Name: visualizing cytoplasmic flow during single-cell wound healing in stentor coeruleus
Uploaded: 2013-12-19T13:45:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus at JoVE.com

This work was supported by NIH grant&#160;R01 GM090305 (WFM).&#160;The procedure described here was developed at the Physiology Course of the Marine Biological Laboratory during the summer of 2012, and we thank the students, instructors, and course directors for many helpful and stimulating discussions.

1. Cell Culture and Collection
<ol>
	<li>Obtain Stentor coeruleus cells either from natural ponds as described by Tartar7 or else from a commercial supplier (see Reagents).</li>
	<li>Maintain cultures by growing in the dark at 20 &#186;C in Modified Stentor Medium (MSM), which is adapted from recipes described by Tartar7 and De Terra12 containing 0.75 mM Na2CO3, 0.15 mM KHCO3, 0.15 mM NaNO3, 0.15 mM KH2PO4, 0.15 mM MgSO4, 0.5 mM CaCl2, and 1.47 mM NaCl.</li>
	<li>Supplement the MSM medium with a carbon source by adding living prey to the medium. Grow cultures of Chlamydomonas reinhardtii TAP medium using standard culture methods13. Prior to feeding, wash Chlamydomonas cells in MSM. Feed 300 ml&#160;Stentor cultures ~3 x 107 Chlamydomonas cells 2-3x per&#160;week.</li>
	<li>Collect Stentor cells for surgery using a pipette. Stentor tend to anchor to the bottom and sides of the culture dish and so, in order to collect cells, agitate the culture by bubbling air through a pipette tip until cells begin to detach and swim freely.</li>
	<li>Capture swimming cells using a pipette equipped with a wide-bore tip to avoid shearing the cells.</li>
	<li>Wash collected cells in fresh MSM.</li>
</ol>
2. Preparation of Tools for Microsurgery
<ol>
	<li>Hold two glass stirring rods over a Bunsen burner or other flame until the ends begin to melt.</li>
	<li>Touch the ends of the two rods together so the molten glass fuses.</li>
	<li>After creating a small droplet of molten glass between the two rods, remove them from the flame, allow to cool for 1-2 sec, and then pull them apart to make a clean break. Ideally, the glass will pull out into thin strings and break smoothly as the glass cools resulting in a needle that is approximately 1-2 cm in length with fine point at the end (Figure 2).</li>
	<li>If the resulting needle is suboptimal the tips may be broken into glass waste and the rods may be reused.</li>
</ol>
3. Mounting Cells for Microsurgery and Observation (Normal Surgery)
<ol>
	<li>Prepare a square Vaseline walled chamber using a square rubber stamp coated in Vaseline and pressed onto a glass coverslip.</li>
	<li>Add MSM containing 2% Methyl Cellulose to the Vaseline square.</li>
	<li>Place the coverslip in a plastic adaptor, which allows the coverslip to be placed onto a microscope with a standard slide holder (Figure 3).</li>
	<li>Add washed cells from step 1.6 to the MSM plus methyl cellulose to impede the motility of the Stentor cells so that they stay in place during surgery.</li>
	<li>Arrange cells in the methyl cellulose so that they are evenly spaced and are pushed close enough to the surface of the coverslip to be in focus during imaging.</li>
</ol>
4. High Resolution Imaging of Wound Healing in Live Stentor
To simultaneously image a Stentor cell at high resolution while cutting it surgically under low magnification, construct a &#34;double decker&#34; microscope with a dissecting scope attached to the eyepiece head of the inverted scope so that it is focused on the top of the slide while the inverted scope is focused from beneath the slide.
<ol>
	<li>Put a slide on the inverted scope (use either a Zeiss Axiovert 200M or an Olympus IX80).</li>
	<li>Remove dissecting microscope body from its stand and determine approximate position above inverted microscope eyepiece for placing dissecting scope to allow the slide to be visualized at low magnification.</li>
	<li>Modify a cardboard box to have the same thickness as the desired spacing between inverted scope eyepiece head and dissecting scope, then tape into inverted scope using lab tape which can be easily removed when experiment is completed. Adjust the thickness of the box according to the exact configuration of the eyepiece head of the inverted microscope and on the size of the dissecting microscope so that the sample will be in the field of view without needing to move the dissecting scope after it is attached.</li>
	<li>Holding dissecting scope against box, move it until the slide is in focus, then tape onto the box again using lab tape. The final configuration is shown in Figure 4.</li>
	<li>Image cells through inverted scope using DIC optics and a 20X air objective lens. Use a long working distance DIC condenser to provide enough space to perform the microsurgery from above.</li>
	<li>Acquire images at a frame rate of 2 images/sec&#160;using a 512 x 512 CCD camera.</li>
	<li>Begin acquisition before starting to cut the cells so as to capture the initial cytoplasmic flow pattern and observe all stages of the wounding and wound healing process.</li>
	<li>Terminate acquisition when cell has resumed normal appearance and/or active motility, in order to visualize and measure cytoplasmic movements throughout the regeneration process.</li>
</ol>
5. Analyzing Cytoplasmic Flow During Wound Healing
Measure cytoplasmic flow using particle image velocimetry (PIV). This technique is routine in the study of fluid dynamics, and can generate flow-fields in the absence of individually trackable particles in the fluid. PIV has been used to measure flows of cytoplasm based on imaging subcellular vesicles and other features visible in the cytoplasm by transmitted light microscopy14.
<ol>
	<li>Download PIVLab, a MATLAB package for PIV, from the web site <a href="http://pivlab.blogspot.com/">http://pivlab.blogspot.com/</a>.</li>
	<li>Load the tiff image stack for each video sequence into the PIV program within MATLAB.</li>
	<li>Manually generate a mask for each image stack to mask out regions of the raw data outside the body of Stentor.</li>
	<li>Set PIVLab to use the fast-Fourier transform setting and a bin size of 64&#160;x 64&#160;pixels, which will bin the pixels in each frame into larger square pixels.</li>
	<li>Run PIVLab to compute cross-correlations between the pixels in each frame with nearby pixels in the next frame to yield a time-dependent flow vector centered at each binned pixel, which has the same time resolution as our raw data.</li>
	<li>Compute time averages of the resulting vector field output to generate streamlines for comparison between data taken with the Stentor before cutting and during wound healing, and calculate histograms of flow velocities (to study flow strength) and change of velocity at a given pixel over time (to study flow fluctuations).</li>
	<li>For image sequences with Stentor cleaved in half and cytoplasm visibly leaking into the surrounding buffer, calculate total flux of cytoplasm back into the cell body proper by multiplying the average flow velocity by the cross-sectional area of the base of the extracellular bubble of cytoplasm.</li>
</ol>

<ol>
	<li>Sonnemann, K. J., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair:+toward+understanding+and+integration+of+single-cell+and+multicellular+wound+responses.">Wound repair: toward understanding and integration of single-cell and multicellular wound responses.</a> Annu. Rev. Cell Dev. Biol. 27, 237-264 (2011).</li><li>Idone, V., Tam, C., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-way+traffic+on+the+road+to+plasma+membrane+repair.">Two-way traffic on the road to plasma membrane repair.</a> Trends Cell Biol. 18 (11), 552-559 (2008).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca2+-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes.</a> Cell. 106 (2), 157-169 (2001).</li><li>Foissner, I., Wastenays, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characean+internodal+cell+as+a+model+system+for+studying+wound+healing.">The characean internodal cell as a model system for studying wound healing.</a> J. Microsc. 247 (1), 10-12 (2012).</li><li>Bement, W. M., Mandato, C. A., Kirsch, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-induced+assembly+and+closure+of+an+actomyosin+purse+string+in+Xenopus+oocytes.">Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.</a> Curr. Biol. 9 (11), 579-587 (1999).</li><li>Kim, H. G., Klotchkova, T. A., Kang, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+without+a+cell+membrane:+regeneration+of+protoplasts+from+disintegrated+cells+of+the+marine+green+alga+Bryopsis+plumosa.">Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa.</a> J. Cell Sci. 114 (11), 2009-2014 (2001).</li><li>Tartar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of Stentor. , 434 (1961).</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+proportionate+structures+in+Stentor.">Regeneration of proportionate structures in Stentor.</a> Biol. Bull. 2 (6), 311-328 (1901).</li><li>Heidemann, S. R., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+regional+approach+to+cell+mechanics.">Towards a regional approach to cell mechanics.</a> Trends Cell Biol. 14 (4), 160-166 (2004).</li><li>Kasza, K. E., Rowat, A. C., Liu, J., Angelini, T. E., Brangwynne, C. P., Koenderinck, G. H., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+as+a+material.">The cell as a material.</a> Curr. Opin. Cell Biol. 19 (1), 101-107 (2007).</li><li>Mitchison, T. J., Charras, G. T., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+a+poroelastic+cytoplasm+for+the+dynamics+of+animal+cell+shape.">Implications of a poroelastic cytoplasm for the dynamics of animal cell shape.</a> Sem. Cell Dev. Biol. 19 (3), 215-223 (2008).</li><li>De Terra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Stentor+coeruleus+on+Colpidium+campylum.">Culture of Stentor coeruleus on Colpidium campylum.</a> J. Eukar. Microbiol. 13 (3), 491-492 (1966).</li><li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydomonas+Sourcebok.">The Chlamydomonas Sourcebok.</a> 1, (2009).</li><li>Vallotton, P., Ponti, A., Waterman-Storer, C. M., Salmon, E. D., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery,+visualization,+and+analysis+of+actin+and+tubulin+polymer+flow+in+live+cells:+a+fluorescent+speckle+microscopy+study.">Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study.</a> Biophys. J. 85 (2), 1289-1306 (2003).</li><li>Miyake, A., Harumoto, T., Iio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defence+function+of+pigment+granules+in+Stentor+coeruleus.">Defence function of pigment granules in Stentor coeruleus.</a> Eur. J. Protistol. 37 (1), 7-88 (2001).</li></ol>

The authors declare that they have no competing financial interests.

The most important application of this method will be to use it to determine the molecular pathways that regulate cytoplasmic flow during wound healing in Stentor. A wide range of chemical inhibitors are available that target motor proteins as well as components of the cytoskeleton. Inhibitors of calcium signaling would also be likely candidates. It would also be of interest to remove or reduce the pigment granules (extrusomes) containing the blue pigment Stentorin, whose release from the cell is triggered under stress, and ask how the exocytotic release of Stentorin may affect flow of nearby cytoplasm. The pigment can be depleted using a simple bleaching treatment15 which could be done just prior to cutting. There does not appear to be any simple way to perform the wound healing assay in a parallel fashion since the cells need to be cut and imaged one at a time. Thus using a single microscope, it would be possible to operate on and measure cells at a rate of roughly 1 per 0.5 hr, so that during a single day of work it should be possible to obtain enough data points for a single compound to determine if it has a statistically significant effect on the process. While the method is thus not in any sense high throughput, it will be absolutely feasible to screen though a selected set of candidate compounds specific for a few carefully chosen target pathways.
A major limitation of the current approach is the relatively crude method for assembling the double-decker microscope. Assuming that the inverted microscope used for imaging is a general use instrument that other workers need to use for other purposes, an important consideration is the ease with which the apparatus can be disassembled and reassembled. While the present method using lab tape is indeed rapid and easily disassembled, it does require careful alignment by the user during assembly each time. An important modification would therefore be to construct a customized removable holder with slots for the eyepiece head of the inverted scope and the body of the dissecting scope. This should be easy to fabricate using modern 3D printer technology.
A second limitation of the approach is the reliance on DIC imaging of intracellular organelles as markers of flow. Because the identity of these organelles observed in the microscope is not currently known, it is not possible to know their size or potential interactions with other cellular components. It might therefore be advantageous to inject extrinsic tracers such as fluorescent beads and use them to track the flow. However this would require an extra step - microinjection - before the experiment could be done, thus further slowing the assay.
The final key limitation is the reliance on a single image plane for data acquisition. The primary importance of this assay is that it goes beyond simply asking whether a wound heals or not, and allows detailed analysis of the flow pattern to be obtained. In this regard, it will be extremely important to extend the imaging to three dimensional imaging. The current method images a single focal plane and thus when the PIV analysis is performed, the result is a slice through the full three dimensional flow field. This is informative but incomplete. By acquiring 3D images at each time step, it should be possible to obtain a fully three dimensional flow field. This will be particularly critical for observing exactly how the cytoplasm moves in the immediate vicinity of the wound site.

Regeneration and wound healing are important biological processes whose mechanisms remain areas of active investigation. Indeed, the ability to heal wounds and regenerate damaged parts is one of the features that sets living things apart from inanimate objects. Normally, regeneration is thought to be mediated by differentiation of stem cells into various cell types needed to rebuild a damaged or severed animal part, while wound healing is viewed in terms of new cells invading a wound to rebuild an intact epithelium. Thus both processes appear to be mediated by collectives of individual cells, and seem therefore to be quintessentially multicellular processes. It is therefore potentially surprising that individual cells are, in some cases, able to repair wounds and regenerate lost components1. Wound healing within single cells can involve multiple pathways including actin-myosin purse string contraction around the wound and membrane patching by rapid fusion of exocytotic vesicles1-3. Although many cell types are capable of some degree of wound healing4-6, perhaps the most dramatic example is the giant ciliate Stentor coeruelus7, shown in Figure 1. Individual Stentor cells are 1 mm long, and are covered with rows of cilia. Stentor cells are cone-shaped with an oral apparatus, consisting of thousands of cilia, at one end, and a narrow hold-fast structure at the other end. Stentor was a classical model organism for studying regeneration because if any part of the cell was cut off, it could regenerate7. For example, in a classic series of experiments, Thomas Hunt Morgan8 showed that a Stentor cell could be cut in half and each half would regenerate a normally proportioned cell in a matter of hours8. The molecular pathways responsible for regeneration in Stentor remain completely unknown.
The ability of Stentor cells to serve as a regeneration model system relies on the amazing ability of these cells to heal themselves after being cut. Why doesn&#39;t the cytoplasm of a Stentor cell leak out when the cell is cut? Wound healing is not due to immediate contraction of the cell cortex, because it is possible to take two Stentor cells, cut them, and then join the two cut cells together in various configurations7. During such grafting experiments, the cytoplasm of the two cells must remain exposed since it fuses when the cells are pushed together. Somehow, the cytoplasm is exposed enough to the surrounding media to allow grafting, but yet it does not flow out of the cell. Is the cytoplasm of Stentor sufficiently elastic that it simply cannot flow out even when a large hole is made in the plasma membrane? Do cells increase the viscosity of cytoplasm locally around a wound to prevent the cytoplasm from flowing out? Cytoplasm is a highly complex active material9-11, and the Stentor wound healing response represents a unique opportunity to study cytoplasmic flows and mechanics during single cell wound healing. However, the use of surgical methods to induce wounding in Stentor poses a technical challenge. In order to cut a 1 mm long cell using glass needles, the conventional surgical paradigm, it is necessary to operate at low magnification with long working distance. Under these imaging conditions, it is not possible to track the flow of cytoplasm, and all one can determine is whether the cytoplasm remains inside the cell or not. The real question we would like to ask is how the movement and viscosity of cytoplasm may change during cutting and healing. This requires high resolution imaging. Here we present an adaptation of Tartar&#39;s protocol for cutting Stentor7 that permits the cutting to be done under lower magnification optics to see the whole cell while simultaneously observing cytoplasm flow at high resolution, allowing this system to be used as a platform for exploring the mechanisms of single-cell wound healing.

<table border="1">
	<tbody>
		<tr>
			<th>Material Name</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th></th>
		</tr>
		<tr>
			<td>Stentor coeruleus live culture</td>
			<td>Carolina Biological</td>
			<td>131598</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Cellulose, 1,500 cP</td>
			<td>Sigma</td>
			<td>M0387</td>
			<td></td>
		</tr>
	</tbody>
</table>
EQUIPMENT:
<table border="1">
	<tbody>
		<tr>
			<th>Material Name</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th></th>
		</tr>
		<tr>
			<td>Soft glass stirring rod 5 in long</td>
			<td>Fisher Scientific</td>
			<td>11-380A</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dish 4.5 in 250 ml&#160;glass</td>
			<td>Carolina Biological</td>
			<td>741004</td>
			<td></td>
		</tr>
		<tr>
			<td>IX81 inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 200M inverted microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 2000 Stereo Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Evolve 512 x 512 EMCCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo 20X objective</td>
			<td>Olympus</td>
			<td>UPLSAPO</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing cytoplasmic flow during single-cell wound healing in stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Using the protocols described above, it was found that the cytoplasm of Stentor is remarkably dynamic, both before and after cutting. Video 1 shows the extensive flow of cytoplasm present in un-cut cells. Despite our original expectation that Stentor&#39;s ability to survive cutting would involve a stiffening or gelling of cytoplasm, in fact when Stentor cells are cut with enough violence to draw the cytoplasm out of the cell, the cytoplasm remains highly dynamic and manages to flow back into the cell in a highly directed fashion (Video 2).
In addition to demonstrating a previously unsuspected degree of cytoplasmic motion during wound healing, the high resolution of imaging in the assay can be used to quantify the flow rates using particle image velocimetry. The ability to make such measurements will allow the effect of chemical inhibitors to be rigorously tested.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig1highres.jpg" src="/files/ftp_upload/50848/50848fig1.jpg" /> 
Figure 1. Morphology of a living Stentor cell. Darkfield image of a living Stentor cell that has been fed with Chlamydomonas as described in Procedure 1.3, visible as green material inside food vacuoles. The oral apparatus through which the cell eats is visible as a rim of cilia at the top of the image.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig2highres.jpg" src="/files/ftp_upload/50848/50848fig2.jpg" /> 
Figure 2. Preparation of glass needle for surgery. (A) Needle is drawn from the end of a solid glass rod. (B) High magnification view of needle tip indicating the length and sharpness necessary for surgery.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig3highres.jpg" src="/files/ftp_upload/50848/50848fig3.jpg" /> 
Figure 3. Custom coverslip holder for microsurgery. In order to allow open access for surgery while allowing optimal imaging conditions, cells are mounted on a coverslip rather than a thick class slide. In order to allow the coverslip to fit on the microscope stage a plastic holder can be fabricated using a 3D printer. The holder has the same dimensions as a standard microscope slide while the depression in the middle matches the dimensions of the coverslip. A hole in one corner allows the coverslip to be popped out of the holder.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig4highres.jpg" src="/files/ftp_upload/50848/50848fig4.jpg" /> 
Figure 4. Double-decker microscope configuration. A dissecting microscope head is mounted on top of the eyepiece head of an inverted microscope. Lab tape allows the apparatus to be rapidly assembled and disassembled when the experiment is completed.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig4highres.jpg" target="_blank">Click here to view larger image</a>.
Video 1. Example of cytoplasmic flow observed in living Stentor cell. Cell imaged with 10X objective with darkfield optics at a rate of 2 frames/sec.
Video 2. Imaging of a Stentor cell as it is cut and disrupted with a glass needle. After cutting a substantial quantity of cytoplasm has been pulled out of the main cell body, but it rapidly flows back into the cell.

Watch this Scientific Journal Video about Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus at JoVE.com

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

visualizing-cytoplasmic-flow-during-single-cell-wound-healing-stentor

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Biology

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Studying Habituation in Stentor coeruleus

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

Studying Habituation in Stentor coeruleus

We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display ...