Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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....

Protokół

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 for the light period: ~50 µmol photons m⁻² s⁻¹ white light) at 20-25 °C for 2 days (Fi....

Wyniki

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 .......

Dyskusje

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 .......

Materiały

Name	Company	Catalog Number	Comments
0.5 mL plastic tube	QSP	502-PLN-Q
15 mL conical tube	SARSTEDT	62.554.502
Adenosine 5'-triphosphate disodium salt hydrate (ATP)	Sima-Aldrich	A2383
Centrifuge	KUBOTA	2800
Chlamydomonas strain CC-125	Chlamydomonas Resource Center		https://www.chlamycollection.org/
Creatine kinase	Merck	CK-RO
Creatine phosphate	Merck	CRPHO-RO
Dithiothreitol (DTT)	Nakalai tesque	14128-46
GEDTA(EGTA)	Dojindo	G002
Hepes	Dojindo	GB70
Igepal CA-630	Sigma-Aldrich	I8896	IUPAC name is octylphenoxypolyethoxyethanol: IGEPAL CA-630 is a substitute for Nonidet P-40 (NP-40); NP-40 is no longer available in Sigma-Aldrich.
MgSO4-7H2O	Nakalai tesque	21002-85
Microscope	Olympus	BX-53
Pasteur pipette	fisher scientific	13-678-20C
Polyethylene glycol, Mr 20,000	Merck	8.18897.1000
Pottasium acetate	Nakalai tesque	28434-25
Sodium Hydroxide	Nacalai	31511-05
Sucrose	FUJIFILM Wako Pure Chemical Corporation	196-00015