Name: reactivation of demembranated cell models in chlamydomonas reinhardtii
Uploaded: 2022-05-06T15:00:00.000+00:00
Duration: 3 min 37 s
Description: Watch this Scientific Journal Video about Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii at JoVE.com

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.&#160;Miyuki Shinohara (Hosei Univ.) for her help in preparing the figures.

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 &#176;C.
1. Cell culture
<ol>
	<li>Culture Chlamydomonas reinhardtii (CC-125) in TAP medium16 in a 12 h/12 h light-dark period (light conditions for the light period: ~50 &#181;mol photons m&#8722;2 s&#8722;1 white light) at 20-25 &#176;C for 2 days (Figure 1). 
	NOTE: The cells must be in a mid-logarithmic growth phase (Movie 1). Long culture (&#62;4 days, in the late-logarithmic growth or stationary phase) decreases the reactivation efficiency of demembranated cell models.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63869/63869fig01.jpg" /> 
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 &#215; 106 cells/mL. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/newMovie1.mp4" target="_blank">Please click here to download this Movie.</a>
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.
<ol>
	<li>Centrifuge ~10 mL of liquid culture at 1000 &#215; g at 20 &#176;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 &#215; 106 cells/mL. When the cell density is lower than this, take enough culture volume to contain ~5 &#215; 107 cells.</li>
	<li>Discard the supernatant, first by decantation and then by a Pasteur pipette, and resuspend the precipitates in ~5 mL of washing buffer.</li>
	<li>Centrifuge cells in the washing buffer at 1000 &#215; g for 3 min at 20 &#176;C.</li>
	<li>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.</li>
	<li>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 &#62;1 mM for stable reactivation17.</li>
	<li>Suspend the remaining cell pellet gently with a pipette, and place the tube on ice again (Figure 3B).</li>
	<li>Take 5-10 &#181;L 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.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63869/63869fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63869/63869fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63869/63869fig04.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/newMovie2.mp4" target="_blank">Please click here to download this Movie.</a>
3. Reactivation of demembranated cell models
<ol>
	<li>Mix 80 &#181;L of reactivation solution, 10 &#181;L of ATP solution, and 10 &#181;L of cell models in a 0.5 mL tube by tapping the tube (Figure 5). 
	NOTE: The final ATP concentration must be &#60;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.
	<ol>
		<li>For reactivating with &#60;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).</li>
	</ol>
	</li>
	<li>Put ~30 &#181;L 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.</li>
	<li>Observe the reactivated cell models under a microscope (Movie 3).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63869/63869fig05.jpg" /> 
Figure 5: Mixing solution by tapping the tube. (A) To 80 &#181;L of the reactivation solution, 10 &#181;L of ATP solution, and 10 &#181;L of cell models were added in sequential order. (B) The solution was mixed by tapping the tube with a finger. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63869/63869fig06.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/newMovie3.mp4" target="_blank">Please click here to download this Movie.</a>

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S., Satir, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction+of+active+sliding+of+microtubules+in+Tetrahymena+cilia.">Direction of active sliding of microtubules in Tetrahymena cilia.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (5), 2045-2049 (1977).</li><li>Fox, L. A., Sale, W. 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The authors have nothing to disclose.

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 &#215; 107 cells are suspended in ~0.5 mL of demembranation buffer (final cell density: 1 &#215; 108 cells/mL). The reactivation rate of the cell models would decrease if the cell model density is much lower than this (e.g., 1 &#215; 106 cells/mL). These cell model-density effects on reactivation are unclear, but the cell extracts probably contain the factors that help reactivation. Of course, the higher the cell density, the more difficult it will be for thorough demembranation. Inadequate demembranation may result in contaminating live cells with cell models. Moving cells can be seenin this case in step 2.7. Contamination of live cells will lead to misinterpretation of the effects of ATP or other components (such as Ca2+) added to the reactivation buffer. This step needs proficiency.
The second critical step is reactivation. Similar to the demembranation step, vigorous pipetting for mixing the solution also induces deciliation. Thus, mixing solutions with tapping tubes is preferable. This step also needs proficiency. In addition, using 0.5 mL tubes is recommended in this step when the volume of the solution is 100 &#181;L (as described in this protocol).
As stated in the Introduction, one advantage of this protocol is that it allows examining the effects of various biological signals in vitro whose concentrations are only transiently elevated in vivo. For example, Ca2+ can be added to the reactivation solution. Because of the chelation effects of EGTA in this case, the final calcium concentration after mixing all the solutions (step 3.1) needs to be carefully calculated. For calculating the final concentration of free Ca2+ in a buffer in the presence of chelators, the program CALCON18 is valid. An example of the calcium-containing reactivation buffer is shown in a previous study19. It is to be noted that a high concentration of Ca2+ (&#62;10&#8722;5 M) induces deciliation20, and thus the effect of Ca2+ on ciliary motility must be tested at lower concentrations as previously described21.
The method suffers certain limitations. The composition of the reactivation buffer does not necessarily reflect the in vivo ionic conditions. So, if some difference in the ciliary motion were observed between the cell models and the live cells, one would need to be careful to determine if this is not an artifact due to the buffer composition. For example, if we experiment with this classic buffer composition, the ciliary beating frequency is lower than expected when the ATP concentration is &#62;1 mM17. A solution to this problem is to increase the Mg2+ concentration and to add adenosine diphosphate (ADP) together with ATP; then, the ciliary beating frequency increases as expected17. Thus, it is necessary to change the buffer composition according to the purpose of the experiment.
The present protocol can be applied to other algae species swimming with cilia. For example, the motility of demembranated Volvox rousseletii, the multicellular Volvocales green alga containing 5,000-10,000 Chlamydomonas-like cells, could be reactivated by adding ATP like C. reinhardtii cell models22. Several modifications were required in this case: polyethylene glycol (PEG) was omitted to preserve the spherical shape of demembranated V. rousseletii, and the concentrations of Igepal CA-630 (detergent) were modified due to a gradient in the detergent sensitivity from the anterior pole to the posterior pole, being highest at the former and lowest at the latter. With modifications, this protocol was also applied to the smallest multicellular Volvocales species, four-celled Tetrabaena socialis23. If the present experiment is to be tried with a new organism, optimization will be necessary by modifying the types and the concentrations of the components in the demembranation and reactivation buffers, particularly detergents and PEG. Depending on the size and morphology of the organism, centrifugation settings need to be tuned. Perfusion is also an option for demembranation instead of centrifugation22.

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-Gy&#246;rgyi 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&#39;s breaststroke5. Ciliary motion is driven by dynein, a minus-end directed microtubule-based motor protein6,7. 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.

<table><tbody><tr><td>0.5 mL plastic tube</td><td>QSP</td><td>502-PLN-Q</td><td></td></tr><tr><td>15 mL conical tube</td><td>SARSTEDT</td><td>62.554.502</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium salt hydrate (ATP)</td><td>Sima-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Centrifuge</td><td>KUBOTA</td><td>2800</td><td></td></tr><tr><td>Chlamydomonas strain CC-125</td><td>Chlamydomonas Resource Center</td><td></td><td>https://www.chlamycollection.org/</td></tr><tr><td>Creatine kinase</td><td>Merck</td><td>CK-RO</td><td></td></tr><tr><td>Creatine phosphate</td><td>Merck</td><td>CRPHO-RO</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Nakalai tesque</td><td>14128-46</td><td></td></tr><tr><td>GEDTA(EGTA)</td><td>Dojindo</td><td>G002</td><td></td></tr><tr><td>Hepes</td><td>Dojindo</td><td>GB70</td><td></td></tr><tr><td>Igepal CA-630</td><td>Sigma-Aldrich</td><td>I8896</td><td>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.</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Nakalai tesque</td><td>21002-85</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX-53</td><td></td></tr><tr><td>Pasteur pipette</td><td>fisher scientific</td><td>13-678-20C</td><td></td></tr><tr><td>Polyethylene glycol, Mr 20,000</td><td>Merck</td><td>8.18897.1000</td><td></td></tr><tr><td>Pottasium acetate</td><td>Nakalai tesque</td><td>28434-25</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Nacalai</td><td>31511-05</td><td></td></tr><tr><td>Sucrose</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>196-00015</td><td></td></tr></tbody></table>

reactivation of demembranated cell models in chlamydomonas reinhardtii

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi 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.

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).&#160;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 deciliation (Figure 4). After mixing with the reactivation buffer and ATP (step 3.1), &#62;50% of cell models became motile (step 3.2). The reactivation rate is increased by gentle and thorough mixing at step 3.1. (% motile cells). Even without the ATP regeneration system (step 3.1.1), the reactivated cells continued to move for ~90 min with the final ATP concentration of 1 mM, before gradually stopping the movement.
Supplementary Table 1: Composition of solutions and buffers used in the study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63869/63869_Supplementary Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

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Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

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.

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in ...

reactivation-of-demembranated-cell-models-in-chlamydomonas-reinhardtii

Nanyang Technological University

Research

JoVE Journal

Biology

2.1K Views.  Hosei University. Deze methode kan helpen om te begrijpen welke factoren de beweeglijkheid van trilharen beïnvloeden. Het belangrijkste voordeel van deze techniek is dat de verschijnselen die slechts tijdelijk in vivo optreden, dergelijke inversie van calciumionconcentraties in trilharen continu in vitro kunnen worden waargenomen. Deze methode draagt specifiek bij aan het onderzoeksveld van beweeglijke trilharen.Het zou moeilijk zijn voor beginners om de demembranatiestap uit te voeren. Daarom wordt he...

Video: Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Genetics

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscience

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Methods for the Study of Regeneration in Stentor

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.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

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