Abstract
We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution.
Abbreviations
- AFs:
-
Actin filaments
- BSA:
-
Bovine serum albumin
- CK:
-
Cytokeratin
- IFs:
-
Intermediate filaments
- MTs:
-
Microtubules
- PBS:
-
Phosphate-buffered saline
- TnTs:
-
Tunneling nanotubes
References
Abounit S, Delage E, Zurzolo C (2015) Identification and characterization of tunneling nanotubes for intercellular trafficking. Curr Protoc Cell Biol 67:12.10.11-21. https://doi.org/10.1002/0471143030.cb1210s67
Ady JW, Desir S, Thayanithy V, Vogel RI, Moreira AL, Downey RJ, Fong Y, Manova-Todorova K, Moore MA, Lou E (2014) Intercellular communication in malignant pleural mesothelioma: properties of tunneling nanotubes. Front Physiol 5:400. https://doi.org/10.3389/fphys.2014.00400
Antanavičiūtė I, Rysevaitė K, Liutkevičius V, Marandykina A, Rimkutė L, Sveikatienė R, Uloza V, Skeberdis VA (2014) Long-distance communication between laryngeal carcinoma cells. PLoS ONE 9(6):e99196. https://doi.org/10.1371/journal.pone.0099196
Burgert A, Letschert S, Doose S, Sauer M (2015) Artifacts in single-molecule localization microscopy. Histochem Cell Biol 144(2):123–131. https://doi.org/10.1007/s00418-015-1340-4
Hase K, Kimura S, Takatsu H, Ohmae M, Kawano S, Kitamura H, Ito M, Watarai H, Hazelett CC, Yeaman C, Ohno H (2009) M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol 11(12):1427–1432. https://doi.org/10.1038/ncb1990
Hua K, Ferland RJ (2017) Fixation methods can differentially affect ciliary protein immunolabeling. Cilia 6:5. https://doi.org/10.1186/s13630-017-0045-9
Huber F, Boire A, López MP, Koenderink GH (2015) Cytoskeletal crosstalk: when three different personalities team up. Curr Opin Cell Biol 32:39–47. https://doi.org/10.1016/j.ceb.2014.10.005
Kreft ME, Romih R, Sterle M (2002) Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder. Cell Biol Int 26(1):63–74. https://doi.org/10.1006/cbir.2001.0829
Kreft ME, Sterle M, Veranič P, Jezernik K (2005) Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation. Histochem Cell Biol 123(4–5):529–539. https://doi.org/10.1007/s00418-005-0770-9
Kreft ME, Di Giandomenico D, Beznoussenko GV, Resnik N, Mironov AA, Jezernik K (2010) Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells. Biol Cell 102(11):593–607. https://doi.org/10.1042/BC20100024
Lokar M, Iglič A, Veranič P (2010) Protruding membrane nanotubes: attachment of tubular protrusions to adjacent cells by several anchoring junctions. Protoplasma 246(1–4):81–87. https://doi.org/10.1007/s00709-010-0143-7
Mattes B, Scholpp S (2018) Emerging role of contact-mediated cell communication in tissue development and diseases. Histochem Cell Biol 150(5):431–442. https://doi.org/10.1007/s00418-018-1732-3
Moll R, Divo M, Langbein L (2008) The human keratins: biology and pathology. Histochem Cell Biol 129(6):705–733. https://doi.org/10.1007/s00418-008-0435-6
Onfelt B, Nedvetzki S, Benninger RK, Purbhoo MA, Sowinski S, Hume AN, Seabra MC, Neil MA, French PM, Davis DM (2006) Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol 177(12):8476–8483
Resnik N, Prezelj T, De Luca GMR, Manders E, Polishchuk R, Veranič P, Kreft ME (2018) Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells. Sci Rep 8(1):17133. https://doi.org/10.1038/s41598-018-35370-y
Romih R, Veranič P, Jezernik K (1999) Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder. Histochem Cell Biol 112(5):375–380
Rustom A, Saffrich R, Markovic I, Walther P, Gerdes HH (2004) Nanotubular highways for intercellular organelle transport. Science 303(5660):1007–1010. https://doi.org/10.1126/science.1093133
Sáenz-de-Santa-María I, Bernardo-Castiñeira C, Enciso E, García-Moreno I, Chiara JL, Suarez C, Chiara MD (2017) Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes. Oncotarget 8(13):20939–20960. https://doi.org/10.18632/oncotarget.15467
Sisakhtnezhad S, Khosravi L (2015) Emerging physiological and pathological implications of tunneling nanotubes formation between cells. Eur J Cell Biol 94(10):429–443. https://doi.org/10.1016/j.ejcb.2015.06.010
Soltys BJ, Borisy GG (1985) Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol 100(5):1682–1689
Sovinski S (2011) Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1. Methods 53(1):27–33
Veranič P, Jezernik K (2002) Trajectorial organisation of cytokeratins within the subapical region of umbrella cells. Cell Motil Cytoskelet 53(4):317–325. https://doi.org/10.1002/cm.10077
Veranič P, Romih R, Jezernik K (2004) What determines differentiation of urothelial umbrella cells? Eur J Cell Biol 83(1):27–34. https://doi.org/10.1078/0171-9335-00351
Veranič P, Lokar M, Schütz GJ, Weghuber J, Wieser S, Hägerstrand H, Kralj-Iglic V, Iglic A (2008) Different types of cell-to-cell connections mediated by nanotubular structures. Biophys J 95(9):4416–4425. https://doi.org/10.1529/biophysj.108.131375
Vielkind U, Swierenga SH (1989) A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry 91(1):81–88
Višnjar T, Chesi G, Iacobacci S, Polishchuk E, Resnik N, Robenek H, Kreft M, Romih R, Polishchuk R, Kreft ME (2017) Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models. Sci Rep 7(1):12842. https://doi.org/10.1038/s41598-017-13103-x
Wang X, Gerdes HH (2012) Long-distance electrical coupling via tunneling nanotubes. Biochim Biophys Acta 8:2082–2086. https://doi.org/10.1016/j.bbamem.2011.09.002
Wang X, Bukoreshtliev NV, Gerdes HH (2012) Developing neurons form transient nanotubes facilitating electrical coupling and calcium signaling with distant astrocytes. PLoS ONE 7(10):e47429. https://doi.org/10.1371/journal.pone.0047429
Weisenberg RC (1972) Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177(4054):1104–1105. https://doi.org/10.1126/science.177.4054.1104
Whelan DR, Bell TD (2015) Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters. Sci Rep 5:7924. https://doi.org/10.1038/srep07924
Wittig D, Wang X, Walter C, Gerdes HH, Funk RH, Roehlecke C (2012) Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes. PLoS ONE 7(3):e33195. https://doi.org/10.1371/journal.pone.0033195
Zakout YM, Salih MM, Ahmed HG (2010) The effect of fixatives and temperature on the quality of glycogen demonstration. Biotech Histochem 85(2):93–98. https://doi.org/10.3109/10520290903126883
Zani BG, Edelman ER (2010) Cellular bridges: Routes for intercellular communication and cell migration. Commun Integr Biol 3(3):215–220
Zupančič D, Mrak Poljšak K, Kreft ME (2018) Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research. Cell Biol Int 42(4):411–424. https://doi.org/10.1002/cbin.10910
Acknowledgements
This research work was supported by the Slovenian Research Agency (ARRS) (Grant No. J3-7494 and P3-0108) and MRIC UL IP-0510 Infrastructure program. We express gratitude to Sanja Čabraja, Linda Štrus, Nada Pavlica Dubarič and Sabina Železnik for their technical assistance.
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Resnik, N., Erman, A., Veranič, P. et al. Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes. Histochem Cell Biol 152, 311–317 (2019). https://doi.org/10.1007/s00418-019-01806-3
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DOI: https://doi.org/10.1007/s00418-019-01806-3