Name: measurement of microtubule dynamics by spinning disk microscopy in monopolar mitotic spindles
Uploaded: 2019-11-15T12:00:00
Description: Watch this Scientific Journal Video about Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles at JoVE.com

We thank the members of the Light Microscopy Facility, Max-Planck Institute of Experimental Medicine, for their expert advice and support.

1. Seeding of HeLa Cells
<ol>
	<li>Prepare 2 mL of 5 &#956;g/mL fibronectin solution in phosphate buffered saline (PBS) and add 450 &#956;L of it into each well of a 4 well chambered coverslip (#1.5). Incubate the slide for 15 min at 37 &#176;C and 5% CO2.</li>
	<li>Rinse asynchronously growing HeLa cells with Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) and incubate with trypsin-EDTA (0.05%: 0.02%; w:v) for 5 min at 37 &#176;C. Stop the enzymatic reaction by the addition of Roswell Park Memorial Instit...

Here, we describe a modification of a method first established by Ertych et al.44. Along with several other modifications, we combine this technique of MT dynamics analysis with dual spinning disk confocal imaging. The use of the dual spinning disk improves the resolution of growing MTs while reducing phototoxicity36. We further reduce the photobleaching and laser light-induced damage of the cells by switching to a longer wavelength fluorescent reporter. The tdTomato fluore...

Microtubules (MTs) are highly dynamic structures found in virtually all eukaryotic cells and in some bacteria1. Together with actin and intermediate filaments, they sculpt the cytoskeleton2,3. Cell division4, molecule transport5, flagellar beating6, the sensation of the surrounding environment through primary cilium7, hearing (kinocilium)8,9, embryogenesis10,11,12, invasion and metastasis13,14, and even memory formation15,16,17,18, and many other processes primarily rely on MTs. Participation of MTs in all these events would be impossible without their remarkable ability to rapidly switch between growth (polymerization) and shrinkage (depolymerization). This property is described as dynamic instability19. MT dynamicity is altered in many pathological conditions20,21,22. Hence, determining the nature of this property can help to understand disease mechanisms and subsequently their treatment.
A long list of methods has been developed for MT dynamics analysis, most of which are based on imaging techniques23. Initially, wide field light microscopes were used for observing the formation of tubulin polymers in vitro24. The discovery of end-binding (EB)-proteins that collect at MT plus-ends and the development of methods to fluorescently label proteins made it possible to observe the behavior of MTs directly in living cells with wide field and confocal fluorescence microscopes25,26,27. One EB-protein is end-binding protein 3 (EB3)28; by overexpressing and tracking EB3 fused to a fluorescent protein, MT plus-end assembly rates can be determined29,30.
Confocal laser scanning fluorescence microscopy (CLSM) is frequently used to follow MT dynamics. However, this imaging technique poses a high risk of phototoxicity and photobleaching, two undesirable processes for live cell and dim sample imaging31. In order to obtain a better signal-to-noise ratio, the laser power and the exposure duration should be high enough while not damaging the samples, and this requires sacrificing resolution in exchange for speed. A suitable alternative to CLSM is spinning disk microscopy32. This imaging modality is based on the use of a Nipkow disk33, which consists of a moving disk bearing an array of pinholes, and works equivalently to many CLS microscopes imaging the same sample simultaneously34. Therefore, the light from the laser will illuminate several regions in the sample simultaneously but retain the confocal nature. The Nipkow disk, therefore, allows obtaining images similar to CLSM but faster and using less laser power. The Nipkow disk was further improved by Yokogawa Electric, which introduced a second disk with an array of microlenses on it that individually direct light into a respective pinhole, further reducing phototoxicity and photobleaching35. Thus, spinning disk laser scanning microscopy became a method of choice for live cell imaging, and it makes it possible to obtain images with high signal-to-noise ratio at a high speed31,36, which is crucial for resolving signals such as those from the fast-growing MT ends.
MT dynamics differ temporarily. For example, the mitotic MTs are more dynamic than the interphase ones37,38. Similarly, differences in the growth rate and shrinkage have been observed even within the same cell cycle phase, such as mitosis39,40. Therefore, to avoid false data collection, the measurement of MT dynamics should be limited to a narrow time-window during the cell cycle. For example, measurement of MT dynamics in prometaphase can be achieved by treating the cells with dimethyl&#173;enastron (DME), a monastrol analogue that inhibits the motor kinesin Eg541 and prevents the formation of the bipolar mitotic spindle42. Inhibition of cells at prometaphase with Eg5 inhibitor DME and other monastrol derivatives does not affect the MT dynamics43,44,45, which makes DME a useful tool for studying MT dynamics both in fixed and live cells44.
Here we combine the method of MT dynamics analysis in prometaphase cells described by Ertych et al.44 with dual spinning disk imaging. This method allows measurement of the MT dynamics in prometaphase cells collected from a single focal plane with a higher imaging rate, yet without photobleaching and minimal phototoxicity. Furthermore, as a fluorescent reporter, we use tandem dimer Tomato fluorescent protein (tdTomato) which has improved brightness and photostability in comparison to the green fluorescent protein (EGFP) and is excited with lower energy light46. Therefore, tdTomato requires less laser power for excitation and is less phototoxic. Altogether, we further improve the method by reducing the phototoxicity and improving the resolution and postprocessing required for the MT dynamics analysis. Additionally, we create a basis for future modifications of the method by combining it with other synchronization techniques.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dimethylenastron</td>
			<td style="width:123px;">Merck</td>
			<td style="width:344px;">324622</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM w/o phenol red</td>
			<td>Gibco</td>
			<td>31053-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum</td>
			<td>Biochrom</td>
			<td>S0415</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fibronectin Bovine Plasma</td>
			<td>Merck</td>
			<td>F4759</td>
			<td>Sterile powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Stable glutamine substitutive</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">jetPRIME</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EB3-TdTomato</td>
			<td>Addgene</td>
			<td>plasmid #50708</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Merck</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin/EDTA solution</td>
			<td>Biochrom</td>
			<td>L2143</td>
			<td>0.05% / o.02 % w/o calcium and magnesium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#181;-slide</td>
			<td>Ibidi</td>
			<td>80426</td>
			<td>4-well slide with #1.5 coverslip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eclipse Ti Inverted microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective</td>
			<td>Nikon</td>
			<td>MRD01991</td>
			<td>CFI Apo TIRF 100XC Oil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ACAL Laser Excahnger</td>
			<td>Nikon</td>
			<td></td>
			<td>Laser box. 405, 458, 488, 514, 561 and 647nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spinning disk module</td>
			<td>Andor</td>
			<td>CSU-W</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Camera</td>
			<td>Andor</td>
			<td>iXon Ultra 888</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Environmental Chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>Dark chamber equipped with CO2 supply, tmeperature control and humidifier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HeLa Cells</td>
			<td>DSMZ</td>
			<td>ACC-57</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NIS Elements v4</td>
			<td>Nikon</td>
			<td></td>
			<td>Spinning disk microscope. Acquisition Software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>Computing environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td>Statistical analysis and display software</td>
		</tr>
	</tbody>
</table>

measurement of microtubule dynamics by spinning disk microscopy in monopolar mitotic spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Following the given protocol outlined in Figure 1A, the pEB3-tdTomato plasmid was transiently expressed in asynchronously growing HeLa cells. The cells were synchronized 48 h after the transfection at prometaphase through DME treatment (Figure 1B). This step ensured that the measurement of MT dynamics was always done at the same phase of the cell cycle. The time-lapse movies were further processed and analyzed with U-Track v2.2.0 as described in its supplementar...

Watch this Scientific Journal Video about Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles at JoVE.com

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

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