Name: a graphical user interface for software-assisted tracking of protein concentration in dynamic cellular protrusions
Uploaded: 2017-07-11T14:00:00
Description: Watch this Scientific Journal Video about A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions at JoVE.com

The authors acknowledge funding from the DFG (EXC-1003 to MG).

1. Cell Culture
<ol>
	<li>Culture HeLa or COS cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 4.5 g/L D-glucose, L-alanine-L-glutamine dipeptide, pyruvate, 10% fetal bovine serum, and 10 units/ml of penicillin/streptomycin. Culture neurons in culture-media without L-glutamine, glutamic acid or aspartic acid, supplemented with 0.5 mM L-alanine-L-glutamine dipeptide, serum-free neuronal supplements and 10 units/ml of penicillin/streptomycin.</li>
	<li>Once 40% confluency is reached, transfect cells with c...

<ol>
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Here we present a detailed protocol for tracking filopodial growth dynamics and analysis of relative protein concentrations in these dynamic structures via the convex-hull algorithm. Using the software, up to 3 channels can be compared pair-wise in a single run, whereby the relative concentrations of two channels (i.e. proteins) is determined throughout the extension/retraction cycle and stored as image and data files in separate folders. In addition to the routine operations, the software also provides a number...

Spatio-temporal control of actin regulatory proteins is associated with filopodium dynamics 1,2. Tracking spatially resolved protein concentration along the whole filopodial length through time is thus crucial to advance our understanding of the mechanisms underlying initiation, elongation, stabilization or collapse of these dynamic structures 3,4. Unlike protein analysis in the cytosol, where many cell shape changes occur at a larger scale, filopodia are dynamic micro structures that constantly buckle 5 and bend, thus precluding analysis using a simple approach such as a line-scan.
Different software solutions for tracking filopodial shape are available 6,7,8,9. Likewise, software for ratiometric tracking of protein dynamics within the cell body has been developed 10,11. To combine automated tracking of filopodial shape and spatio-temporal protein analysis, we recently developed an image analysis software based on the convex-hull algorithm 12. This novel analysis method, which is operated via a graphical user interface (GUI), combines for the first time, relative protein concentration along the filopodial length and growth velocity, thus allowing the accurate measurement of spatio-temporal protein distribution independent of movement of these dynamic structures 12.
The idea behind the software (source code is freely available, see below) is that one of the vertices of the convex hull will coincide with the tip of the filopodium (Figure 1A). By looking in the subsequent frame for the nearest vertex of the convex-hull, the moving tip can be tracked throughout the whole movie. Once the tip is detected in each frame, its position is used to draw an axis by joining the tip with a reference point at the base of the filopodium (Figure 1B). Finally, using equidistant nodal points, whose positions are determined by the median pixel with maximum intensity along the line orthogonal to the axis, are used to determine a backbone that follows the filopodial shape. Taking advantage of this adaptive backbone, a kymograph is generated to trace filopodial growth and protein concentrations for up to three channels along the filopodial length (Figure 1C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55653/55653fig1.jpg" /> 
Figure 1: Working Principle of the Image Analysis Software. (A) The algorithm behind the software. In Step1 the user specifies the reference (base) and the vertex (the tip) of the filopodium. In Step 2-1 the backbone of the filopodium is obtained using the median pixel with maximum intensity value. In Step 3 the backbone is used for spatial protein intensity profile. In Step 2-2 the software automatically tracks the tip in the subsequent frame. The whole procedure iterates. (B) Snapshot of the algorithm with real filopodium introducing important elements such as the convex hull that is being used for tracking. (C) Overview of parameters that can be measured with the algorithm. This figure has been modified from reference 12. <a href="http://cloudflare.jove.com/files/ftp_upload/55653/55653fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The image analysis software is operated in Matlab (referred to as programming software) via a graphical user interface. To maximize flexibility and robustness for the particular experimental setting, the user can adjust a series of tracking parameters (e.g. permitted bending angle and inter-frame movement) and also make some corrections to the movies (e.g. cropping, rotation, removal of unwanted objects) (Figure 2A and Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GUI</td>
			<td>No.</td>
			<td>Mandatory</td>
			<td colspan="5">Description</td>
			<td>Name (in GUI)</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1a</td>
			<td>Y</td>
			<td colspan="5">Loading stacked .tiff file representing cell body (with box checked in) or create superimposed cell body from channels</td>
			<td>CellBody</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1b</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 1</td>
			<td>Protein 1</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1c</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 2</td>
			<td>Protein 2</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1d</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 3</td>
			<td>Protein 3</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1e</td>
			<td>N</td>
			<td colspan="5">Resets everything to preloaded stacked image files</td>
			<td>Reset</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2a</td>
			<td>Y</td>
			<td colspan="5">Scroll bar to determine the initial frame for analysis in GUI window #2</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2b</td>
			<td>Y</td>
			<td colspan="5">Scroll bar to determine the final frame for analysis in GUI window #2</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2c</td>
			<td>Y</td>
			<td colspan="5">Scroll bar representing current frame</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2d</td>
			<td>N</td>
			<td colspan="5">The grey value of the pixels below which all pixels will be set to zero</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2e</td>
			<td>N</td>
			<td colspan="5">The grey value of the pixels above which all pixels will be set to maximum values</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2f</td>
			<td>N</td>
			<td colspan="5">Set the intensity values of the pixels specified by &#60;2e&#62; &#38; &#60;2f&#62;</td>
			<td>Set</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2g</td>
			<td>N</td>
			<td colspan="5">Play the intensity-adjusted movie</td>
			<td>Play</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2h</td>
			<td>N</td>
			<td colspan="5">Crop Image</td>
			<td>Crop</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2i</td>
			<td>N</td>
			<td colspan="5">Rotate Image</td>
			<td>Rotate</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2j</td>
			<td>N</td>
			<td colspan="5">Delete regions in the whole stack</td>
			<td>Delete regions</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>3a</td>
			<td>Y</td>
			<td colspan="5">Click to open the &#8216;Analysis Window&#8217; (GUI window #2)</td>
			<td>Tracking Window</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>3b</td>
			<td>Y</td>
			<td colspan="5">Enter the size of a pixel in microns</td>
			<td>Enter Pixel size</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>4</td>
			<td>Y</td>
			<td colspan="5">Click to generate the boundary/edge image of the superimposed cell body</td>
			<td>Boundary</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>5</td>
			<td>Y</td>
			<td colspan="5">Click to select the base and tip of the filopodia</td>
			<td>Referernce</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6a</td>
			<td>Y</td>
			<td colspan="5">Enter the number of segments or nodes</td>
			<td>No of segments</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6b</td>
			<td>Y</td>
			<td colspan="5">Enter the scan length (perpendicular to axis)</td>
			<td>Scan Width</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6c</td>
			<td>Y</td>
			<td colspan="5">Enter the length above which filopodia starts bending</td>
			<td>Accurate Meas after</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6d</td>
			<td>Y</td>
			<td colspan="5">Enter the radius of the tip detection circle (i.e. area where the vertex can be localized in the next frame)</td>
			<td>Radius of tip detection</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6e</td>
			<td>Y</td>
			<td colspan="5">Enter the maximum angle the filopodium can bend from the vertical axis</td>
			<td>Angle Threshold</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6f</td>
			<td>N</td>
			<td colspan="5">Add reference points for base and tip for that specific frame</td>
			<td>Select reference</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6g</td>
			<td>N</td>
			<td colspan="5">Enter the length above which filopodia starts bending for that specific frame</td>
			<td>Accurate Meas after</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6h</td>
			<td>N</td>
			<td colspan="5">Enter the radius of the detection circle for that specific frame</td>
			<td>Radius of tip detection</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6i</td>
			<td>N</td>
			<td colspan="5">After entering all the parameters for the specific frame click to store the values to memory and file for further reference</td>
			<td>Add</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6j</td>
			<td>N</td>
			<td colspan="5">Click to delete the set manually parameters for that frame</td>
			<td>Delete</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6k</td>
			<td>N</td>
			<td colspan="5">Click to delete all parameters stored manually using the &#8216;optional features panel&#8217; for all frames</td>
			<td>Reset</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6l</td>
			<td>N</td>
			<td colspan="5">Check in before tracking to store all tracking results in memory for future reference</td>
			<td>History trace</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>7</td>
			<td>Y</td>
			<td colspan="5">Click to start tracking</td>
			<td>Track&#38;Analyze</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8a</td>
			<td>N</td>
			<td colspan="5">Click to start tracking protein channel intensity</td>
			<td>Analyze Protein Intensities</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8b</td>
			<td>N</td>
			<td colspan="5">Check in to track protein channel intensity along the filopodial length</td>
			<td>Whole filopodia</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8c</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the reference protein or protein A</td>
			<td>ProteinA</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8d</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the protein B</td>
			<td>ProteinB</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8e</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the protein C</td>
			<td>ProteinC</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8f</td>
			<td>N</td>
			<td colspan="5">Check in to track average protein intensity in the tip</td>
			<td>Leading tip</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8g</td>
			<td>N</td>
			<td colspan="5">Enter the length of the tip</td>
			<td>Tip Length</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8h</td>
			<td>N</td>
			<td colspan="5">Enter the minimum distance from base above which the tip starts forming</td>
			<td>threshold</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8i</td>
			<td>N</td>
			<td colspan="5">Click to save the leading tip analysis results to file</td>
			<td>Push Button</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9a</td>
			<td>N</td>
			<td colspan="5">Click to initiate ratiometric protein analysis</td>
			<td>Compare</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9b</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein B with respect to A</td>
			<td>log10(B/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9c</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein C with respect to A</td>
			<td>log10(C/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9d</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein B with respect to A at the tip</td>
			<td>log10(B/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9e</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein C with respect to A at the tip</td>
			<td>log10(C/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>10a</td>
			<td>N</td>
			<td colspan="5">Choose other color-map (default: Jetplot)</td>
			<td>Color Map</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>10b</td>
			<td>N</td>
			<td colspan="5">Edit the colormap</td>
			<td>Edit colormap</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of All Functions Present in the GUI Windows #1 and #2.
Once this is accomplished, the program creates a convex hull and automatically tracks the tip throughout the movie. Parameters extracted from the movie, such as a ratiometric kymograph, growth velocity, and filopodial length are displayed and also stored in the work folder as images and as data files. Other parameters such as filopodial lifetime, growth rate and retraction rate can then be extracted and further analyzed from the stored data files (Figure 2B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55653/55653fig2.jpg" /> 
Figure 2: Graphical User Interface for using the Image Analysis Software. (A) GUI Window #1 is used for loading and processing images. The program can load up to 3 protein channels, whereby 2 channels are compared pair-wise. The window comes with mandatory (blue) and optional features (green) for pre-processing the images prior to tracking (B) GUI Window #2 is used for tracking the filopodium as well as spatio-temporal and ratio-metric protein analysis. Again, optional features are marked in green. This figure has been modified from reference 12. <a href="http://cloudflare.jove.com/files/ftp_upload/55653/55653fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we present a detailed protocol for sample preparation and software handling. We start with detailed instructions on culturing cells and acquiring movies optimized for image analysis. This section on data acquisition is followed by a detailed description for operating the image analysis software. Throughout the protocol, we introduce critical steps and optional features that should be considered when collecting and processing data. Finally, we analyze filopodia from different model systems with the image analysis software, before closing with a comparison of the described image analysis software with other programs available for filopodial quantification and a discussion on limitations and future direction.

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">DMEM</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:204px;">31966-021</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">10% Fetal bovine serum</td>
			<td style="width:204px;">Biochrom AG</td>
			<td style="width:204px;">L11-044</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Lipofectamine 2000</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:204px;">11668-027</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">1% penicillin/streptomycin</td>
			<td style="width:204px;">Biochrom AG</td>
			<td style="width:204px;">12212</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Neurobasal Medium</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:204px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">B27</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:204px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">HEPES (1M stock solution)</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:204px;">15630</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Citrine-N1</td>
			<td style="width:204px;">Addgene</td>
			<td style="width:204px;">54593</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Labtech</td>
			<td>Thermo</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Glutamax-I</td>
			<td>Thermo</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Hela</td>
			<td>Leibniz Institute DSMZ</td>
			<td>ACC-57</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">COS 7</td>
			<td>Leibniz Institute DSMZ</td>
			<td>ACC-60</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">3T3 cells</td>
			<td>Leibniz Institute DSMZ</td>
			<td>ACC-59</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Microscope</td>
			<td>Nicon Eclipse</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Camera</td>
			<td>Andor</td>
			<td>DU888 Ultra</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Confocal Unit</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:204px;">Pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
	</tbody>
</table>

a graphical user interface for software-assisted tracking of protein concentration in dynamic cellular protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

Using COS cells transfected with a marker for filamentous actin (f-tractin18, red) and a cytosolic reference (green), we found actin-rich filopodial protrusions (Figure 3A, top panel). Time series showed that filopodia rapidly extend and retract (Figure 3A, middle panel). Using the image analysis software, we then traced individual filopodia. Comparison of filopodial length measured by hand vs. the image a...

Watch this Scientific Journal Video about A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions at JoVE.com

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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