The ability to heal wounds and regenerate damaged structures is essential for an organism’s survival. Multicellular organisms mostly rely on cell division to patch wounds and regenerate lost structures with newly proliferated cells, but when a single cell is damaged, whether it be a free-living unicellular organism or a cell within a multicellular tissue, it must be able to recognize and repair that damage without being able to rely on other cells. Now here is this challenge more dramatic than in the giant unicellular ciliate Stentor coeruleus, for when cutting into pieces, each piece will fully regenerate into a healthy, full-sized individual. Stentor cells are a millimeter long with a wine glass shape and have a complex and intricate ultrastructure. Stentor is binucleate ciliates with two morphologically distinct nuclei. The micronuclei is used for germline reproduction and the macronucleus is transcriptionally active throughout the cell cycle. Stentor has an oral pouch, a cilia-lined pore to intake food at its wide anterior and a holdfast, the structure by which the cell attaches to a surface, at its posterior. Connecting these two is a series of microtubule rows called cortical rows that resemble pinstripes. The oral pouch and the holdfast can each fully regenerate if removed, and a bisected cell can regenerate two normal-looking cells. The molecular mechanism for how Stentor regenerates missing parts is a complete mystery. This study focuses on regeneration of the oral apparatus, which consists of a circular band of cilia-based structures known as the membranellar band, connected to an oral pouch located at a defined position. During feeding, the membranellar band creates a fluid flow to bring food to the anterior end of the cell, where it is engulfed through the oral pouch.
Regeneration in Stentor coeruleus can be induced by sucrose shock. This leads to the shedding of the oral apparatus, which is comprised of the oral pouch and membranellar band (Fig. 1A). After sucrose shocking, Stentor look tear-drop shaped and stay stationary for approximately 3 h. After 3 or 4 h of regeneration, Stentor begins to form a membranellar band in the middle of the cell body, initially oriented parallel to the body axis. The membranellar band grows simultaneously towards the top and bottom of the cell. At the top of the cell, the membranellar band will continue growing across the top. After 6 or 7 h of regeneration, the posterior end of the membranellar band will begin to curl to form the oral pouch and a physical indentation of the cell surface can be seen. Within the last 2 h of regeneration, the oral pouch will be moved to the top of the cell along with the membranellar band. Stentor usually completes regeneration within 8 h.
The process of regeneration shows striking similarities to the process of cell division. When a Stentor cell divides asymmetrically along its vertical axis, the anterior daughter cell retains the oral apparatus from the mother cell and the posterior daughter inherits a de novo oral apparatus that forms just prior to cytokinesis. This de novo creation of an oral apparatus during regeneration proceeds through a series of morphological steps virtually identical to those seen during the creation of a new oral pouch during division, namely, the formation of a membranellar band parallel to the body axis, curling of the band, and formation of the oral pouch. During division, the macronucleus undergoes a dramatic shape change from a moniliform string of small spherical nodes into a short tube, when then re-elongates just prior to mitosis. This same nuclear shape change also takes place during regeneration, further suggesting a similarity of the two processes.
The similarity between regeneration and division has also been reported at the transcriptional level, based on studies of the RNA transcriptome during regeneration. Genes encoding elements of the cell division and cell cycle regulatory machinery, including Aurora kinases, are differentially expressed during the later stages of regeneration compared to the earlier stages of regeneration. Such similarities suggest that there may be some common regulatory mechanisms involved in both regeneration and cell division. Since Aurora kinase signaling indicates that a spindle is properly assembled, a similar mechanism could be at work in Stentor to signal the correct assembly of one or more structures during regeneration. But it is also possible that the similarity has nothing to do with regeneration and instead plays some other role. For example, the micronuclei undergo mitosis during both cell division and regeneration, so perhaps the transcriptional changes in cell cycle-related genes have only to do with the micronuclear mitosis and not regeneration itself. However, if the cell cycle machinery really does play a role in regeneration, then inhibition of proteins that regulate the timing of cell division may also affect the timing of regeneration in Stentor. Here we show that two well-characterized Aurora kinase A+B inhibitors slow or stop regeneration in Stentor, providing the first direct experimental evidence that Stentor may harness the cell division machinery to regulate the sequential process of regeneration.