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On Stentor growth, regeneration, vaults, and proteins

  • Author(s): Lin, Athena
  • Advisor(s): Marshall, Wallace F
  • et al.
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.

The molecular mechanism for how Stentor regenerates is a complete mystery, however, the process of regeneration shows striking similarities to the process of cell division. On a morphological level, the process of creating a second mouth in division or a new oral apparatus in regeneration have the same steps and occur in the same order. On the transcriptional level, genes encoding elements of the cell division and cell cycle regulatory machinery, including Aurora kinases, are differentially expressed during regeneration. This suggests that there may be some common regulatory mechanisms involved in both regeneration and cell division. 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 that affect the timing of regeneration. ZM447439 slows down regeneration by at least one hour. PF03814735 completely suppresses regeneration until the drug is removed. Here we provide the first direct experimental evidence that Stentor may harness the cell division machinery to regulate the sequential process of regeneration.

Despite its conservation through many species, Vault’s function as a ribonucleic protein remains unknown. Many theories including signaling, immunity, and drug resistance have been questioned. Here we explored their role in Stentor. Knockdown of vaults have no effect on regeneration in Stentor. Knockdown of vaults did not increase sensitivity to holospora, a ciliate parasite.

Stentor coeruleus is a useful model organism to study single-cell regeneration. They have a distinctive cell shape and large size. They can stretch to 1mm in length. At their anterior end, they have a membranellar band consisting of tightly woven layers of long cilia that beat in synchrony. They have an oral apparatus where they phagocytose unfortunate microorganisms caught in their flow. On the posterior end, they have a holdfast to attach themselves to underwater surfaces (Figure one). And, when cut in half, they can regenerate. The process of regeneration takes eight hours and their transcriptome during regeneration has been characterized. The next step to understanding their regeneration is to have an account of proteins so that we can see the building blocks of Stentor. Because of the size of Stentor, we are able to dissect them into parts and look for enrichment and depletion of proteins in each part. We analyzed over 4000 proteins, providing an inventory of proteins that Stentor will use for regeneration, as well as the proteins required to make a new membranellar band.

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