Endotel Hücreleri Arasında Nanotüp Tünellemenin ve Organel İletiminin Görüntülenmesi

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

Endotel Hücreleri Arasında Nanotüp Tünellemenin ve Organel İletiminin Görüntülenmesi

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.

Keywords:

Actin filaments, Endothelial cells, Endosome, Tunneling nanotube,

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