आसंजन और एस cerevisiae में अग्रवाल आक्रमण के लिए परख

Summary

हम खमीर और आक्रामक और pseudohyphal भेदभाव के एक उपाय के रूप में अगर आक्रमण आसंजन के लिए एक गुणात्मक परख का वर्णन. यह सरल परख विभिन्न म्यूटेंट के आक्रामक phenotype के रूप में अच्छी तरह के रूप में प्रभाव पर्यावरण cues का आकलन और खमीर भेदभाव पर रास्ते संकेतन के लिए इस्तेमाल किया जा सकता है.

Abstract

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8 The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar. Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

Protocol

आवश्यक भुखमरी की स्थिति (0.2% ग्लूकोज के साथ अनुसूचित जाति बनाम 2% ग्लूकोज के साथ अनुसूचित जाति, उदाहरण के लिए) के साथ सिंथेटिक मीडिया प्लेटों पर ब्याज की बढ़ती संस्कृतियों के 200ul रखो यदि संस्कृतियों का घनत्व भी ए…

Discussion

खमीर कोशिकाओं को पोषक तत्वों की उपलब्धता और पर्यावरण की स्थिति के अनुसार विभिन्न भेदभाव मोड भुखमरी और तनाव की स्थिति, विभिन्न पोषक तत्व तनाव के तहत filamentation, और flocculation के तहत बीजाणु गठन सहित, प्रदर्शित करते हैं. एस cere…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Moticam 350	Camera	Motic	discontinued (new model: Moticam 352)	A relatively cheap camera that attaches to eye pieces of microscopes and captures digital images for PC or Mac.