Are WHI5 mRNA levels comparable across all cell cycle stages? Yes, WHI5 mRNA molecules are indeed found in very similar amounts throughout the cell cycle. COMPARE.EDU.VN provides detailed comparisons to help you understand the nuances of scientific research. This finding, supported by live cell WHI5 mRNA quantification, aligns with previous studies, suggesting consistent transcription of WHI5 in the G1 phase. Explore mRNA expression, cell cycle regulation, and gene transcription with confidence.
1. Introduction to WHI5 mRNA Expression
WHI5 plays a crucial role in cell cycle regulation in yeast. Understanding its expression patterns can provide valuable insights into cell growth and division. This study investigates whether WHI5 mRNA levels are comparable across all stages of the cell cycle, offering a detailed look at gene transcription dynamics.
1.1. The Role of WHI5 in Cell Cycle Regulation
WHI5 acts as a cell cycle inhibitor, coordinating growth and division in yeast. Its activity is particularly important in the G1 phase, where it helps regulate the transition to the S phase. Understanding WHI5 expression is vital for comprehending the mechanisms that govern cell proliferation.
1.2. Why Compare WHI5 mRNA Levels Across Cell Cycle Stages?
Comparing WHI5 mRNA levels across different cell cycle stages helps determine whether its expression is consistent or varies depending on the stage. This information is crucial for understanding the regulatory mechanisms that control cell cycle progression.
2. Methodology for Monitoring WHI5 mRNA
To monitor single WHI5 mRNA molecules, the PP7 tagging system was employed. This method allows for the real-time observation of transcription initiation and elongation.
2.1. The PP7 Tagging System
The PP7 tagging system involves inserting multiple copies of an RNA hairpin at the C-terminal of WHI5. This hairpin is recognized by the PP7 bacteriophage coat protein, which is fused to a fluorescent protein (yeGFP).
2.2. Creating the Whi5-PP7tag Strain
A whi5::WHI5-12xPP7hairpin-KanMX strain, referred to as Whi5-PP7tag, was created by inserting 12 copies of the RNA hairpin at the WHI5 C-terminal of wild-type (WT) BY4741 yeasts. This strain allows for the specific labeling and observation of WHI5 mRNA molecules.
2.3. Visualizing WHI5 mRNA with 2x-yeGFP-PP7
Both WT and Whi5-PP7tag cells were transformed with a plasmid expressing a 2x-yeGFP-PP7 coat protein fusion. In the Whi5-PP7tag strain, the yeGFP dimers bind to the Whi5-12xPP7 hairpin tag, labeling each single WHI5 mRNA molecule with 24-yeGFPs, which form a bright spot.
3. Imaging Techniques: sN&B Microscopy
Scanning Number and Brightness (sN&B) microscopy was used to image asynchronous log-phase cultures. This technique helps in characterizing the molecular nature of the bright spots in Whi5mRNA-24x-yeGFP cells.
3.1. How sN&B Microscopy Works
sN&B microscopy involves raster scanning cells and measuring the fluorescence intensity and brightness. This data is then used to compute cell-averaged brightness and stoichiometry.
3.2. Observations from sN&B Imaging
WT and Whi5-PP7tag cells without yeGFP showed no fluorescence beyond auto-fluorescent background. Clear yeGFP expression was observed in yeGFP and Whi5mRNA-24x-yeGFP cells. Bright and highly mobile WHI5 mRNA spots were observed only in Whi5mRNA-24x-yeGFP cells, present in cells at all cell cycle stages.
3.3. Quantitative Data from sN&B Microscopy
The measured fluorescence brightness was 0.29 in Whi5-PP7tag cells, 0.39 in yeGFP cells, and 0.78 in Whi5mRNA-24x-yeGFP cells. This data allowed for the computation of background-corrected brightness and stoichiometry, confirming the presence of WHI5 mRNA molecules.
4. Confocal Imaging for WHI5 mRNA Spot Counting
Standard confocal imaging was used to accurately count WHI5 mRNA spots in individual cells. This method allowed for the collection of light from the entire cell volume while minimizing imaging time.
4.1. Optimizing Imaging Parameters
The imaging parameters were optimized to collect light from the entire cell volume, minimize imaging time to prevent double counting, and increase the signal-to-noise ratio to avoid missing detections.
4.2. Image Analysis Using WEKA Segmentation
Data were analyzed using the trainable WEKA segmentation plugin in ImageJ. This machine learning tool helps in pixel classification and segmentation of WHI5 mRNA spots.
4.3. Distinguishing True Detections from Artifacts
yeGFP negative control cells were used during WEKA training to ensure that WHI5 mRNA-independent yeGFP spatial fluctuations and random clustering did not yield false detections.
5. Results: WHI5 mRNA Expression Across Cell Cycle Stages
The study found that WHI5 mRNA is expressed in similar amounts across all cell cycle stages. This conclusion is supported by both confocal imaging and sN&B microscopy data.
5.1. Number of WHI5 mRNA Spots Detected
0–5 WHI5 mRNA spots were detected in Whi5mRNA-24x-yeGFP cells at all cell cycle stages. The average number of false WHI5 mRNA detections was 0.20, 0.21, and 0.57 in small unbudded, large unbudded/small budded, and medium-to-large budded control cells, respectively.
5.2. Correcting for False Detections
The false detections-corrected average numbers of WHI5 mRNA molecules in sample cells were 1.17, 1.33, and 1.39, corresponding to a slight increase in WHI5 mRNA in large unbudded and small-budded cells compared to small unbudded cells.
5.3. Localization of WHI5 mRNA
WHI5 mRNA localized both to the nucleus and cytosol. This finding suggests that WHI5 mRNA is actively transported and translated in both compartments.
6. Comparing WHI5 mRNA Expression with Existing Data
The results obtained in this study correlate closely with those from fixed cells or at population level. This consistency strengthens the conclusion that WHI5 mRNA levels are relatively constant throughout the cell cycle.
6.1. Consistency with smFISH Data
The findings are in line with smFISH data-based quantification, which also shows similar WHI5 mRNA levels across cell cycle stages. This agreement validates the PP7 tagging system as a reliable method for monitoring mRNA expression.
6.2. Comparison with Previous Studies
The results align with previous studies that suggest WHI5 is transcribed in G1, and WHI5 mRNA molecules are found in very similar amounts throughout the cell cycle. This consistency reinforces the understanding of WHI5 regulation.
6.3. Implications for Cell Cycle Regulation
The consistent expression of WHI5 mRNA across all cell cycle stages implies that the regulation of WHI5 activity is likely controlled at the protein level rather than at the transcriptional level.
7. Detailed Methods: Strains and Culture Conditions
The methods used in this study are described in detail to ensure reproducibility and transparency. This section covers the construction of strains, cell culture conditions, and imaging protocols.
7.1. Strains Construction
All strains were built in the BY4741 background using LiAc transformation. The 12xPP7-KanMX tag sequence was PCR-amplified from the pDZ617-pKAN-12xPP7-V4 plasmid with homology arms for the WHI5 C-terminal region to guide homologous recombination.
7.2. Cell Culture and Media
For imaging, cells were grown to saturation overnight in SC-URA + 2%glucose medium at 30°C in a rotary incubator, then diluted 100-fold in fresh SC + 2%glucose medium 6 hours prior to imaging.
7.3. Preparing Cells for Imaging
Cells were prepared for imaging using specific protocols to prevent inadvertent nutrient shifts. These protocols ensure that the cells are in optimal condition for sN&B and confocal imaging.
8. sN&B and Confocal Imaging Protocols
Detailed protocols for sN&B and confocal imaging are provided to ensure that the experiments can be replicated accurately. These protocols cover the imaging parameters and data acquisition methods.
8.1. sN&B Imaging Protocol
sN&B imaging was performed using raster scans of the same 3030 µm (256256 pixels) FOVs, using an excitation power of 1–2 µW at 488 nm wavelength and a 64 µs pixel dwell time.
8.2. Confocal Imaging Protocol
Cells were imaged on a Zeiss LSM800 Airyscan confocal microscope equipped with a 63× oil objective. 7878 µm (512512 pixels) FOVs were excited with a 488 nm laser at 6% power and maximal speed.
8.3. Adjustments for Nuclear Staining
For experiments with nuclear staining, the Hoechst 33342 dye was used, and adjustments were made to the imaging parameters to compensate for the signal loss due to filtering.
9. Data Analysis and WHI5 mRNA Quantification
The methods used for data analysis and WHI5 mRNA quantification are described in detail. This section covers the use of the trainable WEKA software and the manual scoring of WHI5 mRNA spots.
9.1. Using WEKA for Image Segmentation
Data were analyzed in ImageJ using the trainable WEKA software. Z-stacks were projected using maximal intensity projection for each pixel, and the resulting 2D images were segmented in WEKA using the default parameters.
9.2. Training the WEKA Plugin
To train the plugin, bright spots and other types of cell regions were manually segmented from both sample and control FOVs. This training ensures a clear separation between true WHI5 mRNA detections and artifacts.
9.3. Manual Scoring and Cell Cycle Stage Association
The segmented “class 1” spots (“true” WHI5 mRNA detections) were manually counted in each individual cell and associated with the cell cycle stage as visually determined by bud morphology.
10. Conclusion: Significance of Consistent WHI5 mRNA Levels
The study concludes that WHI5 mRNA is expressed in similar amounts across all cell cycle stages. This finding has significant implications for understanding cell cycle regulation and gene expression dynamics.
10.1. Summary of Key Findings
The key findings of this study include the consistent expression of WHI5 mRNA across all cell cycle stages, the localization of WHI5 mRNA in both the nucleus and cytosol, and the validation of the PP7 tagging system for monitoring mRNA expression.
10.2. Implications for Understanding Cell Cycle Regulation
The consistent expression of WHI5 mRNA suggests that the regulation of WHI5 activity is likely controlled at the protein level. This understanding can guide future research into the mechanisms that govern cell proliferation.
10.3. Future Research Directions
Future research directions may include investigating the post-translational modifications of WHI5 and their effects on cell cycle progression. Additionally, exploring the interactions between WHI5 and other cell cycle regulators could provide further insights into the complex mechanisms that control cell division.
11. FAQ: Frequently Asked Questions About WHI5 mRNA Expression
This section addresses frequently asked questions about WHI5 mRNA expression and its role in cell cycle regulation.
11.1. What is WHI5 and what does it do?
WHI5 is a cell cycle inhibitor in yeast that helps coordinate growth and division. It is particularly important in the G1 phase, where it regulates the transition to the S phase.
11.2. Why is it important to study WHI5 mRNA levels?
Studying WHI5 mRNA levels helps determine whether its expression is consistent or varies depending on the cell cycle stage. This information is crucial for understanding the regulatory mechanisms that control cell cycle progression.
11.3. What is the PP7 tagging system?
The PP7 tagging system is a method used to monitor single mRNA molecules. It involves inserting multiple copies of an RNA hairpin at the C-terminal of a gene, which is then recognized by a fluorescently labeled protein.
11.4. How was WHI5 mRNA monitored in this study?
WHI5 mRNA was monitored using the PP7 tagging system, combined with sN&B microscopy and confocal imaging techniques.
11.5. What were the main findings of the study?
The main finding was that WHI5 mRNA is expressed in similar amounts across all cell cycle stages.
11.6. Where is WHI5 mRNA located within the cell?
WHI5 mRNA was found to be localized both in the nucleus and the cytosol.
11.7. How does this study compare with previous research?
The results of this study correlate closely with those from fixed cells or at population level, reinforcing the conclusion that WHI5 mRNA levels are relatively constant throughout the cell cycle.
11.8. What are the implications of these findings for cell cycle regulation?
The consistent expression of WHI5 mRNA suggests that the regulation of WHI5 activity is likely controlled at the protein level rather than at the transcriptional level.
11.9. What are some potential future research directions?
Potential future research directions include investigating the post-translational modifications of WHI5 and their effects on cell cycle progression, as well as exploring the interactions between WHI5 and other cell cycle regulators.
11.10. Where can I find more information about cell cycle regulation?
More information about cell cycle regulation can be found in scientific journals, textbooks, and reputable online resources. COMPARE.EDU.VN also offers detailed comparisons and analyses of scientific topics.
12. Glossary of Terms
This glossary defines key terms used in the article to ensure clarity and understanding.
12.1. WHI5
A cell cycle inhibitor in yeast that helps coordinate growth and division, particularly in the G1 phase.
12.2. mRNA
Messenger RNA, a type of RNA that carries genetic information from DNA to the ribosomes for protein synthesis.
12.3. Cell Cycle
The series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two new daughter cells.
12.4. G1 Phase
The first phase of the cell cycle, during which the cell grows and prepares for DNA replication.
12.5. S Phase
The phase of the cell cycle during which DNA is replicated.
12.6. PP7 Tagging System
A method used to monitor single mRNA molecules by inserting multiple copies of an RNA hairpin that is recognized by a fluorescently labeled protein.
12.7. sN&B Microscopy
Scanning Number and Brightness microscopy, a technique used to measure the fluorescence intensity and brightness of cells.
12.8. Confocal Microscopy
A type of light microscopy that uses a spatial pinhole to eliminate out-of-focus light, creating clearer images.
12.9. WEKA
A machine learning software used for data analysis and image segmentation.
12.10. smFISH
Single-molecule fluorescence in situ hybridization, a technique used to detect and quantify mRNA molecules in fixed cells.
13. References
This section lists the references cited in the article, providing sources for further reading and verification.
13.1. List of Cited Articles
- Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science. 2011;332(6028):475–8.
- Dorsey S, Tollis S, Cheng J, Black L, Notley S, Tyers M, et al. G1/S transcription factor copy number is a growth-dependent determinant of cell cycle commitment in yeast. Cell Syst. 2018;6(5):539–54.
- Digman MA, Dalal R, Horwitz AF, Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J. 2008;94(6):2320–32.
- Arganda-Carreras I, Kaynig V, Rueden C, Eliceiri KW, Schindelin J, Cardona A, et al. Trainable Weka segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics. 2017;33(15):2424–6.
- Swaffer MP, Kim J, Chandler-Brown D, Langhinrichs M, Marinov GK, Greenleaf WJ, et al. Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size. Mol Cell. 2021;81(23):4861–75.
- Qu Y, Jiang J, Liu X, Wei P, Yang X, Tang C. Cell cycle inhibitor Whi5 records environmental information to coordinate growth and division in yeast. Cell Rep. 2019;29(4):987–94.
- Tollis S, Singh J, Thattikota Y, Palou R, Ghazal G, Coulombe-Huntington J, et al. The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast. PLoS Biol. 2022;20(3): e3001548.
13.2. Additional Resources
- Addgene plasmid #72237: pDZ617-pKAN-12xPP7-V4
- Addgene plasmid #72234: pDZ536-pURA-ADE3p-PP7-PS-2x-yeGFP
14. Further Reading: Exploring Related Topics
This section suggests additional resources for readers interested in exploring related topics in cell cycle regulation and gene expression.
14.1. Recommended Articles and Books
- “Molecular Biology of the Cell” by Alberts et al.
- “Cell Cycle Control” by Murray and Hunt
- “Genes IX” by Lewin
14.2. Online Resources
- National Center for Biotechnology Information (NCBI)
- PubMed
- ScienceDirect
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