Abstract
ABSTRACTReceptor clustering is known to trigger signalling events that contribute to critical changes in cellular functions. Faithful imaging of such clusters by means of fluorescence microscopy relies on the application of adequate cell fixation methods prior to immunolabelling in order to avoid artefactual redistribution by the antibodies themselves. Previous work has highlighted the inadequacy of fixation with paraformaldehyde (PFA) alone for efficient immobilisation of membrane-associated molecules, and the advantages of fixation with PFA in combination with glutaraldehyde (GA). Using fluorescence microscopy, we here highlight how inadequate fixation can lead to the formation of artefactual clustering of receptors in lymphatic endothelial cells, focussing on the transmembrane hyaluronan receptors LYVE-1 and CD44, and the homotypic adhesion molecule CD31, each of which displays their native diffuse surface distribution pattern only when visualised with the right fixation techniques, i.e. PFA/GA in combination. Fluorescence recovery after photobleaching (FRAP) confirms that the artefactual receptor clusters are indeed introduced by residual mobility. In contrast, we observed full immobilisation of membrane proteins in cells that were fixed and then subsequently permeabilised, irrespective of whether the fixative was PFA or PFA/GA in combination. Our study underlines the importance of choosing appropriate sample preparation protocols for preserving authentic receptor organisation in advanced fluorescence microscopy.
Highlights
The cell uses the membrane bilayer and the underlying cytoskeleton as a scaffold to organise surface proteins or receptors so that they are capable of responding to various extracellular cues (Helmreich, 2003; Kusumi and Sako, 1996; Lippincott-Schwartz et al, 2001)
For the purpose of these studies we used primary human dermal lymphatic endothelial cells (HDLECs) super transfected with human LYVE-1 by lentiviral transduction, and the non HA-blocking LYVE-1 mAb 8C (Prevo et al, 2001)
Artefactual LYVE-1 clustering in PFA fixed cells For preliminary comparison of LYVE-1 surface distribution before and after antibody induced crosslinking, we labelled human LYVE-1 (hLYVE-1) transfected HDLEC monolayers with Oregon Green® (OG) 488 conjugates of either monovalent F(ab) fragments or intact bivalent LYVE-1 mAb followed by 1% (w/v) PFA fixation, and imaged the cells by conventional confocal microscopy
Summary
The cell uses the membrane bilayer and the underlying cytoskeleton as a scaffold to organise surface proteins or receptors so that they are capable of responding to various extracellular cues (Helmreich, 2003; Kusumi and Sako, 1996; Lippincott-Schwartz et al, 2001). Changes in the spatial organisation of these molecules such as clustering are often considered as signs of cellular activation Investigations of anomalous diffusion dynamics can highlight such molecular interactions (Kusumi et al, 2005; Honigmann et al, 2014) but do not permit direct visualisation of clusters One remedy for this problem is to ‘freeze’ cells in a specific state by chemical fixation, preserving the true state of receptor clusters for visualisation by optimal immunolabelling. In recent years, owing to advanced fluorescence microscopy and labelling techniques, there has been a growing appreciation of the potential for artefacts during imaging of receptor clustering using immunolabelling, and in particular molecular redistribution induced by the processes used for sample preparation (Tanaka et al, 2010; Schnell et al, 2012; Zhou et al, 2015; Whelan and Bell, 2015)
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