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Abstract As single cells, ciliates build, duplicate, and even regenerate complex cortical patterns by largely unknown mechanisms that precisely position organelles along two cell‐wide axes: anterior–posterior and circumferential (left–right). We review our current understanding of intracellular patterning along the anterior–posterior axis in ciliates, with emphasis on how the new pattern emerges during cell division. We focus on the recent progress at the molecular level that has been driven by the discovery of genes whose mutations cause organelle positioning defects in the model ciliateTetrahymena thermophila. These investigations have revealed a network of highly conserved kinases that are confined to either anterior or posterior domains in the cell cortex. These pattern‐regulating kinases create zones of cortical inhibition that by exclusion determine the precise placement of organelles. We discuss observations and models derived from classical microsurgical experiments in large ciliates (includingStentor) and interpret them in light of recent molecular findings inTetrahymena. In particular, we address the involvement of intracellular gradients as vehicles for positioning organelles along the anterior‐posterior axis.
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SMORES: a simple microfluidic operating room for the examination and surgery of Stentor coeruleus
Abstract Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery ofStentor coeruleus(SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery onStentoras our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.
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- PAR ID:
- 10500467
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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