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* These authors contributed equally
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 "tunneling nanotubes" (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 µm. 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-β1-42 (oAβ) 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 oAβ-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and β-III tubulin-immunostained membrane conduits.
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 µm in length and their diameters vary between 50 nm to 1 µm2,3. 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 ....
NOTE: SH-SY5Y cells cultured in DMEM/F-12 media were differentiated with 10 µM retinoic acid for 7 days and treated with 1 µM oAβ oligomers for 2 h at 37 °C (5% CO2). After treatment, the cells were fixed with Karnovsky'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.......
Here, we identify and characterize oAβ-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 (
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 β-tubulin-positive, close-ended, developing neurites hover between two distant cells and resem.......
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's (Jawaharlal Nehru Centre for Advanced Scientific Research, India) confocal facility and B. Suma for the confocal microscopy in JNCASR.
....Name | Company | Catalog Number | Comments |
35 mm dish with 14 mm well size made of #1.5 cover slip | Cellvis | D35-14-1.5-N | Imaging dish used to seed cells for staining experiments |
Aβ (1-42) 1 mg | AnaSpec | #64129 | Oligomers of amyloid beta to treat the cells |
Alexa flour 488 Goat Anti-rabbit IgG (H+L) | Invitrogen | A11070 | Secondary antibody for phospho-PAK1 |
Biological Safety Cabinet | Thermo Scientific (MSC Advantage) | 51025411 | Provide aspetic conditions duirng cell culture |
CO2 Incubator | Thermo Electron Corporation (Heraeus Hera Cell 240) | 51026556 | For growing cells at or near body temperature |
Confocal Laser Scanning Microscope | ZEISS (Carl Zeiss) | LSM 880 | Able to generate three-dimensional images of large specimen at super-resolution |
DABCO [1,4-Diazobicyclo-(2,2,2) octane] | Merck | 8034560100 | Anti-bleaching reagent |
DAPI (4′,6-diamidino-2-phenylindole) | Sigma | D9542-1MG | Neuclear stainer |
DMEM media | Gibco | 11965092 | Used for the preparation of 100uM of Aβ (1-42) |
DMEM/F12 (1:1 mixture of DMEM and Ham’s F12) | Gibco | 12500062 | Culture media for SH-SY5Y |
DMSO (Dimethyl sulphide) Cell culture grade | Cryopur | CP-100 | Cell culture grade used as dissolving agent for Retinoic acid |
DMSO (Dimethyl sulphide) Molecular grade | Himedia | MB058 | Used as one of the dissolving agent for the lyophilized Aβ (1-42) |
Fetal Bovine Serum | Gibco | 16000044 | Major supplement for Culture media (US origin) |
Formalin Fixative (Neutral buffered 10%) | Sigma Aldrich | HT5014-120ML | Component in the Karnovsky's fixative solution |
Glutaraldehyde (Grade I, 25% in H2O) | Sigma | G5882 | Component in the Karnovsky's fixative solution |
HFIP (1,1,1,3,3,3-hexafluoro-2-propanol ) solution | TCI | H024 | Used to dissolve Aβ (1-42) 1 mg |
Image Processing/ Analysis Software: FIJI (ImageJ) | National Institute of Health (NIH) | Used to process/analyze the images and to differentiate the TNTs from neurites using its plugin named "volume viewer". | |
Lyophilizer | Christ, Alpha | 2.4 LDplus | 0.05 mg aliquots of Aβ (1-42) can be stored in -20 °C after lyophilization only |
Penicillin-Streptomycin-Neomycin Mixture | Thermo fisher Scientific | 15640055 | Antibiotic mixture |
Phalloidin-iFlor 555 | Abcam | ab176756 | F-actin binding stain |
Phospho-PAK1 (Thr423) /PAK2 (Thr402) [Rabbit] | CST | #2601 | Primary antibody |
Polyclonal Antibody to Tubulin Beta 3 (TUBb3) | Cloud clone | PAE711Hu01 | Primary antibody |
Retinoic acid | Sigma-Aldrich | R2625-50MG | Differentiating reagent |
Saponin | Merck | 8047-15-2 | Detergent used in the Incubation buffer in immunostaining |
Water bath sonicator (Quart, Drain valve Heater) | Ultrasonic Cleaner | 3.0 L/3.2 | Sonicator used to dissolve Aβ (1-42) stock, after DMSO adding to it during the preparation of 100 µM Aβ (1-42) |
ZEN Microscopy software | ZEISS (Carl Zeiss) | Imaging software to acquire confocal microscopy images with smart automation |
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