something.) used on the internet and it was from Thailand. It was full English except for the English and Thailand. Langauge text on the case. I am sure it would depend on the company making the game for if it would have English or ONLY their native language.
I am sure a lot of non English speaking countries have the English option. EA was the only publisher with fully-localized games available in Thailand. Since the closure of EA Thailand, all EA titles are being marketed by EA Singapore, who has ordered a stop to all localization work. If I remember correctly, the last localized EA game in Thailand was Burnout Paradise: The Ultimate Box. Luckily, I was able to play it in English thanks to a registry hack.
The Thai versions of Crysis and Spore cannot be reverted to English though. That's strange, I bought The Ultimate Box a few weeks ago here in Thailand and it didn't even have a Thai option. The install and game were entirely in English. Not complaining of course, but strange that there wasn't even the option to have it in Thai.
One good thing about buying games here is that they are a lot cheaper, but unfortunately I can't buy anything on Steam because they won't let me use my American credit card here. Stardock's Impulse works fine though. Actually, you can get Crysis to work in English, but it's a ridiculously involved process. I was able to get it working by cracking open a.cab file, extracting the sounds and replacing them with ones I got from the English language demo. That only partially worked (up until the point the demo ended) but I just got somebody to upload their.cab file from an English version of the game on rapidshare and was able to get the game working. Bogaty I opted to download a pirated ISO just for the English files and put in a key from my genuine Thai copy.
A bit of a waste of disk space, but a lot simpler. That's strange, I bought The Ultimate Box a few weeks ago here in Thailand and it didn't even have a Thai option. The install and game were entirely in English. Not complaining of course, but strange that there wasn't even the option to have it in Thai. One good thing about buying games here is that they are a lot cheaper, but unfortunately I can't buy anything on Steam because they won't let me use my American credit card here. Stardock's Impulse works fine though. Bdever32 It was the opposite on mine!
No language options at all, and the installer just gave me the game in Thai. Probably depends on regional settings or something. And yeah, the retail prices are brilliant. Even better than those serial number stores from Europe. Steam download speeds in Thailand suck though. EA Download Manager, on the other hand, is very fast.
Haven't tried Impulse yet.
The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut.
Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D–associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis. INTRODUCTION When deprived of nutrients, diploid yeast cells exit the mitotic cell cycle and undergo sporulation ( ). Unlike cytokinesis in vegetative (mitotically dividing) cells, in which a single plasma membrane is divided in two, sporulating cells in meiosis II initiate de novo synthesis of four new membranes, each of which encapsulates a haploid nucleus and essential cellular components. As the four “haploid” lobes of the nucleus separate, the process of spore morphogenesis begins at the spindle pole bodies (SPBs) implanted in the nuclear envelope (;; ). Prospore membrane (PSM) development proceeds through a series of transitions, beginning as SPB-proximal disks and forming spheres upon closure ( ).
During subsequent spore wall synthesis, mannan and β-1,3-glucan layers are deposited in the lumen formed by the double-bilayer PSM ( ). The outermost two layers, rich in chitosan ( ) and dityrosine ( ), form after breakdown of the outer PSM leaflet and endow spores with resistance to environmental assaults ( ). Septins were first identified in Saccharomyces cerevisiae and are found throughout nonplant eukaryotes (; ). Five septin proteins expressed in vegetative cells (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) assemble into hetero-octamers that interact with the plasma membrane and anneal into a filamentous network (,;; ). Filamentous septin rings at the site of vegetative cytokinesis, the mother–bud neck, act as scaffolds to recruit chitin synthases ( ) and components of the actomyosin ring (;;; ).
Septin rings are also associated with a diffusion barrier function that compartmentalizes regions of the cortex ( ). By contrast, roles for septins in sporulation are poorly understood.
Expression of CDC3, CDC10, and the two sporulation-specific septins SPR3 and SPR28 is highly induced during sporulation via the transcription factor Ndt80 (;; ). The products of these genes colocalize with Cdc11 in PSM-associated higher-order structures (;;;; ), whereas Cdc12 and Shs1 are largely evicted from existing complexes and appear to be replaced by Spr3 and Spr28, respectively ( ). Septin assemblies probably interact with the inner PSM leaflet, because vegetatively synthesized Cdc10 molecules are inherited by mature spores and are recycled for use upon spore germination (; ); were they on the outer leaflet, they would be left behind in the ascal cytoplasm. Others previously found only mild sporulation defects in septin mutants, leading to the idea that septins are unimportant for this process ( ).
Here we carefully analyze PSM dynamics and spore wall composition in mutant cells to identify specific functional requirements for septins during budding yeast sporulation. RESULTS AND DISCUSSION In previous studies, the magnitude of effects of septin mutations on sporulation varied with strain background (; ).
Overall sporulation efficiency was calculated as the frequency of asci per total number of cells. We constructed homozygous deletions of SPR3 or SPR28 in two nearly isogenic strain backgrounds with very different sporulation efficiencies. BY4743 sporulates with relatively low efficiency (37%), but alteration of single nucleotides at three loci in the BY4743 genome (to create an “SKBY” strain) is able to reconstitute nearly all of the high sporulation efficiency of the SK1 strain background (;; ). In both strain backgrounds, sporulation efficiency was reduced drastically (up to 10-fold) in spr3∆/∆ mutants and spr28∆/∆ mutants. Defects in ascus production, spore number per ascus, and prospore membrane morphogenesis in septin-mutant cells.
(A) Overall sporulation efficiencies and spore number per ascus for WT, spr3∆/∆, or spr28∆/∆ cells in two. Septin-mutant cells that were able to form asci typically did not preserve all four meiotic products in the form of tetrads. Triads, dyads, or monads were the most common classes of mutant asci. Loss of even a single copy of SPR3, SPR28, CDC3, or CDC10 significantly decreased overall efficiency and spore number per ascus ; by contrast, CDC3 and CDC10 are haplosufficient in vegetative cells ( ). These data demonstrate that sporulation is particularly dependent on septin expression and agree with the results of unbiased screening in which BY4743 spr3∆/∆ or cdc10∆/∆ mutants displayed severe decreases in sporulation efficiency (; ). Variations in genetic background among different lab strains are known ( BUD4; ) or likely ( SSD1; ) to influence the severity of vegetative defects accompanying septin mutation; these or other differences may explain why other studies reported milder sporulation phenotypes. In addition, we used a medium and temperature determined empirically to be optimal for wild-type (WT) BY4743 sporulation, which could involve a particular dependence on septin function.
However, unbiased analysis of natural isolates of S. Cerevisiae identified as a candidate regulator of sporulation efficiency a polymorphism within the CDC10 promoter ( ), which we noticed lies within the Ndt80 binding-site consensus sequence (CACAAA T;; polymorphic base in bold).
Thus, rather than representing minor players in budding yeast sporulation, alterations in septin gene expression during sporulation may in fact drive evolutionary differences in gametogenesis. When defects occur early in sporulation, the two haploid nuclei adjacent to the two “newest” SPBs are the most likely to be encased in PSMs and spore walls ( ), leading to the preferential inheritance in dyads of one of each of any heterozygous alleles at centromere-linked loci (i.e., “nonsister” dyads NSDs). By contrast, defects arising after PSM biogenesis produce dyads with a random pair of meiotic products (; ). We found that NSDs were strongly enriched in spr3∆/∆ and spr28∆/∆ mutant asci (Supplemental Figure S1). Thus defects in septin-mutant cells arise as soon as PSM biogenesis begins. To visualize PSMs in septin-mutant cells, we used a fusion of green fluorescent protein (GFP) to Spr1, which localizes to the lumen between the two PSM bilayers and eventually resides in the mature spore wall (; ).
After 24 h in sporulation medium, all PSMs within a given WT cell were similar in size and phase of growth. In stark contrast, the growth of PSMs in spr3∆/∆ and spr28∆/∆ cultures was severely perturbed. PSMs within a single ascus were differently sized and asynchronous in development, and some PSMs collapsed into bright foci. A large proportion (∼30%) of spr3∆/∆ mutants had PSMs in which Spr1-GFP was unevenly distributed around the prospore perimeter; this was almost never observed in WT cells. Another subset of spr3∆/∆ and spr28∆/∆ mutant cells displayed a characteristic of abnormally permeable spore walls ( ): Spr1-GFP, which normally remains restricted to the spore periphery, “leaked” out into the ascal cytoplasm.
Of importance, although mammalian septins help to coordinate cytokinesis with mitotic chromosome segregation ( ), in yeast, even severe defects in the fidelity of meiotic chromosome segregation do not perturb PSM biogenesis (;; ). Indeed, coexpression of a fluorescently tagged histone with Spr1-GFP confirmed that four postmeiosis II nuclear “lobes” formed normally in septin mutants, but PSMs often failed to develop around individual lobes. Finally, monitoring chromosome segregation by fluorescently labeling both a centromere-linked locus (using the tetO/TetR-GFP system; ) and SPBs (with DsRed-tagged Spc110) in spr3∆/ SPR3 heterozygous cells revealed that the tagged chromosome segregated properly in each of 34 dyads (24 of which were shown by this assay to be NSDs; unpublished data). Thus defects in meiosis per se cannot explain the PSM defects in septin mutants. Three-dimensional (3D) renderings created from serial images through the z-plane revealed that in cells with the “membrane extension” defect, a significant degree of PSM extension occurred before collapse, creating dense, torpedo-shaped structures.
Negative-stain electron microscopy (EM) confirmed that in septin mutants, PSMs developed asynchronously and were frequently misdirected in ways that clearly corresponded to the distinct defects seen by light microscopy. PSMs that had either collapsed onto or grown in a direction away from the nucleus likely correspond to the Spr1-GFP signals that appeared as small foci in two-dimensional images and as torpedo-shaped structures in 3D reconstructions. Prospores in which multiple layers of membrane were stacked on one side, and others in which the width of the lumen varied widely, likely correspond to the “uneven distribution” Spr1-GFP category. Together, these images indicate that septins are critical for proper PSM growth—particularly for ensuring a constant lumen size and maintaining normal PSM distance from the nuclear envelope—and are consistent with the observed decreases in sporulation efficiency and spore number per ascus. These defects are also highly reminiscent of defects in forespore membrane (equivalent to the PSM) orientation in septin-mutant fission yeast cells ( ).
Many spr3∆/∆ and spr28∆/∆ prospores that had apparently succeeded in PSM morphogenesis nonetheless had spore walls that lacked the trilaminar appearance of WT spores (; ). PSM biogenesis is necessary but not sufficient to ensure proper spore wall deposition. Indeed, spr3∆/∆ and spr28∆/∆ spores within tetrads contained significantly less dityrosine and chitosan than WT but deposited equivalent amounts of inner spore wall layer mannan. The acquisition of spore refractility in light microscopy occurs just before chitosan synthesis ( ); because the spores we analyzed were refractile, this places the earliest spore wall defect in septin-mutant cells at the chitosan stage. Thus septin-mutant prospores inefficiently deposit the outer spore wall layers, which are primarily responsible for resistance to environmental insults ( ).
We found that, whereas WT spores retained their refractility to transmitted light, spore walls in spr3∆/∆ and spr28∆/∆ asci disappeared after exposure to either of two ecologically relevant stressors, freeze–thaw and high heat. Examination of Spr1-GFP revealed that wall disappearance was accompanied by apparent PSM collapse. Thus impaired wall biogenesis in the absence of septins is manifested at the organismal level as increased susceptibility to harsh environmental conditions.
Spore wall defects in septin-mutant cells. (A) EM micrographs as in. (B) Representative images of spores of the indicated genotypes stained with fluorescein–Con A (mannan), eosin Y (chitosan), or autofluorescence in unstained cells visualized. For yeast cells, the crops of insects represent a refuge from the environment ( ), but reemergence requires passage through the intestinal tract. Previous research suggested that whereas vegetative S. Cerevisiae cells excreted by Drosophila are mostly inviable, spores with properly formed walls are resistant to this killing process (;; ).
To ask whether the wall defects we observed in septin-mutant spores compromise survival during insect digestion, we fed starved, WT Drosophila a mix of WT and spr3∆/∆ sporulation cultures (adjusted to 50% total spr3∆/∆ spores) and examined the cells deposited in their feces. As expected, each fecal deposit was primarily composed of yeast, among which asci were clearly visible ( and Supplemental Figure S2B). To determine whether the survivors were vegetative cells or spores, we tested clones arising from resuspended feces spread on a plate for heterozygosity at the MAT, MET17, and LYS2 loci, genes that were heterozygous in both starting diploid strains.
Whereas spontaneous mitotic loss of heterozygosity is rare, meiosis ensures that each spore inherits a single allele. Seventy percent (16 of 23) of surviving clones had a single allele at one or more of these loci (, and Supplemental Figure S2). Twenty-two percent (2 of 9) of MAT a/α diploids had only met17∆ or lys2∆, similar to the 25% expected from interascus mating. Thus nearly all survivors were spores. Crucially, although ∼60% of cells and 50% of spores entering the Drosophila gut carried the spr3∆ allele, only one of the 23 surviving clones did (4%) , indicating strong selection during insect digestion against spores produced by spr3∆/∆ cells. The leading-edge protein (LEP) complex found at the “lip” of the growing PSM is believed to guide proper orientation of PSM extension (; ).
Among known LEP components, Ssp1 is essential for PSM closure and recruitment of the LEP Ady3 ( ). Defects in ssp1∆/∆ mutants are reminiscent of those we describe for septin mutants, including torpedo-shaped prospores, PSMs that extend in the wrong directions, and incompletely synthesized walls ( ). Ssp1 has even been proposed to be a “divergent septin” that performs septin-like functions during sporulation ( ). Septins are able to form higher-order structures in ssp1∆/∆ cells ( ), but their association with PSMs is perturbed (Supplemental Figure S3). Whether septins are required for efficient LEP localization was unknown.
Ssp1 does not tolerate standard GFP fusions ( ), so we used Ady3-GFP as a reporter for LEP localization. In WT cells, Ady3-GFP localized primarily to rings, usually four per ascus , as expected (; ). In cells lacking SPR3 or SPR28, on the other hand, Ady3-GFP was most frequently found in numerous small puncta. Indistinguishable aberrant Ady3 “precursor structures” have been described for other mutants (; ). Of note, dyads formed in ady3∆ mutants incorporate a random pair of meiotic products, consistent with a primary defect in wall production, not PSM biogenesis (; ). The NSDs and more severe PSM defects that we found in septin mutants thus suggest that proper localization of Ssp1 (which does not require Ady3; ) is also perturbed when septins are mutated, providing a plausible rationale for at least some of the PSM morphogenesis and spore wall defects we observed in septin-mutant cells.
LEP complex mislocalization in septin mutants and septin mislocalization upon ectopic LEP expression. (A) Representative images of cells of the indicated genotypes expressing Ady3-GFP. Arrowheads, presumptive rings. (B) As in, for SPR3+/∆. If Ssp1 mislocalization underlies PSM and wall defects in septin-mutant cells, then Ssp1 overexpression might drive proper Ssp1 localization in septin-mutant cells and improve sporulation. When overexpressed in SPR3+/∆ heterozygotes, Spr3 increased sporulation efficiency and spore number per ascus , as expected, but Ssp1 expressed in an identical manner had no effect on sporulation and instead induced severe defects in vegetative septin localization and function (i.e., elongated cellular morphologies; ).
Although Ssp1 can form rare cytosolic rings in vegetative cells ( ), it did not colocalize with the ectopic septin structures in our Ssp1-expressing cells. Thus the requirement for septins in Ssp1 function cannot be overcome solely by increasing Ssp1 levels, and functional Ssp1–septin interactions are likely indirect. Cells lacking the PSM-associated protein Sma1 form few spores because PSM extension does not proceed far beyond the SPB (; ). As in septin mutants, LEPs mislocalize in sma1∆/∆ cells (; ). To our surprise, in sma1∆/∆ diploids carrying Spr1-GFP and Cdc10–red fluorescent protein (RFP) plasmids, a large proportion of cells achieved PSM extension, proper Cdc10-RFP localization, and formation of mature spores. However, within each culture, there was also a population of cells that exhibited the expected PSM extension defect, and these cells always lacked Cdc10-RFP. This result suggested that the presence of extra, plasmid-encoded Cdc10 in sma1∆/∆ cells promoted extension of the otherwise stunted PSMs.
Experiments with distinct plasmids and controls showed that extra copies of CDC3 or CDC10, but not of CDC12 or the empty vector, restored spore-forming ability to sma1∆/∆ mutants but had no effect on sporulation in mutants lacking SSP1 or any of a number of other genes implicated in this process (Supplemental Figure S3; unpublished data). A CDC10 plasmid failed to improve sma1∆/∆ sporulation in the absence of SPR3 (no visible spore among 280 apparent asci), indicating that the CDC10 rescue required assembly of Spr3-containing septin complexes. Additional studies will be required to understand at a mechanistic level how septin function can be altered by increasing the expression of only a single subunit of a complex believed to function as a stoichiometric hetero-oligomer.
Septin overexpression rescues LEP localization and sporulation in PSM-extension-deficient sma1∆/∆ cells. (A) As in, but from cultures of WT or sma1∆/∆ cells expressing Spr1-GFP with or without Cdc10-RFP. Without extra copies of CDC10, Ady3-GFP in sma1∆/∆ cells localized almost exclusively to aberrant “cap”-like PSM structures near the SPBs (; ). By contrast, a CDC10 plasmid restored Ady3-GFP localization to the characteristic LEP rings in a sizeable subset of sma1∆/∆ cells. Sma1∆/∆ rescue by CDC10 could reflect improved ability of the PSM to extend in an LEP-independent manner. However, we believe that these findings are more consistent with a model in which septins promote PSM extension by facilitating proper LEP localization. In summary, we conclude that septins are critical factors in budding yeast gametogenesis.
Without them, PSM biogenesis is severely perturbed, and walls that are produced do not provide the stress resistance that is key for this stage of the yeast life cycle. Intriguingly, septins are also required for gametogenesis in mammals ( ), but the precise roles that septins play remain unclear. Our study provides a foundation for investigations of additional mechanistic details of septin functions during gametogenesis in yeast and beyond. Sporulation conditions Diploid strains were grown in liquid yeast extract/peptone/dextrose (YPD) medium to saturation. A 250-μl amount of the culture was pelleted, washed twice with water, and resuspended in 2 ml of sporulation medium (1% potassium acetate, 0.05% dextrose, 20 mg/ml leucine, 40 mg/ml uracil). Cells carrying plasmids were grown in synthetic defined dextrose medium lacking appropriate nutrients for 1 d and then switched to YPD for 18 h before washing and transferring to sporulation medium.
The CDC3, CDC12, and non–RFP-tagged CDC10 plasmids used in the sma1∆/∆ rescue experiments were pMVB100, pMVB66, and pLA10, respectively; the empty vector was pRS316. Cells carrying plasmids with glutathione S-transferase (GST)–hexahistidine (His 6)-tagged open reading frames under control of the GAL1/10 promoter were prepared the same, way except they were switched to YP medium with 2% raffinose for 5 h and then to YP with 1% galactose and 1% raffinose for 18 h before being washed and transferred to sporulation medium. Cultures were incubated at room temperature (∼22°C). Depending on strain background (SKBY or BY4743), cultures were sporulated for 2–5 d before examination.
Sporulation efficiency and spore counting Mature spore walls are refractile when viewed by transmitted light. Spores were thus identified as refractile spheres 1–5 μm in diameter. The accuracy of this scoring method was validated in two ways: 1) using a strain expressing a fluorescently tagged histone to identify the four-lobed meiosis II nucleus (unpublished data) and 2) using fluorescent signals generated from the Spr1-GFP plasmid, whose expression is restricted to sporulating cells (; unpublished data). At least 200 cells were scored for each culture. Microscope image acquisition All single-focal-plane images were acquired using an EVOSfl inverted fluorescence microscope (ThermoFisher Scientific, Waltham, MA) with 10× dry, 40× dry, and 60× oil objectives. Filter cubes were as follows: 4′,6-diamidino-2-phenylindole (DAPI; excitation 357/44 nm, emission 447/60 nm), Texas red (excitation 585 nm, emission 624 nm), yellow fluorescent protein (excitation 500 nm, emission 542 nm), and GFP (excitation 470/22 nm, emission 510/42 nm). Images were cropped, contrast adjusted, and inverted in Photoshop (Adobe, San Jose, CA).
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Magenta-cyan merged images were generated in ImageJ ( ) by preparing a standard red-green merged image from raw white-on-black images and then converting to RGB and inverting the image. For z-stack and 3D projections, images were acquired using a Nikon (Melville, NY) Ti-E microscope with a 1.45 numerical aperture 100× CFI Plan Apo objective, piezoelectric stage (Physik Instrumente, Auburn, MA), spinning-disk confocal scanner unit (CSU10; Yokogawa, Tokyo, Japan), 488- and 561-nm lasers (Agilent Technologies, Santa Clara, CA), and an electron-multiplying charge-coupled device camera (iXon Ultra 897; Andor Technology, Belfast, United Kingdom) using NIS Elements software (Nikon). Image z-stacks were created by capturing consecutive images at 0.3-μm intervals. The z-stack and 3D stack projections were created using Fiji software ( ).
Image stacks were imported as 16-bit files, cropped, and compiled to make either a z-stack projection or 3D renderings. The z-stacks modeled as 3D renderings were cropped to size, filtered with a gaussian blur of 1.0, converted to an 8-bit image, and projected using the 3D viewer plug-in for Fiji. Electron microscopy BY4743-background cells were grown to saturation in 5 ml of YPD. A 1-ml amount of saturated culture was pelleted, washed twice with water, and inoculated into 25 ml of sporulation medium. Cultures were sporulated for 30 h and prepared as described previously ( ).
Cells were collected by filtration, and the resulting paste was rapidly frozen under high pressure in a BAL-TEC HPM-010 high-pressure freezer (Technotrade International, Manchester, NH). The frozen cells were freeze substituted into acetone containing 2% osmium tetroxide and 0.1% uranyl acetate at –80°C and then warmed to room temperature. Samples were embedded in Spurr’s resin per manufacturer’s instructions (Electron Microscopy Sciences, Port Washington, PA). Embedded cells were serially sectioned at a thickness of 50 nm, and the sections were stained with 2% uranyl acetate in 70% methanol and lead citrate. Sections were viewed on a Philips CM10 electron microscope (Philips, Eindhoven, Netherlands), and images were collected with a Gatan BioScan digital camera by using the Digital Micrograph software package (Gatan, Pleasanton, CA). Analysis of cell wall composition Relative dityrosine fluorescence was calculated from images taken using the DAPI filter. Images were acquired at 70% intensity for 1 s.
For chitosan analysis, spores were pelleted, washed twice with McIlvaine’s buffer (0.2 M sodium phosphate, 0.1 M citric acid, pH 6.0) and resuspended in McIlvaine’s buffer and 30 μl of eosin Y (5 mg/ml in water; VWR 22816; ). Samples were incubated for 10 min in darkness at room temperature. After incubation, spores were pelleted, washed twice, and resuspended in the buffer before imaging with the GFP filter.
For mannan analysis, spores were pelleted and resuspended in 50 μl of 5 mg/ml 20T Zymolyase (0832092; MP Biomedicals, Santa Ana, CA) to remove the cell wall. Spores were incubated at 30°C for 10 min. Samples were again pelleted and resuspended in water with 0.1 mg/ml concanavalin A (Con A) coupled to fluorescein (C827; Life Technologies, Carlsbad, CA) before imaging with the GFP filter. For all images, using the line scan function in Fiji, an 8 pixel–wide line was drawn from outside of the ascus, across two spores, and back out of the cell. Maximum fluorescence (the spore walls) was subtracted from minimum fluorescence (slide background) to yield a background-corrected value. Spore survival during passage through Drosophila A 25-ml amount of molten 2% agar made in water was poured into a 100 mm × 15 mm Petri dish (FB0875712; Fisherbrand; Fisher Scientific, Pittsburgh, PA).
The sporulation efficiency of cultures of WT (strain BY4743 carrying plasmid pSPR1-GFP) or spr3∆/∆ cells (strain MMY0200) was calculated by examining aliquots microscopically (WT, 67 spores/157 cells; spr3∆/∆, 39 spores/197 cells) and used to prepare a mixture of cultures containing 50% WT and 50% spr3∆ spores (66% spr3∆/∆ culture, 33% WT culture). A 100-μl amount of this mixture was spotted in the center of the polymerized, cooled agar. Approximately 50 adult male flies were transferred to a vial containing a half-circle of 7.0-cm Whatman Qualitative #3 filter paper (1003070) and 500 μl of sterile water. After 6.5 h at 25°C, 30 flies were transferred directly to the Petri dish containing agar and yeast. After overnight incubation at 25°C, flies were anesthetized with CO 2 gas and removed. The lid of the plate was scanned with a Typhoon imager (600 PMT, 532-nm laser, no emission filter; GE Healthcare, Pittsburgh, PA) to determine the overall number of fecal deposits, several of which were then visualized with 10× and 40× objectives on the EVOSfl microscope. Each of the deposits was resuspended in 1 μl of sterile water, and these were pooled together.
An aliquot of 5 μl was spread on the surface of a YPD agar plate. Note that of 50 individual asci that were micromanipulated to defined positions in a grid pattern on the plate, only one could be dissected, and none of these isolated spores or the 49 undissected asci produced a colony after 7 d incubation at 30°C, suggesting that survival and ascus wall removal are very inefficient.
Of the ∼25 colonies that emerged from the unmanipulated fecal spread, 23 were picked and analyzed for marker phenotypes on appropriate media. In addition, multiplex colony PCR ( ) was performed to determine the genotype at MAT. For the two reactions that failed, mating type was determined by mixing the clones with other clones of known, complementary genotypes at MET17 and LYS2 and selecting for diploids prototrophic for methionine and lysine.
Immunofluorescence Immunofluorescence was performed as described elsewhere ( ), with the following modifications. Diploid cells made by mating MMY0184 and BY4742 and carrying plasmids for SPR3, SSP1, or URA3 overexpression were grown in synthetic dropout medium lacking uracil overday and switched to YP medium with 2% raffinose for 5 h and then to YP with 1% galactose and 1% raffinose for 18 h before spheroplasting. Spheroplasts were fixed in 4% formaldehyde for 15 min at 30°C and mounted on glass coverslips coated with poly- l-lysine (P8920; Sigma-Aldrich, St.
After blocking, cells were incubated for 1 h with anti-His 6 monoclonal antibody (mAb; Y1011; Ubiquitin-Proteasome Biotechnologies, Aurora, CO) diluted 1:1000 in 50 mM Tris, pH 8, 150 mM NaCl, 1% nonfat dry milk, 0.5 mg/ml bovine serum albumin, and 0.1% Tween 20 (buffer B). Coverslips were washed five times in buffer B and then incubated for 1 h with Alexa Fluor 594 goat anti-mouse mAb (1:2000). Cells were washed five times with buffer B and mounted onto slides with mounting medium (90% glycerol). We thank Fred Winston, JoAnne Engebrecht, Michael Knop, Mark Johnston, and Jeremy Thorner for sharing reagents, Jeff Moore for reagents and use of the confocal microscope, Tom Giddings for electron microscopy, members of Tania Reis’s lab for help with the Drosophila experiments, and Courtney Johnson and Rachel Schaefer for technical assistance. Research reported here was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Awards R00GM086603 (to M.A.M.) and T32GM008730 (to L.R.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.