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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The in vitro reactivation of motile cells is a crucial experiment in understanding the mechanisms of cell motility. The protocol describes reactivating the demembranated cell models of Chlamydomonas reinhardtii, a model organism to study cilia/flagella.

Abstract

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Györgyi demonstrated in the mid-20th century, in vitro reactivation of demembranated cells has been a traditional and potent way to examine cell motility. The fundamental advantage of this experimental method is that the composition of the reactivation solution may be easily changed. For example, a high-Ca2+ concentration environment that occurs only temporarily due to membrane excitation in vivo can be replicated in the lab. Eukaryotic cilia (a.k.a. flagella) are elaborate motility machinery whose regulatory mechanisms are still to be clarified. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism in the research field of cilia. The reactivation experiments using demembranated cell models of C. reinhardtii and their derivatives, such as demembranated axonemes of isolated cilia, have significantly contributed to understanding the molecular mechanisms of ciliary motility. Those experiments clarified that ATP energizes ciliary motility and that various cellular signals, including Ca2+, cAMP, and reactive oxygen species, modulate ciliary movements. The precise method for demembranation of C. reinhardtii cells and reactivation of the cell models is described here.

Introduction

The in vitro reactivation of demembranated motile cells is a valuable tool for studying the molecular basis for the regulatory mechanism of cell motility. Szent-Györgyi first demonstrated in vitro contraction of rabbit skeletal muscle fibers extracted with 50% glycerol by adding adenosine triphosphate (ATP)1. This experiment was the first to prove that ATP energizes muscle contraction. The methodology was soon applied to the study of ATP-energized ciliary/flagellar motility, such as sperm flagella2, Paramecium cilia3, and Chlamydomonas reinhardtii cilia (al....

Protocol

A wild-type strain of Chlamydomonas reinhardtii, CC-125, was used for the present study. CC-125 was obtained from Chlamydomonas Resource Center (see Table of Materials) and maintained on a Tris-acetate-phosphate (TAP)16, 1.5% agarose medium at 20-25 °C.

1. Cell culture

  1. Culture Chlamydomonas reinhardtii (CC-125) in TAP medium16 in a 12 h/12 h light-dark period (light conditions f.......

Representative Results

The demembranation and reactivation process in C. reinhardtii wild type strain (CC-125) is shown here. The culture 2 days after inoculation became a light green color (step 1.1) (Figure 1). Cells were collected (step 2.1), washed (step 2.2), and demembranated (step 2.5). After demembranation, all cell models became immotile (step 2.7). The demembranated cilia (called axonemes) remain attached to the cell body, which shows that the immotility of the cell models is not caused by .......

Discussion

There are two critical steps in this protocol. The first is a process known as demembranation, which needs to be carried out gently but thoroughly. Deciliation (i.e., detaching of cilia from the cell body) is induced by vigorous pipetting or vortexing, rendering the cell models immobile even after the addition of ATP. Typically, 5 × 107 cells are suspended in ~0.5 mL of demembranation buffer (final cell density: 1 × 108 cells/mL). The reactivation rate of the cell models would decrease if .......

Acknowledgements

This study was supported by grants from the Japan Society for the Promotion of Science KAKENHI (https://www.jsps.go.jp/english/index.html) to N.U. (19K23758, 21K06295) and K.W. (19H03242, 20K21420, 21H00420), from the Ohsumi Frontier Science Foundation (https://www.ofsf.or.jp/en/) to K.W., and from the Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials (http://alliance.tagen.tohoku.ac.jp/english/) to N.U. and K.W. We thank Ms. Miyuki Shinohara (Hosei Univ.) for her help in preparing the figures.

....

Materials

NameCompanyCatalog NumberComments
0.5 mL plastic tubeQSP502-PLN-Q
15 mL conical tubeSARSTEDT62.554.502
Adenosine 5'-triphosphate disodium salt hydrate (ATP)Sima-AldrichA2383
CentrifugeKUBOTA2800
Chlamydomonas strain CC-125Chlamydomonas Resource Centerhttps://www.chlamycollection.org/
Creatine kinaseMerckCK-RO
Creatine phosphateMerckCRPHO-RO
Dithiothreitol (DTT)Nakalai tesque14128-46
GEDTA(EGTA)DojindoG002
HepesDojindoGB70
Igepal CA-630Sigma-AldrichI8896IUPAC name is octylphenoxypolyethoxyethanol: IGEPAL CA-630 is a substitute for Nonidet P-40 (NP-40); NP-40 is no longer available in Sigma-Aldrich.
MgSO4-7H2ONakalai tesque21002-85
MicroscopeOlympusBX-53
Pasteur pipettefisher scientific13-678-20C
Polyethylene glycol, Mr 20,000Merck8.18897.1000
Pottasium acetateNakalai tesque28434-25
Sodium HydroxideNacalai31511-05
SucroseFUJIFILM Wako Pure Chemical Corporation196-00015

References

  1. Szent-Gyorgyi, A. Free-energy relations and contraction of actomyosin. Biological Bulletin. 96 (2), 140-161 (1949).
  2. Hoffman-Berling, H. Adenosintriphosphat als betriebsstoff von zellbewegungen. Biochimica et B....

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