More Like this

1. The macroscopic limit to synchronization of cellular clocks in single cells of Neurospora crassa

   https://doi.org/10.1038/s41598-022-10612-2  

   Cheong, Jia Hwei ; Qiu, Xiao ; Liu, Yang ; Al-Omari, Ahmad ; Griffith, James ; Schüttler, Heinz-Bernd ; Mao, Leidong ; Arnold, Jonathan ( April 2022 , Scientific Reports)

   We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a “big chamber” microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent.

    

   more » « less
2. Microfluidic Chamber Device to Test quorum sensing Theory.

   Cheong, J. H. ( October 2020 , MicroTAS2020)

   We report a microfluidic device that mimics an artificial tissue to test the theory of quorum sensing as a method for synchronization of a model fungal system, Neurospora crassa (N. crassa). High synchronicity between cells were observed by calculating the Kuramoto order parameter (K) between different fields of view.The dimensions of the microfluidic chamber allows us to also calculate an upper limit of the radius of a hypothesized quorum sensing signal by using the diffusion approximation for signal travelling within the device. 

   more » « less
3. 3D projection electrophoresis for single-cell immunoblotting

   https://doi.org/10.1038/s41467-020-19738-1  

   Grist, Samantha M. ; Mourdoukoutas, Andoni P. ; Herr, Amy E. ( December 2020 , Nature Communications)

   Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is “electrophoretically projected” into the 3^rddimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm²device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

    

   more » « less
4. Airplug‐Mediated Isolation and Centralization of Single T Cells in Rectangular Microwells for Biosensing

   https://doi.org/10.1002/adtp.201900085  

   Sukumar, Pavithra ; Deliorman, Muhammedin ; Brimmo, Ayoola T. ; Alnemari, Roaa ; Elsori, Deena ; Chen, Weiqiang ; Qasaimeh, Mohammad A. ( October 2019 , Advanced Therapeutics)

   Sorting cells in a single cell per microwell format is of great interest to basic biological studies, biotherapeutics, and biosensing including cell phenotyping. For instance, isolation of individual immune T cells in rectangular microwells has been shown to empower the multiplex cytokine profiling at the single cell level for therapeutic applications. The present study, shows that there is an existing bias in temporal cytokine sensing that originates from random “unpredicted” positions of loaded cells within the rectangular microwells. To eliminate this bias, the isolated cells need to be well‐aligned with each other and relative to the sensing elements. Hence, an approach that utilizes the in situ formation and release of airplugs to localize cells toward the center of the rectangular microwells is reported. The chip includes 2250 microwells (each 500 × 50 × 20 µm³) arranged in nine rows. Results show 20% efficiency in trapping single T cells per microwells, where cells are localized within ± 3% of the center of microwells. The developed platform could provide real‐time dynamic and unbiased multiplex cytokine detection from single T cells for phenotyping and biotherapeutics studies.

    

   more » « less
5. Secreted Proteases Control the Timing of Aggregative Community Formation in Vibrio cholerae

   https://doi.org/10.1128/mBio.01518-21  

   Jemielita, Matthew ; Mashruwala, Ameya A. ; Valastyan, Julie S. ; Wingreen, Ned S. ; Bassler, Bonnie L. ( December 2021 , mBio)

   Sourjik, Victor ; Vogel, Joerg (Ed.)

   ABSTRACT Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae , a multicellular community aggregation program occurs in liquid, during the stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV, control the onset timing of both void formation and aggregation; moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally nonaggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle. IMPORTANCE Bacteria can work as collectives to form multicellular communities. Vibrio cholerae , the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing. Here, we show that, beyond overarching control by quorum sensing, there are two aggregation subprograms. One subprogram, which we call void formation, creates a scaffold within which cells can embed. The second subprogram, which allows bacteria to enter the scaffold, requires motility. We discovered that four extracellular proteases control the timing of both void formation and aggregation. We argue that, by using redundant proteases, V. cholerae ensures the reliable execution of this community formation process. These findings may provide insight into how V. cholerae persists in the marine environment or colonizes the human host, as both lifestyles are central to the spread of the disease cholera. 

   more » « less