Methods for the Study of Regeneration in <em>Stentor</em>

Abstract

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Reorganization of complex ciliary flows around regeneratingStentor coeruleus

“…While diverse chemical treatments lead to a similar oral regeneration response [26], the sucrose shock was found to be the most reproducible-producing the cleanest MB removal, leaving sharp borders at the detachment zone. Here, we follow previously published protocols [11,27] with only slight modifications. Briefly, Stentor were exposed to a 10% sucrose solution for 2-3 min, which elicited synchronous MB shedding in more than 80% of the cases (electronic supplementary material).…”

Reorganisation of complex ciliary flows around regeneratingStentor coeruleus

“…DNA-dependent RNA synthesis is required to supply the new protein required during intermediate stages of 21 regeneration, but not during the later stages. Depending on temperature, medium, and other unknown factors 22 [19], the total regeneration time ranges between 8-10 hours at 20-25 °C.…”

“…of the above staging. Oral regeneration in S. coeruleus was induced by sucrose shock, with slight modifications 34 from published protocols [21,5], which were based on historical works (see supp. materials).…”

Demonstration of RNA interference by bacterial feeding in Stentor polymorphus

“…Cultures were maintained in dishes containing 50mL of sterile filtered water from the pond where the cells were isolated. Cells were fed twice a week with Chlamydomonas reinhardtii (strain cc125, kind gift from the Marshall lab) as has been done previously for Stentor coeruleus [10,22] and kept at 22ºC in ambient room light with indirect sunlight from windows in lab. In this way the cells were under loosely controlled light/dark cycles, as it was previously reported that cells divide at night [13].…”

“…To maintain these organisms long-term in the lab, I established stable cultures of isolated S. polymorphus using the same laboratory culturing methods already used for S. coeruleus [22]. Briefly, S. polymorphus cells were washed in sterile filtered pond water (hereafter referred to as pond water) and ~300 cells were placed in a small glass jar containing 50 mL of pond water.…”