Endotel Hücreleri Arasında Nanotüp Tünellemenin ve Organel İletiminin Görüntülenmesi

Öz

The tunneling nanotube plays a role in intercellular communication. Nanotube tunnels are thought to be formed to regenerate, to survive or to resist stress by eukaryotic cells. Nanotube tunnels, forming bridges between homotypic and heterotypic cells, have shown to transmit signaling molecules such as calcium ion flux, and to transport organelles, pathogens or oncogenic molecules. The basic structure of nanotube tunnels is microfilaments. The actin skeleton triggers the formation of a nanotube tunnel and a bridge between two distant cells under stressing conditions. Cellular aging occurs in endothelial cells in a long-term cell culture. In this study, it is aimed to visualize nanotube tunneling between endothelial cells under cellular aging. Human umbilical cord vascular endothelial cells (HUVECs) were grown in the standard cell culture conditions with repeated passages (P). The actin cytoskeleton and endosomes were labeled with phalloidin and anti-EEA1 antibody respectively, for fluorescence microscopy. Nanotube tunnel lengths were measured in control (P3-4) and experimental (P8-10) HUVECs preparations. The average length for P8-10 was determined to be 30 μm. The endosomes were located together with the actin cytoskeleton in the nanotube tunnel. These findings show that endosomes, cargo-carrier inside the cell, can also transfer substances between HUVECs by nanotube tunneling. As a result, nanotube tunnels, formed between HUVECs of high passages depending on the dynamics of the microfilaments, were found to be functional. Nanotube tunneling, accepted as a new way of communication between cells, can be evaluated as a morphological parameter in studies of stress responses.

Anahtar Kelimeler

• Actin filaments
• Endothelial cells
• Endosome
• Tunneling nanotube

Endotel Hücreleri Arasında Nanotüp Tünellemenin ve Organel İletiminin Görüntülenmesi

Öz

Nanotüp tünelleme hücreler arası iletişimde rol almaktadır. Ökaryotik hücrelerin yenilenmek, hayatta kalmak ya da strese direnmek üzere nanotüp tüneller oluşturduğu düşünülmektedir. Homotipik ya da heterotipik hücreler arasında köprüler oluşturan nanotüp tünellerin kalsiyum iyon akışı gibi sinyal moleküllerini ilettiği, organel, patojen ya da onkojenik molekülleri aktardığı gösterilmiştir. Nanotüp tünellerin temel yapısı mikrofilamentlerdir. Stres oluşturan çevresel etkenler altında aktin iskeletinin nanotüp tünellerin oluşmasını tetiklediği ve birbirinden uzak iki hücre arasında köprü oluşturduğu belirlenmiştir. Uzun-süreli hücre kültürü ortamı endotel hücrelerinde strese neden olmakta ve hücresel yaşlanma oluşmaktadır. Bu çalışmada standart hücre kültürü ortamında tekrarlayan pasajlar (P) ile çoğaltılan insan göbek kordonu damar endotel hücreleri (HUVEC) arasında nanotüp tünellemenin görüntülenmesi amaçlandı. Floresan mikroskop incelemesi için aktin iskeleti ve endozomlar sırası ile falloidin ve anti-EEA1 antikoru ile işaretlendi. Kontrol grubu (P3-4) ve deney grubu (P8-10) HUVEC’ler ile hazırlanan preparatlarda nanotüp tünel uzunlukları ölçüldü. P8-10 için ortalama uzunluk 30 μm olarak belirlendi. Endozomların nanotüp tünel yapısındaki aktin iskeleti ile birlikte konumlandığı gösterildi. Bu bulgular, hücre içinde kargo taşıyan endozomların, nanotüp tünelleme ile HUVEC’ler arasında da madde aktarımı yapabileceğini göstermektedir. Sonuçta tekrarlayan pasajlar ile çoğaltılan HUVEC’ler arasındaki nanotüp tünellerin mikrofilamentlerin dinamiğine bağlı olarak işlevsel olduğu belirlenmiştir. Hücreler arasında yeni bir iletişim yolu olarak kabul gören nanotüp tünelleme, stres cevabının irdelendiği çalışmalarda morfolojik bir parametre olarak değerlendirilebilir.

Anahtar Kelimeler

• Aktin filamenti
• Endotel hücreleri
• Endozom
• Nanotüp tünelleme

Kaynakça

1. 1. Sherer NM. Long-distance relationships: do membrane nanotubes regulate cell-cell communication and disease progression? Mol Biol Cell 2013; 24(8):1095-8.
2. 2. Rustom A, Saffrich R, Markovic I, Walther P, Gerdes H-H. 2004. Nanotubular highways for intercellular organelle transport. Science 303:1007-10.
3. 3. Önfelt B, Nedvetzki S, Benninger RK, et al. Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol 2006, 177:8476.
4. 4. Eugenin EA, Gaskill PJ, Berman JW. Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: a potential mechanism for intercellular HIV trafficking. Cell Immunol 2009; 254:142-8.
5. 5. Sherer NM, Lehmann MJ, Jimenez-Soto LF, Horensavitz C, Pypaert M, Mothes W. Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission. Nat Cell Biol 2007; 9:310-5.
6. 6. Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Köhler K, et al. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 2008; 10(2):211-9.
7. 7. Victoria GS, Zurzolo C. 2017. The spread of prion-like proteins by lysosomes and tunneling nanotubes: implications for neurodegenerative diseases. J Cell Biol 216:2633-44.
8. 8. Kadiu I, Gendelman HE. Human immunodeficiency virus type 1 endocytic trafficking through macrophage bridging conduits facilitates spread of infection. J Neuroimmune Pharmacol 2011; 6(4):658-75.

9. 9. Sowinski S, Alakoskela JM, Jolly C, Davis DM. Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1. Methods 2011; 53(1):27-33.
10. 10. Watkins SC, Salter RD. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity 2005; 23:309-18.
11. 11. Biran A, Perelmutter M, Gal H, Burton DGA, Ovadya Y, Vadai E, et al. Senescent cells communicate via intercellular protein transfer. Genes Dev 2015; 29:791-802.
12. 12. Thayanithy V, Dickson EL, Steer C, Subramanian S, Lou E. Tumorstromal cross talk: direct cell-to-cell transfer of oncogenic microRNAs via tunneling nanotubes. Transl Res 2014; 164:359-65.
13. 13. Wang Y, Cui J, Sun X, Zhang Y. Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ 2011; 18:732-42.
14. 14. Koyanagi M, Brandes RP, Haendeler J, Zeiher AM, Dimmeler S. Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes. Circ Res 2005; 96:1039-1041.
15. 15. Austefjord MW, Gerdes HH, Wang X. Tunneling nanotubes: diversity in morphology and structure. Commun Integr Biol 2014; 7:1-5.
16. 16. Sept D, McCammon JA. Thermodynamics and kinetics of actin filament nucleation. Biophys J. 2001; 81(2):667-74.
17. 17. Tojkander S, Gateva G, Lappalainen P. Actin stress fibers–assembly, dynamics and biological roles. J Cell Sci 2012;125(Pt 8):1855-64.
18. 18. Mattera R, Arighi CN, Lodge R, Zerial M. Bonifacino JS. Divalent interaction of the GGAs with the Rabaptin-5–Rabex-5 complex. EMBO J 2003; 22(1):78-88.
19. 19. Scita G, Di Fiore PP. The endocytic matrix. Nature 2010; 463(7280):464-73.
20. 20. Kimura S, Hase K, Ohno H. The molecular basis of induction and formation of tunneling nanotubes. Cell Tissue Res 2013; 352(1): 67-76.
21. 21. Zhu D, Tan KS, Zhang X, Sun AY, Sun GY, Lee JC. Hydrogen peroxide alters membrane and cytoskeleton properties and increases intercellular connections in astrocytes. J Cell Sci 2005; 118:3695-703.
22. 22. Islam MN, Das SR, Emin MT, Wei M, Sun L, Westphalen K, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med 2012; 18:759-65.
23. 23. Jansens RJ, Tishchenko A, Favoreel HW. Bridging the Gap: Virus Long-Distance Spread via Tunneling Nanotubes. J Virol 2020; 94(8):e02120-19.
24. 24. Yasuda K, Khandare A, Burianovskyy L, Maruyama S, Zhang F, Nasjletti A, et al. Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors: exchange of lysosomal pool. Aging (Albany NY), 2011; 3(6):597 -608.
25. 25. Buachan P, Chularojmontri L, Wattanapitayakul SK. Selected activities of Citrus maxima Merr. fruits on human endothelial cells: enhancing cell migration and delaying cellular aging. Nutrients 2014; 6(4):1618-34.
26. 26. Boisen L, Drasbek KR, Pedersen AS, Kristensen, P. Evaluation of endothelial cell culture as a model system of vascular ageing. Exp Gerontol 2010; 45(10):779-87.
27. 27. M Bektaş, E Hacıosmanoğlu, B Özerman, B Varol, R Nurten, E Bermek. On diphtheria toxin fragment A release into the cytosol—Cytochalasin D effect and involvement of actin filaments and eukaryotic elongation factor 2. Int J Biochem Cell Biol. 2011; 43(9):1365-72.
28. 28. Gerdes HH, Bukoreshtliev NV, Barroso JF. Tunneling nanotubes: a new route for the exchange of components between animal cells. FEBS Lett 2007; 581:2194.
29. 29. Hanna SJ, McCoy-Simandle K, Leung E, Genna A, Condeelis J, Cox D. The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis. Sci Rep 2017; 7: 8547.
30. 30. Delage E, Cervantes DC, Pénard E, Schmitt C, Syan S, Disanza A et al. Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes. Sci Rep 2016; 6: 39632.
31. 31. Astanina K, Koch M, Jüngst C, Zumbusch A, Kiemer AK. Lipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells. Sci Rep 2015; 5:11453.
32. 32. Pedicini L, Miteva KT, Hawley V, Gaunt HJ, Appleby HL, Cubbon RM, et al. Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombinSci Rep 2018; 8(1):7569.
33. 33. Liu K, Ji K, Guo L, Wu W, Lu H, Shan P, et al. Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer. Microvas Res 2014; 92:10-8.
34. 34. Wang X, Yu X, Xie C, Tan Z, Tian Q, Zhu D, et al. Rescue of brain function using tunneling nanotubes between neural stem cells and brain microvascular endothelial cells. Mol Neurobiol. 2016; 53:2480-8.
35. 35. Connor Y, Tekleab S, Nandakumar S, Walls C, Tekleab Y, Husain A et al. Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype. Nat Commun 2015; 6:8671.
36. 36. Errede M, Mangieri D, Longo G, Girolamo F, de Trizio I, Vimercati A, et al. Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis. Fluids Barriers CNS 2018; 15:28.
37. 37. Pasquier J, Guerrouahen BS, Al Thawadi H, Ghiabi P, Maleki M, Abu-Kaoud N, et al. Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J Transl Med 2013; 11:94.
38. 38. Roehlecke C, Schmidt MH. Tunneling Nanotubes and Tumor Microtubes in Cancer. Cancers 2020; 12(4):857.
39. 39. Seyed-Razavi Y, Hickey MJ, Kuffová L, McMenamin PG, Chinnery HR. Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction. Immunol Cell Biol 2013; 91(1):89-95.
40. 40. Alarcon-Martinez L, Villafranca-Baughman D, Quintero H, Kacerovsky JB, Dotigny F, Murai KK, et al. Interpericyte tunnelling nanotubes regulate neurovascular coupling. Nature 2020; 585(7823): 91-5.