Name: detection and quantification of tunneling nanotubes using 3d volume view images
Uploaded: 2022-08-31T14:00:00
Description: Watch this Scientific Journal Video about Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images at JoVE.com

D.K.V and A.R thank the Manipal Academy of Higher Education for the TMA Pai fellowship. We thank the Science and Engineering Research Board of India for SERB-SRG (#SRG/2021/001315), as well as the Indian Council of Medical Research of India (#5/4-5/Ad-hoc/Neuro/216/2020-NCD-I) and the Intramural fund of Manipal Academy of Higher Education, Manipal, India. We thank JNCASR&#39;s (Jawaharlal Nehru Centre for Advanced Scientific Research, India) confocal facility and B. Suma for the confocal microscopy in JNCASR.

NOTE: SH-SY5Y cells cultured in DMEM/F-12 media were differentiated with 10 &#181;M retinoic acid for 7 days and treated with 1 &#181;M oA&#946; oligomers for 2 h at 37 &#176;C (5% CO2). After treatment, the cells were fixed with Karnovsky&#39;s fixative solution and double-immunostained with phospho-PAK1 (Thr423)/PAK2 (Thr402) antibody and F-actin-binding phalloidin. Later, confocal z-stack images were taken using a confocal laser scanning microscope. The TNTs were quantified by manual counting and distinguis...

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Several researchers in the past 2 decades have been trying to understand and characterize the structure of TNTs18. The lack of specific markers hinders progress, and there is an increasing demand for a convenient, standardized method that can be used to identify, characterize, and quantify TNTs. TNTs are defined as F-actin-based membrane conduits that hover between two cells. Studies have shown that &#946;-tubulin-positive, close-ended, developing neurites hover between two distant cells and resem...

Tunneling nanotubes (TNTs) are F-actin-based, primarily open-ended membrane conduits and play a vital role in the intercellular transfer of cargo and organelles1. The unique characteristic of TNTs is that they connect neighboring cells without any contact with the substratum; they are over 10-300 &#181;m in length and their diameters vary between 50 nm to 1 &#181;m2,3. TNTs are transient structures, and their lifetime lasts between a few minutes to several hours. TNTs were first demonstrated in PC12 neuronal cells1; later, numerous studies showed their existence in several cell types in vitro and in vivo4,5. Several studies have revealed the pathological significance of TNTs in various disease models, such as neurodegenerative diseases, cancer, and viral infections6,7,8.
The structural heterogeneities of TNTs have been demonstrated by several studies in various cellular systems9. The differences are based on cytoskeleton composition, the mechanism of formation, and the cargo types transferred10. Primarily, the open-ended, F-actin-positive membrane continuity that hovers between two neighboring cells and transfers organelles is considered to consist of TNTs11. However, the lack of clarity or diversity observed in the formation of TNTs adds to the difficulty in developing TNT-specific markers. Thus, it is difficult to identify TNT structures by conventional detection methods and distinguish membrane nanotubes in terms of open-ended and close-ended protrusions12. However, the characteristic of TNTs to hover as F-actin membrane protrusions between two cells is relatively easier and more feasible to identify using conventional imaging techniques. Other actin-based cellular protrusions such as filopodia and dorsal filopodia cannot hover between two distant cells, particularly when cells are fixed. Of note, close-ended, electrically coupled, developing neurites are often termed TNT-like structures13.
It is known that F-actin plays an important role in TNT formation, and several studies have shown that the F-actin inhibitor cytochalasin D inhibits the formation of TNTs14,15. In contrast, inhibitors of microtubules do not have any effect on TNT formation16. The last 2 decades have seen several reports on the significant role that TNTs play in the spread of pathology and tumor resistance and therapy17. Therefore, there is a never-ending demand for better techniques for TNT characterization.
The lack of specific markers of TNTs and the diversity in morphology and cytoskeletal composition make it difficult to develop a unique method of characterization. Some studies have used automated image detection and TNT quantification techniques18,19. However, there are several advantages of the present 3D volume manual analysis method over automatic image analysis for the detection and quantification of TNTs. Often, trained human eyes can spot these hovering nano-structures more easily than an automated image detection method. Moreover, automatic detection methods could be difficult to implement in laboratories lacking algorithm expertise. The present method could be widely adopted by researchers due to its precision and reproducibility.
In a recent study, we showed that oA&#946; promotes the biogenesis of TNTs in neuronal cells via a PAK1-mediated, actin-dependent, endocytosis mechanism12. oA&#946;-induced TNTs also express activated PAK1 (or phospho-PAK1). We developed a 3D volume view image reconstruction method to distinguish oA&#946;-induced, F-actin- and phospho-PAK1-immunostained TNTs. &#946;-III tubulin-positive, developing neurites often resemble TNT-like hovering structures20. Hence, we further distinguished F-actin-based TNTs from &#946;-III tubulin-positive neurites and other TNT-like protrusions. 3D volume view images have been used to identify TNTs on the basis of their characteristics of hovering over the substratum and staying connected between two neighboring cells. This paper describes the identification and detection of actin-containing membrane conduits or TNTs using confocal z-stack images and, finally, manual quantification of the identified structures from 3D volume view reconstruction images. The presented method cannot distinguish open-ended proper TNTs from close-ended TNT-like structures; this method helps identify TNTs in in vitro 2D cell culture on a flat substratum. However, the method is easy to implement and reproduce and can be widely used for the precise quantification of only actin-based TNTs and to distinguish them from neurites and &#946;-tubulin positive TNT-like structures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm dish with 14 mm well size made of #1.5 cover slip</td>
			<td>Cellvis</td>
			<td>D35-14-1.5-N</td>
			<td>Imaging dish used to seed cells for staining experiments</td>
		</tr>
		<tr>
			<td>A&#946; (1-42) 1 mg</td>
			<td>AnaSpec</td>
			<td>#64129</td>
			<td>Oligomers of amyloid beta to treat the cells</td>
		</tr>
		<tr>
			<td>Alexa flour 488 Goat Anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td>Secondary antibody for phospho-PAK1</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>Thermo Scientific (MSC Advantage)</td>
			<td>51025411</td>
			<td>Provide aspetic conditions duirng cell culture</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Electron Corporation (Heraeus Hera Cell 240)</td>
			<td>51026556</td>
			<td>For growing cells at or near body temperature</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>ZEISS (Carl Zeiss)</td>
			<td>LSM 880</td>
			<td>Able to generate three-dimensional images of large specimen at super-resolution</td>
		</tr>
		<tr>
			<td>DABCO [1,4-Diazobicyclo-(2,2,2) octane]</td>
			<td>Merck</td>
			<td>8034560100</td>
			<td>Anti-bleaching reagent</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542-1MG</td>
			<td>Neuclear stainer</td>
		</tr>
		<tr>
			<td>DMEM media</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Used for the preparation of 100uM of A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>&#160;DMEM/F12 (1:1 mixture of DMEM and Ham&#8217;s F12)</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td>Culture media for SH-SY5Y</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulphide) Cell culture grade</td>
			<td>Cryopur</td>
			<td>CP-100</td>
			<td>Cell culture grade used as dissolving agent for Retinoic acid</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulphide) Molecular grade</td>
			<td>Himedia</td>
			<td>MB058</td>
			<td>Used as one of the dissolving agent for the lyophilized A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>&#160;Major supplement for Culture media (US origin)</td>
		</tr>
		<tr>
			<td>Formalin Fixative (Neutral buffered 10%)</td>
			<td>Sigma Aldrich</td>
			<td>HT5014-120ML</td>
			<td>Component in the Karnovsky&#39;s fixative solution</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (Grade I, 25% in H2O)</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td>Component in the Karnovsky&#39;s fixative solution</td>
		</tr>
		<tr>
			<td>HFIP (1,1,1,3,3,3-hexafluoro-2-propanol ) solution</td>
			<td>TCI</td>
			<td>H024</td>
			<td>Used to dissolve A&#946; (1-42) 1 mg</td>
		</tr>
		<tr>
			<td>Image Processing/ Analysis Software: FIJI (ImageJ)</td>
			<td>National Institute of Health (NIH)</td>
			<td></td>
			<td>Used to process/analyze the images and to differentiate the TNTs from neurites using its plugin named &#34;volume viewer&#34;.</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Christ, Alpha</td>
			<td>2.4 LDplus</td>
			<td>0.05 mg aliquots of A&#946; (1-42) can be stored in -20 &#176;C after lyophilization only</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin Mixture</td>
			<td>Thermo fisher Scientific</td>
			<td>15640055</td>
			<td>Antibiotic mixture</td>
		</tr>
		<tr>
			<td>Phalloidin-iFlor 555</td>
			<td>Abcam</td>
			<td>ab176756</td>
			<td>F-actin binding stain</td>
		</tr>
		<tr>
			<td>Phospho-PAK1 (Thr423) /PAK2 (Thr402) [Rabbit]</td>
			<td>CST</td>
			<td>#2601</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Polyclonal Antibody to Tubulin Beta 3 (TUBb3)</td>
			<td>Cloud clone</td>
			<td>PAE711Hu01</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-50MG</td>
			<td>Differentiating reagent</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Merck</td>
			<td>8047-15-2</td>
			<td>Detergent used in the Incubation buffer in immunostaining</td>
		</tr>
		<tr>
			<td>Water bath sonicator (Quart, Drain valve Heater)</td>
			<td>Ultrasonic Cleaner</td>
			<td>3.0 L/3.2</td>
			<td>Sonicator used to dissolve A&#946; (1-42) stock, after DMSO adding to it during the preparation of 100 &#181;M A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>ZEN Microscopy software</td>
			<td>ZEISS (Carl Zeiss)</td>
			<td></td>
			<td>Imaging software to acquire confocal microscopy images with smart automation</td>
		</tr>
	</tbody>
</table>

detection and quantification of tunneling nanotubes using 3d volume view images

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely &#34;tunneling nanotubes&#34; (TNTs). TNTs are defined as open-ended, lipid bilayer-encircled membrane extensions that mediate continuity between neighboring cells of diameters ranging between 50 nm and 1 &#181;m. TNTs were demonstrated initially in neuronal cells, but successive studies have revealed the existence of TNTs in several cell types and diseases, such as neurodegenerative diseases, viral infections, and cancer. Several studies have referred to close-ended, electrically coupled membrane nanostructures between neighboring cells as TNTs or TNT-like structures.
The elucidation of ultrastructure in terms of membrane continuity at the endpoint is technically challenging. In addition, studies on cell-cell communication are challenging in terms of the characterization of TNTs using conventional methods due to the lack of specific markers. TNTs are primarily defined as F-actin-based, open-ended membrane protrusions. However, one major limitation is that F-actin is present in all types of protrusions, making it challenging to differentiate TNTs from other protrusions. One of the notable characteristics of F-actin-based TNTs is that these structures hover between two cells without touching the substratum. Therefore, distinct F-actin-stained TNTs can conveniently be distinguished from other protrusions such as filopodia and neurites based on their hovering between cells.
We have recently shown that the internalization of oligomeric amyloid-&#946;1-42 (oA&#946;) via actin-dependent endocytosis stimulates activated p21-activated kinase-1 (PAK1), which mediates the formation of F-actin-containing TNTs coexpressed with phospho-PAK1 between SH-SY5Y neuronal cells. This protocol outlines a 3D volume analysis method to identify and characterize TNTs from the captured z-stack images of F-actin- and phospho-PAK1-immunostained membrane protrusions in oA&#946;-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and &#946;-III tubulin-immunostained membrane conduits.

Here, we identify and characterize oA&#946;-induced TNTs in SH-SY5Y neuronal cells by constructing 3D volume views from confocal z-stack images (Figure 1). The cells were double-immunostained with F-actin and phospho-PAK1. Confocal z-stack images of immunostained cells were analyzed to identify TNTs (Figure 2). Further, DIC images were analyzed to verify that the F-actin- and phospho-PAK1-stained TNT structures were membrane conduits between cells (<strong class...

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Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images

Tunneling nanotubes (TNTs) are primarily open-ended F-actin membrane nanotubes that connect neighboring cells, facilitating intercellular communication. The notable characteristic that distinguishes TNTs from other cell protrusions is the hovering nature of the nanotubes between cells. Here, we characterize TNTs by constructing a 3D volume view of confocal z-stack images.

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely ...

The lack of tunneling nanotubes specific markers restrict progress in the field and there is an increasing demand for a method to identify, characterize, and quantify tunneling nanotubes. Automatic detection methods are difficult to implement in without the expertise to develop an algorithm and/or change the existing algorithm, but our manual method is easy to implement with better precision. Long-range intercellular transfer via tunneling nanotubes is implemented in several ways in various disease models, including neurodegenerative pathology, viral spreading, and cancer.Thus, studies on tunneling nanotubes to understand their role in pathogenesis are important. It is possible the tunneling nanotubes might break during the staining process. Avoid using cover slips and mounting on the slides, using glass bottom imaging dishes instead.Modified fixation protocol helps to keep tunneling nanotubes intact. Demonstrating the procedure will be Deepak KV, a PhD student from the laboratory of Sangeeta NAF. To begin, wash the control and oligomeric A-beta treated cells twice with PBS for two minutes before fixation.Prepare Karnovsky's fixative solution by using 2%formalin fixative and 2.5%glutaraldehyde dissolved in 0.1 molar phosphate buffer of pH 7.2. Then fix the cells in the imaging dish by adding Karnovsky's fixative solution for 45 minutes at room temperature. Prepare the incubation buffer by dissolving 0.1 gram of saponin in five milliliters of FBS and diluted by adding 95 milliliters of PBS.Then wash the fixed cells twice using incubation buffer for two minutes. After fixation, add the first antibody against phospho-PAK1, add a dilution of one to 250 in the incubation buffer and incubate overnight at four degrees Celsius in a dark, moist chamber. The next day after 24 hours of incubation, wash the cells twice with the incubation buffer for two minutes.Then add secondary antibody conjugated to Alexa Fluor 488 and Phalloidin 555. Incubate the cells in the dark, moist chamber for 1.5 hours at room temperature. After the incubation, wash the cells twice with incubation buffer for two minutes.Stain the nucleus by adding DAPI in a one to 2000 dilution and incubate for five to 10 minutes at room temperature in the dark. Prepare DABCO mounting medium using 25 milligrams of DABCO in 90%glycerol and 10%PBS. To dissolve properly, adjust the pH to 8.6 using spectrophotometric grade dilute hydrochloric acid and keep the solution on a rocker for mixing.As an anti-bleaching agent, add the DABCO mounting medium directly onto the imaging dish containing the cover slip at the bottom. Wait for at least one to two hours and directly proceed to perform confocal imaging. To identify tunneling nanotubes, capture Z-stack images of immunostained cells using a confocal laser-scanning microscope by first selecting the channels and clicking the required lasers sequentially in the windows track one, track two, and track three in the confocal software.Click the option TPMT below the track one window to take the differential interference contrast images with fluorescence channels. Select the Acquisition tab of the software, click on the Z-stack tab, and wait for a window to open. Then click Live Scan to focus the cells at the bottom of the dish.Select that focused image as the first stack, and then focus up to see the topmost part of the cell, and select that as the last stack. Stop live scanning and click the number next to the Optimal tab to fix the step size of the stacks which determines the number of slices and the intervals based on the thickness of the cells. Take sequential images of three channels of DAPI, FITC, and TRITC with 405 nanometer, 488 nanometer, and 561 nanometer lasers and capture with a pixel dwell time of 1.02 microsecond.Capture images in the DIC channel with fluorescence channels to observe the cell boundary. Open the confocal images saved in czi data format in Fiji software for analysis. Select the Hyperstack option to see each Z-stack and channel of the image.Scroll the channel and Z-stack scroll bars to select the exact stack of a particular channel of interest. Then select the F-actin stained channel first by scrolling the channel bar and manually scroll the Z-stacks to see each stack one by one. Identify the F-actin stained structures that seem to be connecting cells that are visible in the lower parts of the Z-stacks and are close to the surface of the imaging dish with Z equal to two as neurites.Identify the tunneling nanotubes, the F-actin positive hovering cell to cell conduits by scrolling the Z-stacks toward the top from Z equals to four. Look for neurites near the lower parts of the Z-stacks toward the surface and observe that they start disappearing with the scrolling of Z-stacks toward the top at Z equal to six. Identify phospho-PAK1 positive tunneling nanotubes similarly as F-actin positive tunneling nanotubes by analyzing the hovering nature of the conduits from the Z-stacks.As phospho-PAK1 staining is weaker than F-actin staining, look for phospho-PAK1 stained tunneling nanotubes at Z equal to four and at Z equal to six. Further, observe the DIC images to verify that the F-actin and phospho-PAK1 stained tunneling nanotube structures are membrane conduits between cells. In addition, merge F-actin and phospho-PAK1 channels to verify that the identified tunneling nanotubes are F-actin and phospho-PAK1 co-stained structures.To quantify the tunneling nanotubes, count the total cell numbers and the identified tunneling nanotubes manually and represent the numbers as percentages. To distinguish F-actin and beta III tubulin positive tunneling nanotubes like hovering conduits from Z-stack images, merge F-actin and beta III tubulin channels, then analyze the Z-stacks of the merged images. Look for exclusively F-actin stained tunneling nanotubes that are faintly visible at Z equal to three and prominent at Z equal to six and Z equal to nine.Similarly, identify F-actin and beta III tubulin. double-positive tunneling nanotubes like hovering conduits at Z equal to six and Z equal to nine. Identify other F-actin and beta III tubulin stained non-hovering protrusions from the lower parts of the Z-stacks.Measure the diameter of the tunneling nanotubes using the Line tool in Fiji. Verify the scale of measurement by clicking Analyze and then set scale so that distance in pixels is set automatically from the czi images. Measure the diameters of the tunneling nanotubes at the XY plane.Using the Volume Viewer plugin in Fiji which allows 3D re-slicing and threshold-enabled 3D visualization, split the Z-stack images into individual channels. Then crop the single channel Z-stack images to use 3D reconstruction view to highlight one or two tunneling nanotubes or neurites at a time. Pin a single tunneling nanotubes or neurite in the XY plane and mark XZ and YZ access cross sections.Observe the neurites at the bottom of the XZ plane, in the XZ and YZ planes, and the tunneling nanotubes in the upper Z-stacks. Select the individual tunneling nanotubes or neurite to reconstruct the 3D volume view in the XZ plane. In the 3D reconstruction, observe the neurites at the bottom of the Z-plane and the tunneling nanotubes appearing as hovering structures connecting two cells without touching the bottom Z-plane.Confocal Z-stack images of immunostained cells with F-actin and phospho-PAK1 were analyzed to identify tunneling nanotubes. Further, DIC images were analyzed to verify that the F-actin and phospho-PAK1 stained tunneling nanotubes structures were membrane conduits between cells. The cells were double immunostained with F-actin and beta III tubulin and the tunneling nanotube-like F-actin and the tunneling nanotube-like F-actin and beta III tubulin double positive membrane conduits were distinguished from only F-actin positive tunneling nanotubes.The tunneling nanotubes co-expressed with phospho-PAK1 and F-actin were distinguished from other neurites'cell protrusions by constructing 3D volume view images based on their characteristic of hovering between two cells without touching the substratum. Usage of right fixative agent is important as imperfect fixation of the sample may lead to rapid proteolytic degradation of the target proteins and a reduction in the specific immunoreactivity.

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