Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• Klíčová slova
• Bordetella, calcium homeostasis, effector protein BteA, host cell death mechanism, type III secretion system (T3SS),
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• Bordetella bronchiseptica genetika   metabolismus   účinky léků   MeSH
• Bordetella pertussis genetika   patogenita   metabolismus   účinky léků   MeSH
• buněčná smrt * účinky léků   MeSH
• endoplazmatické retikulum metabolismus   účinky léků   MeSH
• homeostáza * MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• sekreční systém typu III metabolismus   genetika   MeSH
• vápník * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• sekreční systém typu III MeSH
• vápník * MeSH

Mitochondrial morphology is an important parameter of cellular fitness. Although many approaches are available for assessing mitochondrial morphology in mammalian cells, only a few technically demanding and laborious methods are available for yeast cells. A robust, fully automated and user-friendly approach that would allow (1) segmentation of tubular and spherical mitochondria in the yeast Saccharomyces cerevisiae from conventional wide-field fluorescence images and (2) quantitative assessment of mitochondrial morphology is lacking. To address this, we compared Global thresholding segmentation with deep learning MitoSegNet segmentation, which we retrained on yeast cells. The deep learning model outperformed the Global thresholding segmentation. We applied it to segment mitochondria in strain lacking the MMI1/TMA19 gene encoding an ortholog of the human TCTP protein. Next, we performed a quantitative evaluation of segmented mitochondria by analyses available in ImageJ/Fiji and by MitoA analysis available in the MitoSegNet toolbox. By monitoring a wide range of morphological parameters, we described a novel mitochondrial phenotype of the mmi1Δ strain after its exposure to oxidative stress compared to that of the wild-type strain. The retrained deep learning model, all macros applied to run the analyses, as well as the detailed procedure are now available at https://github.com/LMCF-IMG/Morphology_Yeast_Mitochondria .

• Klíčová slova
• Deep learning, Mitochondria, Mmi1, Oxidative stress, TCTP, Yeast,
• MeSH
• deep learning MeSH
• fluorescenční mikroskopie metody   MeSH
• mitochondrie * metabolismus   MeSH
• oxidační stres MeSH
• počítačové zpracování obrazu * metody   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny MeSH

Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.

• Klíčová slova
• Actin, Adherens junctions, Calpains, Cell scattering, ERK, Epithelial polarity, Focal adhesions, HGF/SF, Migration, Proteases, Tight junctions, Transepithelial electrical resistance,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná adheze * MeSH
• buňky MDCK MeSH
• CRISPR-Cas systémy MeSH
• epitelové buňky * metabolismus   cytologie   MeSH
• hepatocytární růstový faktor metabolismus   MeSH
• kadheriny metabolismus   MeSH
• kalpain * metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   MeSH
• pohyb buněk MeSH
• proteolýza MeSH
• psi MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-katenin MeSH
• hepatocytární růstový faktor MeSH
• kadheriny MeSH
• kalpain * MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• vápník MeSH

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes - the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines - the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.

• Klíčová slova
• MDCK, Rat2, coherence-controlled holographic imaging/microscopy, dry mass content, holotomography, quantitative phase imaging/microscopy, single-cell segmentation,
• MeSH
• buněčné linie MeSH
• holografie * metody   MeSH
• kvantitativní fázové zobrazování MeSH
• lasery MeSH
• mikroskopie * metody   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown. METHODS: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δxrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy. RESULTS: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy. CONCLUSIONS: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.

• Klíčová slova
• P-bodies, Xrn1, diauxic shift, eisosomes, yeast,
• MeSH
• exoribonukleasy * genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• populační růst MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• exoribonukleasy * MeSH
• messenger RNA MeSH

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

• Klíčová slova
• Actin, Cell cycle, Focal adhesions, MDCK cells, Microscopy-based analysis, Microvilli, RACK1, Rat2 cells,
• MeSH
• buněčné dělení MeSH
• mikroskopie * MeSH
• proliferace buněk MeSH
• proteiny vázající GTP * genetika   MeSH
• psi MeSH
• receptory buněčného povrchu genetika   metabolismus   MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny vázající GTP * MeSH
• receptory buněčného povrchu MeSH

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.

• Klíčová slova
• Cell scattering, Cell-cell adhesions, ERK, Epithelial plasticity, Fra1, HGF/SF,
• MeSH
• adenokarcinom * metabolismus   MeSH
• buněčná adheze MeSH
• epitelové buňky metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• MAP kinasový signální systém fyziologie   MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivované proteinkinasy kinas metabolismus   MeSH
• nádory slinivky břišní * metabolismus   MeSH
• proliferace buněk MeSH
• signální transdukce MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAPK1 protein, human MeSH Prohlížeč
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivované proteinkinasy kinas MeSH
• transkripční faktory MeSH

The formation of stress granules (SGs), membrane-less organelles that are composed of mainly messenger ribonucleoprotein assemblies, is the result of a conserved evolutionary strategy to cellular stress. During their formation, which is triggered by robust environmental stress, SGs sequester translationally inactive mRNA molecules, which are either forwarded for further processing elsewhere or stored during a period of stress within SGs. Removal of mRNA molecules from active translation and their sequestration in SGs allows preferential translation of stress response transcripts. By affecting the specificity of mRNA translation, mRNA localization and stability, SGs are involved in the overall cellular reprogramming during periods of environmental stress and viral infection. Over the past two decades, we have learned which processes drive SGs assembly, how their composition varies under stress, and how they co-exist with other subcellular organelles. Yeast as a model has been instrumental in our understanding of SG biology. Despite the specific differences between the SGs of yeast and mammals, yeast have been shown to be a valuable tool to the study of SGs in translation-related stress response. This review summarizes the data surrounding SGs that are formed under different stress conditions in Saccharomyces cerevisiae and other yeast species. It offers a comprehensive and up-to-date view on these still somewhat mysterious entities.

• Klíčová slova
• P-bodies, mRNA, stress granules, stress response, translation, yeast,
• MeSH
• cytoplazmatická granula * fyziologie   MeSH
• fyziologický stres MeSH
• messenger RNA genetika   MeSH
• Saccharomyces cerevisiae * genetika   MeSH
• savci genetika   MeSH
• stresová tělíska MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• messenger RNA MeSH

Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.

• Klíčová slova
• Rack1, cell adhesion, cell dry mass, cell spreading, coherence-controlled holographic microscopy, extracellular matrix, focal adhesion kinase, front–rear polarity, quantitative phase imaging,
• MeSH
• buněčná adheze genetika   fyziologie   MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adhezní tyrosinkinasy genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• mikroskopie fázově kontrastní MeSH
• pohyb buněk genetika   fyziologie   MeSH
• polarita buněk genetika   fyziologie   MeSH
• receptory pro aktivovanou kinasu C genetika   metabolismus   MeSH
• RNA interference MeSH
• tvar buňky genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• RACK1 protein, rat MeSH Prohlížeč
• receptory pro aktivovanou kinasu C MeSH

Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.

• MeSH
• homeostáze proteinů * MeSH
• proteiny tepelného šoku HSP40 metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• proteiny teplotního šoku metabolismus   MeSH
• refolding proteinů MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transportní systémy aminokyselin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BTN2 protein, S cerevisiae MeSH Prohlížeč
• HsP104 protein, S cerevisiae MeSH Prohlížeč
• HSP42 protein, S cerevisiae MeSH Prohlížeč
• proteiny tepelného šoku HSP40 MeSH
• proteiny tepelného šoku HSP70 MeSH
• proteiny teplotního šoku MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SIS1 protein, S cerevisiae MeSH Prohlížeč
• transportní systémy aminokyselin MeSH