Two Rabs, Ypt3 and Ypt2, regulating the trafficking of Golgi-derived secretory vesicles have key roles in biogenesis of the spore plasma membrane in fission yeast. During sporulation, the Rabs and secretory vesicles relocalize at the meiotic spindle pole body, where spore plasma membrane formation subsequently initiates.
Sporulation, gametogenesis in yeast, consists of meiotic nuclear division and spore morphogenesis. In the fission yeast Schizosaccharomyces pombe, the four haploid nuclei produced after meiosis II are encapsulated by the forespore membrane (FSM), which is newly synthesized from spindle pole bodies (SPBs) in the cytoplasm of the mother cell as spore precursors. Although the coordination between meiosis and FSM assembly is vital for proper sporulation, the underlying mechanism remains unclear. In the present study, we identified a new meiosis-specific protein Npg1, and found that it was involved in the efficient formation of spores and spore viability. The accumulation and organization of the FSM was compromised in npg1-null cells, leading to the error-prone envelopment of nuclei. Npg1 was first seen as internuclear dots and translocated to the SPBs before the FSM assembled. Genetic analysis revealed that Npg1 worked in conjunction with the FSM proteins Spo3 and Meu14. These results suggest a possible signaling link from the nucleus to the meiotic SPBs in order to associate the onset of FSM assembly with meiosis II, which ensures the successful partitioning of gametic nuclei.
In Schizosaccharomyces pombe, ecl family genes are induced by several signals, such as starvation of various nutrients, including sulfur, amino acids, and magnesium, and environmental stress, including heat or oxidative stress. These genes mediate appropriate cellular responses and contribute to the maintenance of cell viability and induction of sexual differentiation. Although this yeast has three ecl family genes with overlapping functions, any environmental conditions that induce ecl3+ remain unidentified. We demonstrate that ecl3+ is induced by phosphate starvation, similar to its chromosomally neighboring genes, pho1+ and pho84+, which respectively encode an extracellular acid phosphatase and an inorganic phosphate transporter. ecl3+ expression was induced by the transcription factor Pho7 and affected by the cyclin-dependent kinase (CDK)-activating kinase Csk1. Phosphate starvation induced G1 arrest and sexual differentiation via ecl family genes. Biochemical analyses suggested that this G1 arrest was mediated by the stabilization of the CDK inhibitor Rum1, which was dependent on ecl family genes. This study shows that ecl family genes are required for appropriate responses to phosphate starvation and provides novel insights into the diversity and similarity of starvation responses.
Dissolving microneedles (DMs) were applied to lidocaine for local anesthesia of the skin. Three DM array chips were prepared where lidocaine was localized at the acral portion of DMs (type 1), loaded in whole DMs (type 2), and lidocaine was loaded both in whole DMs and the chip (type 3). DM chips were 15-mm diameter with 225 DMs, each 500-μm long with a 300-μm diameter base. The lidocaine contents were (type 1) 0.08 ± 0.01 mg, (type 2) 0.22 ± 0.01 mg and (type 3) 8.52 ± 0.49 mg. Lidocaine was released from type 1 and 2 DM array chips within 10 min. Pharmacological activity of DMs were compared to lidocaine cream by the suppression of idiospasm of hair-removed rat skin. Type 1, 2 and 3 DMs showed faster onset time, 5 min, than lidocaine cream. Type 2 and 3 DMs showed stronger anti-idioplasmic activity than type 1 DMs. Pharmacokinetic study showed that tissue lidocaine levels, 62.8 ± 3.6 (type 1), 89.1 ± 9.9 (type 2) and 131.2 ± 10.2(type 3) μg/g wet weight at 5 min after the removal of DM were obtained higher than lidocaine cream, 26.2 ± 12.5 μg/g wet weight. Those results suggest the usefulness of type 2 DMs to obtain fast onset time for the local anesthesia in the skin.
In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.
The fission yeast Schizosaccharomyces pombe employs two main strategies to adapt to the environment and survive when starved for nutrients. The strategies employ sporulation via sexual differentiation and extension of the chronological lifespan.When a cell is exposed to nutrient starvation in the presence of a cell of the opposite sex, the cells undergo fusion through conjugation and sporulation through meiosis.S. pombe spores are highly resistant to diverse stresses and may survive for a very long time. In this minireview, among the various sexual differentiation processes induced by starvation, we focused on and summarized the findings of the molecular mechanisms of spore formation in fission yeast. Furthermore, comparative measurements of the chronological lifespan of stationary phase cells and G 0 cells and the survival period of spore cells revealed that the spore cells survived for a long period, indicating the presence of an effective mechanism for survival. Currently, many molecules involved in sporulation and their functions are being discovered; however, our understanding of these is not complete. Further understanding of spores may not only deepen our comprehension of sexual differentiation but may also provide hints for sustaining life.
Fission yeast Ypt2, an orthologue of the mammalian small GTPase Rab8, is responsible for post-Golgi membrane trafficking. During meiosis, Ypt2 localizes at the spindle pole body (SPB), where it regulates de novo biogenesis of the spore plasma membrane. Recruitment of Ypt2 to the SPB is dependent on its meiosis-specific GDP/GTP exchange factor (GEF), the SPB-resident protein Spo13. Here we have examined the SPB recruitment of Ypt2 by Spo13. The GEF activity of Spo13 was required, but not essential for recruitment. Furthermore, Ypt2 recruitment was regulated in a meiosis-specific manner and partially regulated by the nuclear Dbf2-related (NDR) kinase Sid2, indicating the existence of a novel regulatory mechanism for localization of Rab GTPases during meiosis.
Synaptobrevin, also called vesicle-associated membrane protein (VAMP), is a component of the plasma membrane N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays a key role in intracellular membrane fusion. Previous studies have revealed that, similar to synaptobrevin in other organisms, the fission yeast synaptobrevin ortholog Syb1 associates with post-Golgi secretory vesicles and is essential for cytokinesis and cell elongation. Here, we report that Syb1 has a role in sporulation. After nitrogen starvation, green fluorescent protein (GFP)-Syb1 is found in intracellular dots. As meiosis proceeds, GFP-Syb1 accumulates around the nucleus and then localizes at the forespore membrane (FSM). We isolated a syb-S1 mutant, which exhibits a defect in sporulation. In syb1-S1 mutants, the FSM begins to form but fails to develop a normal morphology. Electron microscopy shows that an abnormal spore wall is often formed in syb1-S1 mutant spores. Although most syb1-S1 mutant spores are germinated, they are less tolerant to ethanol than wild-type spores. The syb1-S1 allele carries a missense mutation, resulting in replacement of a conserved cysteine residue adjacent to the transmembrane domain, which reduces the stability and abundance of the Syb1 protein. Taken together, these results indicate that Syb1 plays an important role in both FSM assembly and spore wall formation. Members of the soluble N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) family contribute to transport specificity by regulating interactions between membrane vesicles and their appropriate target membranes (1). SNARE proteins exist as complementary sets of v-SNAREs, found on vesicle membranes, and t-SNAREs, found on target membranes. Recent classification, however, takes into account the structural features of SNARE proteins, subdividing them into RSNAREs and Q-SNAREs (2).There are approximately 40 SNAREs in an animal cell, and each associates with a particular organelle in the biosyntheticsecretory or endocytic pathway (3). A v-SNARE is a single polypeptide chain, whereas a t-SNARE complex is composed of two or three proteins. The v-SNAREs and t-SNAREs have characteristic helical domains, and when a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the helical domains of the other to form a stable four-helix bundle. The resulting trans-SNARE complex locks the two membranes together.SNAREs have been well characterized in neurons, where they mediate the docking and fusion of synaptic vesicles at the nerve terminal's plasma membrane (PM) during the process of neurotransmitter release. The SNARE complex responsible for docking synaptic vesicles at the PM of nerve terminals consists of three proteins. The transmembrane proteins v-SNARE synaptobrevin (also called vesicle-associated membrane protein [VAMP]) and t-SNARE syntaxin each contribute one ␣-helix to the complex (4, 5), whereas the peripheral membrane protein t-SNARE SNAP-25 contributes two ␣-helices to the four-helix bun...
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