{"title":"Interview With Adrian Candelas. Winner of the French Society for Cell Biology (SBCF) Thesis Award 2024","authors":"Paul Trevorrow, Adrian Candelas","doi":"10.1111/boc.12009","DOIUrl":"https://doi.org/10.1111/boc.12009","url":null,"abstract":"","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 3","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143638766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mathieu Pinot, Marie André, Chantal Roubinet, Céline Bruelle, Roland Le Borgne
The recent development of a wide variety of genetically encoded photoconvertible fluorescent proteins has made it possible to study unprecedented dynamic processes by monitoring sub-populations of cells or labeled proteins. The use of photoconvertible fluorescent proteins, such as Eos, KAEDE, mMaple3, Dendra2 is a major advance. However, the conditions of their use in vivo and the inherent potential side-effects remain poorly characterized. Here, we used Drosophila pupal notum to characterize in vivo the conditions for photoconversion (PC) at the subcellular level. We compared the ability to photoconvert proteins exhibiting distinct localization and dynamics, namely, cytosolic and transmembrane proteins fused to photoconvertible probes and expressed at physiological levels. We report that the restriction of PC to a predefined region of interest depends on the mobility of the tagged protein, the power of the PC laser and the number of iterations. We characterized the axial spreading inherent to one-photon microscopy, which results in a PC cone that limits probe tracking on the z-axis. We discussed how the use of a two-photon laser can overcome this issue. We detail biases in the use of photoconvertible probes and propose strategies to circumvent them. Overall, our study provides a framework to study protein behavior at the subcellular level in living organisms.
{"title":"Advantages and Limitations of Photoconvertible Probes to Study Subcellular Dynamics in Epithelial Cells","authors":"Mathieu Pinot, Marie André, Chantal Roubinet, Céline Bruelle, Roland Le Borgne","doi":"10.1111/boc.12008","DOIUrl":"https://doi.org/10.1111/boc.12008","url":null,"abstract":"<p>The recent development of a wide variety of genetically encoded photoconvertible fluorescent proteins has made it possible to study unprecedented dynamic processes by monitoring sub-populations of cells or labeled proteins. The use of photoconvertible fluorescent proteins, such as Eos, KAEDE, mMaple3, Dendra2 is a major advance. However, the conditions of their use in vivo and the inherent potential side-effects remain poorly characterized. Here, we used <i>Drosophila</i> pupal notum to characterize in vivo the conditions for photoconversion (PC) at the subcellular level. We compared the ability to photoconvert proteins exhibiting distinct localization and dynamics, namely, cytosolic and transmembrane proteins fused to photoconvertible probes and expressed at physiological levels. We report that the restriction of PC to a predefined region of interest depends on the mobility of the tagged protein, the power of the PC laser and the number of iterations. We characterized the axial spreading inherent to one-photon microscopy, which results in a PC cone that limits probe tracking on the <i>z</i>-axis. We discussed how the use of a two-photon laser can overcome this issue. We detail biases in the use of photoconvertible probes and propose strategies to circumvent them. Overall, our study provides a framework to study protein behavior at the subcellular level in living organisms.</p>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 3","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.12008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143638765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We present here the summary of two cell biology-related conferences held in Mexico in November 2024. Very broad topics were depicted, nevertheless, a focus on mechanotransduction was perceptible in the two events.
{"title":"Conference Report: Cell Biology and Mechanobiology in Mexico","authors":"Tatiana Fioderlisio-Coll, Kheya Sengupta, Mathieu Hautefeuille, Laurent Limozin, Pierre-Henri Puech","doi":"10.1111/boc.12006","DOIUrl":"https://doi.org/10.1111/boc.12006","url":null,"abstract":"<p>We present here the summary of two cell biology-related conferences held in Mexico in November 2024. Very broad topics were depicted, nevertheless, a focus on mechanotransduction was perceptible in the two events.</p>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 3","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.12006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143554805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
RNA plays crucial roles in cellular organization and metabolism, and modulating its activity is essential for maintaining cellular functions. RNA activity, involving both catalytic (ribozymes) and translation processes, is controlled via myriad mechanisms involving different binding partners such as proteins and smaller polar solutes. We previously reported that lipid membranes can directly interact with the artificial R3C ribozyme changing its activity, however, the effect of lipids on naturally occurring ribozymes remains unknown.
Results
Here, we report that both catalytic activity as well as RNA integrity can be controlled by the presence of different lipid membranes. Gel-phase lipid membranes decreased the activity of hepatitis delta virus ribozyme and increased the activity of a hammerhead ribozyme. The presence of lipid liquid membrane surfaces triggered RNA degradation with greater degradation occurring in the single-stranded regions of RNA.
Conclusion
The interplay between RNA activity and stability in the presence of different lipid membranes introduces multiple possibilities, where different combinations of ribozyme and lipid membrane composition could produce different effects on activity.
Significance
Taken together, these observations support the hypothesis that the activity of both natural and artificial RNAs can be modulated by lipid membranes which, in turn, provides a foundation for the development of novel riboswitch-like molecules, and lipid membrane-based RNA-biosensors.
{"title":"Effects of lipid membranes on RNA catalytic activity and stability","authors":"Tomasz Czerniak, James P. Saenz","doi":"10.1111/boc.202400115","DOIUrl":"https://doi.org/10.1111/boc.202400115","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Backgound Information</h3>\u0000 \u0000 <p>RNA plays crucial roles in cellular organization and metabolism, and modulating its activity is essential for maintaining cellular functions. RNA activity, involving both catalytic (ribozymes) and translation processes, is controlled via myriad mechanisms involving different binding partners such as proteins and smaller polar solutes. We previously reported that lipid membranes can directly interact with the artificial R3C ribozyme changing its activity, however, the effect of lipids on naturally occurring ribozymes remains unknown.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Here, we report that both catalytic activity as well as RNA integrity can be controlled by the presence of different lipid membranes. Gel-phase lipid membranes decreased the activity of hepatitis delta virus ribozyme and increased the activity of a hammerhead ribozyme. The presence of lipid liquid membrane surfaces triggered RNA degradation with greater degradation occurring in the single-stranded regions of RNA.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>The interplay between RNA activity and stability in the presence of different lipid membranes introduces multiple possibilities, where different combinations of ribozyme and lipid membrane composition could produce different effects on activity.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Significance</h3>\u0000 \u0000 <p>Taken together, these observations support the hypothesis that the activity of both natural and artificial RNAs can be modulated by lipid membranes which, in turn, provides a foundation for the development of novel riboswitch-like molecules, and lipid membrane-based RNA-biosensors.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.202400115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mireia Bosch-Calvet, Alejandro Pérez-Venteo, Alex Cebria-Xart, Marta Garcia-Cajide, Caroline Mauvezin
Background Information
Mitosis is crucial for the faithful transmission of genetic material, and disruptions can result in chromosomal instability (CIN), a hallmark of cancer. CIN is a known driver of tumor heterogeneity and anti-cancer drug resistance, thus highlighting the need to assess CIN levels in cancer cells to design effective targeted therapy. While micronuclei are widely recognized as CIN markers, we have recently identified the toroidal nucleus, a novel ring-shaped nuclear phenotype arising as well from chromosome mis-segregation.
Results
Here, we examined whether increasing nuclear envelope stiffness through lamin A/C overexpression could affect the formation of toroidal nuclei and micronuclei. Interestingly, lamin A/C overexpression led to an increase in toroidal nuclei while reducing micronuclei prevalence. We demonstrated that chromatin compaction and nuclear stiffness drive the formation of toroidal nuclei. Furthermore, inhibition of autophagy and lysosomal function elevated the frequency of toroidal nuclei without affecting the number of micronuclei in the whole cell population. We demonstrated that this divergence between the two CIN biomarkers is independent of defects in lamin A processing.
Conclusions and Significance
These findings uncover a complex interplay between nuclear architecture and levels of CIN, advancing our understanding of the mechanisms supporting genomic stability and further contributing to cancer biology.
{"title":"Nuclear stiffness through lamin A/C overexpression differentially modulates chromosomal instability biomarkers","authors":"Mireia Bosch-Calvet, Alejandro Pérez-Venteo, Alex Cebria-Xart, Marta Garcia-Cajide, Caroline Mauvezin","doi":"10.1111/boc.12001","DOIUrl":"https://doi.org/10.1111/boc.12001","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background Information</h3>\u0000 \u0000 <p>Mitosis is crucial for the faithful transmission of genetic material, and disruptions can result in chromosomal instability (CIN), a hallmark of cancer. CIN is a known driver of tumor heterogeneity and anti-cancer drug resistance, thus highlighting the need to assess CIN levels in cancer cells to design effective targeted therapy. While micronuclei are widely recognized as CIN markers, we have recently identified the toroidal nucleus, a novel ring-shaped nuclear phenotype arising as well from chromosome mis-segregation.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Here, we examined whether increasing nuclear envelope stiffness through lamin A/C overexpression could affect the formation of toroidal nuclei and micronuclei. Interestingly, lamin A/C overexpression led to an increase in toroidal nuclei while reducing micronuclei prevalence. We demonstrated that chromatin compaction and nuclear stiffness drive the formation of toroidal nuclei. Furthermore, inhibition of autophagy and lysosomal function elevated the frequency of toroidal nuclei without affecting the number of micronuclei in the whole cell population. We demonstrated that this divergence between the two CIN biomarkers is independent of defects in lamin A processing.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions and Significance</h3>\u0000 \u0000 <p>These findings uncover a complex interplay between nuclear architecture and levels of CIN, advancing our understanding of the mechanisms supporting genomic stability and further contributing to cancer biology.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.12001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mitochondria, as the central hub of cellular energy metabolism and a critical regulator of signaling pathways, play indispensable roles in spermatogenesis and sperm function. In recent years, the mechanisms by which RNA-binding proteins regulate reproductive development and gametogenesis have emerged as a focal point in mitochondrial biology. Here, we review the latest progresses on the role of mitochondrial translation and its associated ribosomal regulation in sperm formation and activation. In Caenorhabditis elegans, the RNA-binding protein complex AMG-1/SLRP-1 modulates key processes of sperm development by maintaining mitochondrial homeostasis. Furthermore, we explore the distinct roles of mitochondrial translation and metabolic functions in sperm activation and motility. This review summarizes the mechanisms by which mitochondrial ribosomal regulation governs spermatogenesis and sperm function, offering a foundation for future investigations in reproductive biology.
{"title":"Mitochondrial Ribosome Regulation Drives Spermatogenesis and Male Fertility","authors":"Zhanxin Chang, Long Miao, Peng Wang","doi":"10.1111/boc.12007","DOIUrl":"https://doi.org/10.1111/boc.12007","url":null,"abstract":"<div>\u0000 \u0000 <p>Mitochondria, as the central hub of cellular energy metabolism and a critical regulator of signaling pathways, play indispensable roles in spermatogenesis and sperm function. In recent years, the mechanisms by which RNA-binding proteins regulate reproductive development and gametogenesis have emerged as a focal point in mitochondrial biology. Here, we review the latest progresses on the role of mitochondrial translation and its associated ribosomal regulation in sperm formation and activation. In <i>Caenorhabditis elegans</i>, the RNA-binding protein complex AMG-1/SLRP-1 modulates key processes of sperm development by maintaining mitochondrial homeostasis. Furthermore, we explore the distinct roles of mitochondrial translation and metabolic functions in sperm activation and motility. This review summarizes the mechanisms by which mitochondrial ribosomal regulation governs spermatogenesis and sperm function, offering a foundation for future investigations in reproductive biology.</p>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Based on recently published parameters (Rane et al. 2023, JPCB 127, 5046–5054) for (rs)EGFP triplet state formation and decay rates and yields, we consider the power density dependence of triplet state population dynamics and its consequences for the application of green fluorescent proteins in biological single molecule fluorescence microscopy.
Results
We find that under certain conditions, the photon budget of GFP type fluorescent proteins can be linearly dependent on power density and we propose a possible explanation for such a non-Hirschfeld photobleaching behavior. Moreover, illumination with millisecond pulses at sub-kHz rates is shown to improve photostability.
Conclusions
We stipulate that a judicious choice of excitation wavelength should take into account the triplet state absorption spectrum along with the singlet state absorption spectrum. Formulas are given for the estimation of the effects of such choice as function of the experimental parameters.
Significance
The linear photobleaching model as proposed by Hirschfeld 50 years ago with power-independent photon budget is not generally applicable to fluorescent proteins with millisecond-lived triplet states.
基于最近公布的参数（Rane et al. 2023, JPCB 127, 5046-5054），我们考虑了三重态种群动态的功率密度依赖性及其对绿色荧光蛋白在生物单分子荧光显微镜中的应用的影响。结果我们发现在一定条件下，GFP型荧光蛋白的光子收支与功率密度呈线性关系，并对这种非hirschfeld光漂白行为提出了可能的解释。此外，以亚千赫频率的毫秒脉冲照明被证明可以改善光稳定性。结论在选择激发波长时，应同时考虑三重态吸收光谱和单线态吸收光谱。给出了这种选择的影响随实验参数的函数的估计公式。50年前Hirschfeld提出的与功率无关的光子预算线性光漂白模型一般不适用于具有毫秒三重态的荧光蛋白。
{"title":"Impact of triplet state population on GFP-type fluorescence and photobleaching","authors":"Martin Byrdin, Svetlana Byrdina","doi":"10.1111/boc.202400076","DOIUrl":"https://doi.org/10.1111/boc.202400076","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background Information</h3>\u0000 \u0000 <p>Based on recently published parameters (Rane et al. 2023, JPCB 127, 5046–5054) for (rs)EGFP triplet state formation and decay rates and yields, we consider the power density dependence of triplet state population dynamics and its consequences for the application of green fluorescent proteins in biological single molecule fluorescence microscopy.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>We find that under certain conditions, the photon budget of GFP type fluorescent proteins can be linearly dependent on power density and we propose a possible explanation for such a non-Hirschfeld photobleaching behavior. Moreover, illumination with millisecond pulses at sub-kHz rates is shown to improve photostability.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>We stipulate that a judicious choice of excitation wavelength should take into account the triplet state absorption spectrum along with the singlet state absorption spectrum. Formulas are given for the estimation of the effects of such choice as function of the experimental parameters.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Significance</h3>\u0000 \u0000 <p>The linear photobleaching model as proposed by Hirschfeld 50 years ago with power-independent photon budget is not generally applicable to fluorescent proteins with millisecond-lived triplet states.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.202400076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143396836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.
{"title":"Gallbladder cholangiocyte organoids","authors":"Ankita Dutta, Nandita Chowdhury, Shinjini Chandra, Payel Guha, Vaskar Saha, Dwijit GuhaSarkar","doi":"10.1111/boc.202400132","DOIUrl":"https://doi.org/10.1111/boc.202400132","url":null,"abstract":"<p>Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.</p>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.202400132","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143396837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Matthieu Sanial, Ryan Miled, Marine Alves, Sandra Claret, Nicolas Joly, Véronique Proux-Gillardeaux, Anne Plessis, Sébastien Léon
Background Information
The discovery of green fluorescent protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility, and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation.
Methods and Results
In this study, we report that various green, orange, and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading, and migration on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations.
Conclusions and Significance
Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable to or better than antibody-based detection, a better quantification, and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.
{"title":"Direct observation of fluorescent proteins in gels: A rapid, cost-efficient, and quantitative alternative to immunoblotting","authors":"Matthieu Sanial, Ryan Miled, Marine Alves, Sandra Claret, Nicolas Joly, Véronique Proux-Gillardeaux, Anne Plessis, Sébastien Léon","doi":"10.1111/boc.202400161","DOIUrl":"https://doi.org/10.1111/boc.202400161","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background Information</h3>\u0000 \u0000 <p>The discovery of green fluorescent protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility, and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>In this study, we report that various green, orange, and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading, and migration on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions and Significance</h3>\u0000 \u0000 <p>Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable to or better than antibody-based detection, a better quantification, and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.202400161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143379976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tumor hypoxia reshapes the microenvironment, driving progression, invasion, metastasis, and therapy resistance. Patient-derived tumor organoids, formed under three-dimensional conditions, preserve cellular heterogeneity and nutrient gradients, making them ideal for studying hypoxia-induced tumor responses. This study examines hypoxia-induced changes in lung tumor organoids from two patients, focusing on tumor-associated markers, stem cell markers, and migration capabilities.
Results
Our findings demonstrate that hypoxia distinctively modulates the expression of lung cancer markers thyroid transcription factor-1, cytokeratin 7, and ΔNP63 variant. Hypoxia also induces the upregulation of stem cell-associated markers, resulting in an increased proportion of cancer stem cells. Furthermore, hypoxic lung tumor organoids exhibit unique migratory behavior upon reoxygenation, driven by epithelial-mesenchymal transition and the elevated expression of matrix metalloproteinases 7 and matrix metalloproteinases 9, indicating their enhanced invasive potential.
Conclusions
These findings highlight the value of lung tumor organoids as models for studying hypoxia's complex role in lung cancer. Hypoxia significantly modulates lung tumor organoids growth, stemness, and migratory behavior, providing critical insights into tumor progression and therapy resistance.
{"title":"Lung tumor organoids migrate as cell clusters containing cancer stem cells under hypoxic condition","authors":"Yanjiao Li, Jiarong Zou, Yanhua Fang, Jianing Zuo, Ruoyu Wang, Shanshan Liang","doi":"10.1111/boc.202400081","DOIUrl":"https://doi.org/10.1111/boc.202400081","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Tumor hypoxia reshapes the microenvironment, driving progression, invasion, metastasis, and therapy resistance. Patient-derived tumor organoids, formed under three-dimensional conditions, preserve cellular heterogeneity and nutrient gradients, making them ideal for studying hypoxia-induced tumor responses. This study examines hypoxia-induced changes in lung tumor organoids from two patients, focusing on tumor-associated markers, stem cell markers, and migration capabilities.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Our findings demonstrate that hypoxia distinctively modulates the expression of lung cancer markers thyroid transcription factor-1, cytokeratin 7, and ΔNP63 variant. Hypoxia also induces the upregulation of stem cell-associated markers, resulting in an increased proportion of cancer stem cells. Furthermore, hypoxic lung tumor organoids exhibit unique migratory behavior upon reoxygenation, driven by epithelial-mesenchymal transition and the elevated expression of matrix metalloproteinases 7 and matrix metalloproteinases 9, indicating their enhanced invasive potential.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>These findings highlight the value of lung tumor organoids as models for studying hypoxia's complex role in lung cancer. Hypoxia significantly modulates lung tumor organoids growth, stemness, and migratory behavior, providing critical insights into tumor progression and therapy resistance.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143379978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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