Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

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 (also called flagella)⁴ using non-ionic detergents for demembranation.

The unicellular green alga C. reinhardtii is a model organism for studying cilia: it swims with two cilia by beating them like a human's breaststroke⁵. Ciliary motion is driven by dynein, a minus-end directed microtubule-based motor protein^6,7. Ciliary dyneins can be classified into outer-arm dyneins and inner-arm dyneins. Mutants lacking each kind of dynein have been isolated as slow-swimming mutants with different motility abnormalities. Detailed in vitro motility analysis of these mutants has significantly advanced dynein research⁸.

Many important findings have been achieved utilizing this method and its derivatives since the in vitro reactivation experiment of demembranated C. reinhardtii cells (cell models) was established. Reactivation of cell models in a series of Ca²⁺ buffers, for example, showed⁹ that two cilia are differently regulated by submicromolar Ca²⁺, and this asymmetrical cilia control enables the phototactic orientation of C. reinhardtii¹⁰. Furthermore, both cilia show waveform conversion from the forward swimming mode (called asymmetric waveform) to the backward swimming mode (called symmetric waveform that appears for a short period when cells are photo- or mechano-shocked)^11,12. This waveform conversion is regulated by submillimolar Ca²⁺, which was shown by the reactivation of the so-called nucleoflagellar apparatus (a complex containing two cilia, the basal bodies, the structures linking the basal bodies with the nucleus, and the remnant of the nucleus)¹¹ or demembranated axonemes of isolated cilia¹³. Other than Ca²⁺, redox (reduction-oxidation) poise is a signal that regulates the ciliary beating frequency, which was shown by reactivation of cell models in redox buffers containing different ratios of reduced glutathione vs. oxidized glutathione¹⁴. In addition, cyclic adenosine monophosphate (cAMP) asymmetrically regulates two cilia, which was shown by the reactivation of axonemes with photocleavable caged cAMP¹⁵. These in vitro findings, combined with genetic findings, have led to a deeper understanding of the molecular mechanisms of cilia regulation in C. reinhardtii.

A protocol for reactivating the cell models is described here. The method is simple, allows for various modifications, and can be applied to multiple organisms that move with cilia. However, because demembranated cells are fragile, it requires some tips to reactivate the motility of cell models with good efficiency while preventing deciliation.

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 for the light period: ~50 µmol photons m⁻² s⁻¹ white light) at 20-25 °C for 2 days (Figure 1).
   NOTE: The cells must be in a mid-logarithmic growth phase (Movie 1). Long culture (>4 days, in the late-logarithmic growth or stationary phase) decreases the reactivation efficiency of demembranated cell models.

Figure 1: Liquid culture after 2-day culturing. From a TAP-1.5% agar plate, a chunk of wild-type cells to fill the platinum loop was inoculated to ~150 mL TAP liquid medium in a flask. The cell density after 2-day culture was 2.3 × 10⁶ cells/mL. Please click here to view a larger version of this figure.

Movie 1: Swimming of live cells. Cells were observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. Scale bar = 100 µm. Please click here to download this Movie.

2. Preparation of demembranated cell models

NOTE: Before starting the experiment, keep the washing buffer at room temperature and the demembranation, dilution, reactivation buffers, and the ATP solution on ice. The composition of these buffers is provided in Supplementary Table 1.

1. Centrifuge ~10 mL of liquid culture at 1000 × g at 20 °C for 3 min.
   NOTE: Throughout the protocol from this step, do not use autoclaved plasticware (tubes, pipette tips, etc.), decreasing reactivation efficiency. For conical tubes, reused ones are preferable. The cell density after 2-day culture may be typically 1.0-5.0 × 10⁶ cells/mL. When the cell density is lower than this, take enough culture volume to contain ~5 × 10⁷ cells.
2. Discard the supernatant, first by decantation and then by a Pasteur pipette, and resuspend the precipitates in ~5 mL of washing buffer.
3. Centrifuge cells in the washing buffer at 1000 × g for 3 min at 20 °C.
4. Discard the supernatant carefully with a pipette (Figure 2).
   NOTE: Pasteur pipettes are preferable. When using micropipettes, avoid using pipette tips that have been autoclaved.
5. Overlay ~0.5 mL of demembranation buffer on a cell pellet, shake the tube gently by hand to roughly suspend the cells in the buffer, and put the tube on ice (Figure 3A).
   NOTE: It is not necessary to entirely suspend the pellet at this moment. Raise the concentration of MgSO₄ to 15 mM in the demembranation and reactivation buffers when cell models are reactivated, with a final ATP concentration >1 mM for stable reactivation¹⁷.
6. Suspend the remaining cell pellet gently with a pipette, and place the tube on ice again (Figure 3B).
7. Take 5-10 µL of the cell models, dilute 10-fold with the dilution buffer, and observe under the microscope (Movie 2) to confirm all the cell models are demembranated and not swimming (Figure 4).
   NOTE: If some cells are still swimming (alive), add the non-ionic detergent used in the demembranation buffer directly to the cell model solution to the final concentration of ~0.15%. Alternatively, repeat steps 2.1-2.5 with the demembranation solution containing 0.15% of detergent.

Figure 2: Discarding the supernatant. The rest of the supernatant was carefully removed with a Pasteur pipette after the supernatant was removed by decantation of the tube. Please click here to view a larger version of this figure.

Figure 3: Demembranation. (A) After overlaying 0.5 mL of demembranation solution onto the cell pellet, the solution was mixed by a hand to suspend the cells roughly. (B) After mixing, the rest of the cell pellet was entirely suspended in the solution by a Pasteur pipette. Please click here to view a larger version of this figure.

Figure 4: Effects of demembranation. (A) A live cell stuck on a glass slide. (B) A cell model stuck on a glass slide. Note that in the cell model, the cilia became slightly thinner. The images were observed under a microscope with a 20x objective lens and an oil-immersion dark-field condenser. Scale bar = 10 µm. Please click here to view a larger version of this figure.

Movie 2: Confirmation of demembranation. Cell model suspension was observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. No cell was swimming. Scale bar = 100 µm. Please click here to download this Movie.

3. Reactivation of demembranated cell models

1. Mix 80 µL of reactivation solution, 10 µL of ATP solution, and 10 µL of cell models in a 0.5 mL tube by tapping the tube (Figure 5).
   NOTE: The final ATP concentration must be <3 mM because ciliary beating frequency, a parameter for ciliary motion, increases with ATP concentration and saturates at 2-3 mM of ATP¹⁷.
   CAUTION: Mixing by pipetting or vortexing causes deciliation and decreases reactivation efficiency.
   1. For reactivating with <0.2 mM of ATP, add an ATP-regeneration system, such as 70 U/mL of creatine kinase and 5 mM of creatine phosphate (see Table of Materials).
      NOTE: The reactivation solution is prepared at a higher concentration (1.125x, Supplementary Table 1) than the dilution solution so that after mixing the ATP dissolved in water, the contents reach the following final concentrations: 30 mM of Hepes (pH 7.4), 5 mM of MgSO₄, 1 mM of dithiothreitol (DTT), 1 mM of EGTA, and 50 mM of potassium acetate (see Table of Materials).
2. Put ~30 µL of the mixed solution onto a glass slide and gently place a coverslip on it with spacers to avoid mechanical shock to the cell models (Figure 6).
   NOTE: White petroleum or double-sided adhesive tapes can be used to make the spacers.
3. Observe the reactivated cell models under a microscope (Movie 3).

Figure 5: Mixing solution by tapping the tube. (A) To 80 µL of the reactivation solution, 10 µL of ATP solution, and 10 µL of cell models were added in sequential order. (B) The solution was mixed by tapping the tube with a finger. Please click here to view a larger version of this figure.

Figure 6: Making spacers on coverslip edges. (A) A thin layer of white petroleum was applied to the back of a hand. (B) A small amount of white petroleum was scraped off with an edge of a coverslip. (C) A spacer was made on the edge of a coverslip. (D) Another spacer was made on the opposite edge. Please click here to view a larger version of this figure.

Movie 3: Swimming of reactivated cell models. The motility of the cell models was reactivated by adding ATP at a final concentration of 1 mM, and observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. Scale bar = 100 µm. Please click here to download this Movie.

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 ...

Materials

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