Super-Resolution Imaging of Tight and Adherens Junctions: Challenges and Open Questions.

Hannes Gonschior,Volker Haucke,Martin Lehmann

doi:10.3390/ijms21030744

Abstract

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

Highlights

Cell contacts are formed on the one hand by the adherens junction (AJ) which provides mechanical adhesion, and on the other by the tight junction (TJ) which primarily acts as a barrier to solutes and water and as a fence that separates the apical and basolateral plasma membrane domains
TJs were first observed by transmission electron microscopy (TEM) as apical membrane contacts extending over 200–500 nm with electron-dense areas forming a continuous belt-like attachment by fusing the outer membrane leaflets of two neighboring cells into a ≈3 nm thick structure [3]
Due to the inherent limitations of EM with respect to defining molecular and structural dynamics many key questions remain unsolved yet: for example, how are the individual protein components like channel and barrier forming claudins of TJs or the different components of AJs organized within the junction and how does their interaction contribute to the overall functionality? How are junctions remodeled, e.g., by strand reformation, endocytosis, during cell division, or upon injury? To answer these questions, it is indispensable to understand the nanoscale organization of the molecular components of TJs and AJs in fixed cells and tissue and, most importantly, in living preparations

Summary

Adherens Junctions and Tight Junctions

Epithelial and endothelial cells line multiple organs to segregate internal and external compartments. It is indispensable to understand the nanoscale organization of the molecular components of TJs (claudins, TAMPs, JAMs, ZOs) and AJs (cadherins, catenins, vinculin, actin-binding proteins) in fixed cells and tissue and, most importantly, in living preparations. Due to the inherent limitations of EM with respect to defining molecular and structural dynamics many key questions remain unsolved yet: for example, how are the individual protein components like channel and barrier forming claudins of TJs or the different components of AJs organized within the junction and how does their interaction contribute to the overall functionality? Such an approach requires multi-color imaging at nanoscale resolution by super-resolution microscopy (SRM). We provide an overview on recent advances and limitations in SRM, summarize important new insight in junction research from recent studies and provide an outlook for future research

Resolution Limit

Results

Discussion and Outlook