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.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<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...

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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 ...

What we present here is a robust and simple method to analyze microtubule dynamics using live-cell spinning disk confocal imaging in cells synchronized in prometaphase, and images are afterwards analyzed on a MATLAB-based platform. The use of red-shifted fluorescent protein in combination with spinning disk microscopy reduces phototoxicity. Therefore, a larger number of cells can be imaged within the same preparation.Microtubule dynamicity is altered in a large number of pathological conditions. Therefore, understanding the regulation and the behavior of microtubule dynamics in those processes can help us understanding the mechanism of disease, and eventually, developing therapies to compensate for them. And the method is also upscaleable, so it could potentially be applied in a drug discovery effort.The method can be modified by changing fluorescence desynchronization protocol and obtaining cells from different phases of the cell cycle. This can be useful when screening drugs against microtubules and identifying defect on dividing and non-dividing cells. It is necessary to optimize the transfection protocol and seeding density for each cell type.The expression level of EB3 should be low, in order to detect single growing microtubules. Start by rinsing asynchronously growing HeLa cells in DPBS, then incubate them with trypsin-EDTA for five minutes, at 37 degrees Celsius. Stop the trypsinization by adding RPMI-1640 medium supplemented with 10%heat inactivated FCS at a three to one ratio, the added trypsin-EDTA.Calculate the concentration of the cells according to manuscript directions, and seed 50, 000 cells into each well of a prepared, chambered coverslip. Return the coverslip to the incubator, and grow cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide. On the next day, remove the cells from the incubator, and drop-wise add 100 microliters of transfection mixture into each well.Return the cells to the incubator, and after four hours of incubation supplement the cells with fresh medium and additionally incubate for 20 hours. Prepare a 2.5 micromolar solution of dimethylenastron, or DME, in phenol red free DMEM, supplemented with 10%FCS and two millimolar L-glutamine. Replace the growth medium in the chambered coverslip with the DME growth medium, and return the cells to the incubator.After 3.5 hours of incubation, transfer the cells to the microscope by mounting the chambered coverslip into an environmental chamber set to 37 degrees Celsius and 5%carbon dioxide, with dark panels for imaging. Then continue the incubation for a total of four hours. Perform the time lapse imaging on an inverted microscope with a 100X, 1.49 numerical aperture, oil immersion objective, a dual disk confocal system, and a reliable auto focus system.Define the imaging parameters as described in the text manuscript. Find a cell in prophase, and focus in the Z-plane corresponding to the center of the monopolar mitotic spindle. Then acquire images every 5 seconds, over a total of one minute, with no binning and no illumination between exposures.Start by loading the numerical analysis software and adding the u-track V2.2.0 folder into the software search path, called movie selector GUI, from the command window. Then import the raw files generated by the image acquisition software. Manually enter the numerical aperture of the objective and the time interval used for imaging.Once all the images are loaded, save the entered time lapse series as a movie by selecting save as movie list, and the u-track option on the right side of the dialogue window. Select microtubule plus-ends from the pop-up menu, and click okay, which opens a new dialogue window to determine the parameters of the three steps of the analysis. For step one, click settings and select comet detection from the drop down menu.Define the parameters for the difference of Gaussian filter and the watershed segmentation, as described in the manuscript. Then select apply settings to all movies and click apply. For step two, select the microtubule plus-end dynamics, and use the setting options to define the values for linking, gap closing, merging, and splitting, and Kalman filter functions, according to the text manuscript.For problems with dimensionality, choose two from the drop down menu, and use five frames for maximum gap to close, and three frames for minimum length of track segments from first step. As before, select apply settings to all movies and click apply. For step three, choose microtubule dynamics classification as a track analysis method, and define the parameters through the setting button.Select the boxes for remove tracks at the beginning and end of the movie, and make statistics histograms. From the drop down list, select using two to three frames before forward gap, and 95th percentile of forward gap speed distribution, for forward and backward reclassification, respectively. Once all parameters are defined, select apply check/uncheck to all movies, and run all movies boxes from the control panes u-track window, then press run.A message is displayed once the movie processing is complete. The pEB3-tdTomato plasmid was transiently expressed in HeLa cells. The cells were synchronized with DME treatment.Time lapse movies of microtubule growth were analyzed, and the resulting growth speed and dynamicity were plotted. The parameters described to affect the analysis, such as maximum gap length and maximum shrinkage factor, were modified for the same set of time lapse movies. The corresponding values of growth speed and dynamicity were calculated.While the resulting growth speed was not significantly affected, the dynamicity was different when the maximum gap length was modified. In all three cases, the detection of microtubule subtracks was similar, yet the reconstruction of the full MT trajectories was mostly affected when maximum gap length was set to 15. In order to assess whether imaging conditions interfered with microtubule behavior, the first and second halves of the time lapse series were analyzed separately, and the corresponding growth speed and dynamicity were compared.As expected, no significant differences were detected. It is important to keep in mind, that because the cells are synchronized in prometaphase, the density of microtubule is very high. Therefore, one has to set the parameters of the analysis very carefully, not to misidentify different microtubule.The measurement of microtubule dynamics with this method can be compared with studies of other biological targets directly, indirectly, regulating microtubules.

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

Abstract