Clathrin-coated vesicles form a unique net-like structure in liver sinusoidal endothelial cells by assembling along undisrupted microtubules |
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Authors: | Falkowska-Hansen Berit Falkowski Martin Metharom Pat Krunic Damir Goerdt Sergij |
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Institution: | Department of Dermatology, University Medical Center Mannheim, Ruprecht-Karls University Heidelberg, Theodor-Kutzer-Ufer 1-3, 68163 Mannheim, Germany. B.Falkowska-Hansen@DKFZ-Heidelberg.de |
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Abstract: | Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected. |
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Keywords: | AP adaptor protein DMEM Dulbecco's modified Eagles' medium CCV clathrin-coated vesicles CHC clathrin heavy chain CLC clathrin light chain CME clathrin-mediated endocytosis EEA1 early endosome antigen 1 EGF epidermal growth factor FCS fetal calf serum FITC fluorescein isothiocyanate FITC-AHGG fluorescein isothiocyanate labelled heat aggregated γ-globulin HUVECs human umbilical vein endothelial cells LSECs liver sinusoidal endothelial cells PBS phosphate-buffered saline PC parenchymal cells SEC sinusoidal endothelial cells TBS Tris-buffered saline |
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