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1. Developing immortal cell lines from Xenopus embryos , four novel cell lines derived from Xenopus tropicalis

   https://doi.org/10.1098/rsob.220089  

   Gorbsky, Gary J.; Daum, John R.; Sapkota, Hem; Summala, Katja; Yoshida, Hitoshi; Georgescu, Constantin; Wren, Jonathan D.; Peshkin, Leonid; Horb, Marko E. (July 2022, Open Biology)

   The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO 2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus . 

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2. Cell lysis via acoustically oscillating sharp edges

   https://doi.org/10.1039/C9LC00498J  

   Wang, Zeyu; Huang, Po-Hsun; Chen, Chuyi; Bachman, Hunter; Zhao, Shuaiguo; Yang, Shujie; Huang, Tony J. (December 2019, Lab on a Chip)

   In this article, we demonstrate an acoustofluidic device for cell lysis using the acoustic streaming effects induced by acoustically oscillating sharp-edged structures. The acoustic streaming locally generates high shear forces that can mechanically rupture cell membranes. With the acoustic-streaming-derived shear forces, our acoustofluidic device can perform cell lysis in a continuous, reagent-free manner, with a lysis efficiency of more than 90% over a range of sample flow rates. We demonstrate that our acoustofluidic lysis device works well on both adherent and non-adherent cells. We also validate it using clinically relevant samples such as red blood cells infected with malarial parasites. Additionally, the unique capability of our acoustofluidic device was demonstrated by performing downstream protein analysis and gene profiling without additional washing steps post-lysis. Our device is simple to fabricate and operate while consuming a relatively low volume of samples. These advantages and other features including the reagent-free nature and controllable lysis efficiency make our platform valuable for many biological and biomedical applications, particularly for the development of point-of-care platforms. 

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3. Cell behaviors underlying Myxococcus xanthus aggregate dispersal

   https://doi.org/10.1128/msystems.00425-23  

   Murphy, Patrick; Comstock, Jessica; Khan, Trosporsha; Zhang, Jiangguo; Welch, Roy; Igoshin, Oleg A. (September 2023, mSystems)

   Rodríguez-Verdugo, Alejandra (Ed.)

   ABSTRACT The soil bacteriumMyxococcus xanthusis a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a secondM. xanthusstrain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle ofM. xanthus. IMPORTANCEUnderstanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacteriumMyxococcus xanthusforms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions. 

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4. The cell morphological diversity of Saccharomycotina yeasts

   https://doi.org/10.1093/femsyr/foad055  

   Chavez, Christina_M; Groenewald, Marizeth; Hulfachor, Amanda_B; Kpurubu, Gideon; Huerta, Rene; Hittinger, Chris_Todd; Rokas, Antonis (December 2023, FEMS Yeast Research)

   Abstract The ∼1 200 known species in subphylum Saccharomycotina are a highly diverse clade of unicellular fungi. During its lifecycle, a typical yeast exhibits multiple cell types with various morphologies; these morphologies vary across Saccharomycotina species. Here, we synthesize the evolutionary dimensions of variation in cellular morphology of yeasts across the subphylum, focusing on variation in cell shape, cell size, type of budding, and filament production. Examination of 332 representative species across the subphylum revealed that the most common budding cell shapes are ovoid, spherical, and ellipsoidal, and that their average length and width is 5.6 µm and 3.6 µm, respectively. 58.4% of yeast species examined can produce filamentous cells, and 87.3% of species reproduce asexually by multilateral budding, which does not require utilization of cell polarity for mitosis. Interestingly, ∼1.8% of species examined have not been observed to produce budding cells, but rather only produce filaments of septate hyphae and/or pseudohyphae. 76.9% of yeast species examined have sexual cycle descriptions, with most producing one to four ascospores that are most commonly hat-shaped (37.4%). Systematic description of yeast cellular morphological diversity and reconstruction of its evolution promises to enrich our understanding of the evolutionary cell biology of this major fungal lineage. 

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5. Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

   https://doi.org/10.1128/mBio.02517-20  

   Hu, Xiaoyu; O’Shaughnessy, William J.; Beraki, Tsebaot G.; Reese, Michael L. (December 2020, mBio)

   Boyle, Jon P. (Ed.)

   ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma . We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network. 

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