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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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.......

Representative Results

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 (

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
35 mm dish with 14 mm well size made of #1.5 cover slipCellvisD35-14-1.5-NImaging dish used to seed cells for staining experiments
(1-42) 1 mgAnaSpec#64129Oligomers of amyloid beta to treat the cells
Alexa flour 488 Goat Anti-rabbit IgG (H+L)InvitrogenA11070Secondary antibody for phospho-PAK1
Biological Safety CabinetThermo Scientific (MSC Advantage)51025411Provide aspetic conditions duirng cell culture
CO2 IncubatorThermo Electron Corporation (Heraeus Hera Cell 240)51026556For growing cells at or near body temperature
Confocal Laser Scanning MicroscopeZEISS (Carl Zeiss)LSM 880Able to generate three-dimensional images of large specimen at super-resolution
DABCO [1,4-Diazobicyclo-(2,2,2) octane]Merck8034560100Anti-bleaching reagent
DAPI (4′,6-diamidino-2-phenylindole)SigmaD9542-1MGNeuclear stainer
DMEM mediaGibco11965092Used for the preparation of 100uM of Aβ (1-42)
 DMEM/F12 (1:1 mixture of DMEM and Ham’s F12)Gibco12500062Culture media for SH-SY5Y
DMSO (Dimethyl sulphide) Cell culture gradeCryopurCP-100Cell culture grade used as dissolving agent for Retinoic acid
DMSO (Dimethyl sulphide) Molecular gradeHimediaMB058Used as one of the dissolving agent for the lyophilized Aβ (1-42)
Fetal Bovine SerumGibco16000044 Major supplement for Culture media (US origin)
Formalin Fixative (Neutral buffered 10%)Sigma AldrichHT5014-120MLComponent in the Karnovsky's fixative solution
Glutaraldehyde (Grade I, 25% in H2O)SigmaG5882Component in the Karnovsky's fixative solution
HFIP (1,1,1,3,3,3-hexafluoro-2-propanol ) solutionTCIH024Used 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".
LyophilizerChrist, Alpha2.4 LDplus0.05 mg aliquots of Aβ (1-42) can be stored in -20 °C after lyophilization only
Penicillin-Streptomycin-Neomycin MixtureThermo fisher Scientific15640055Antibiotic mixture
Phalloidin-iFlor 555Abcamab176756F-actin binding stain
Phospho-PAK1 (Thr423) /PAK2 (Thr402) [Rabbit]CST#2601Primary antibody
Polyclonal Antibody to Tubulin Beta 3 (TUBb3)Cloud clonePAE711Hu01Primary antibody
Retinoic acidSigma-AldrichR2625-50MGDifferentiating reagent
SaponinMerck8047-15-2Detergent used in the Incubation buffer in immunostaining
Water bath sonicator (Quart, Drain valve Heater)Ultrasonic Cleaner3.0 L/3.2Sonicator used to dissolve Aβ (1-42) stock, after DMSO adding to it during the preparation of 100 µM Aβ (1-42)
ZEN Microscopy softwareZEISS (Carl Zeiss)Imaging software to acquire confocal microscopy images with smart automation

References

  1. Rustom, A., Saffrich, R., Markovic, I., Walther, P., Gerdes, H. H. Nanotubular highways for intercellular organelle transport. Science. 303 (5660), 1007-1010 (2004).
  2. Desir, S., et al. Chemotherapy-i....

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