The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

Shireen Mentor,David Fisher,Khayelihle Brian Makhathini

doi:10.3390/ijms23020742

Abstract

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Highlights

Academic Editor: Hyunsoo ChoThe brain microvascular endothelial cells (BECs) play a critical role as an interdependent network constituting the basic angioarchitecture of the blood-brain barrier (BBB) [1].The functionality of the BBB is closely related to the regulation of permeability and entails paracellular (PC) sealing by intercellular tight junction (TJ) protein complexes as a central feature of the barrier’s physical function
The mechanics that are required for two juxtaposed BEC membranes to align in a manner that enables TJ interaction with its counterparts on adjacent cells have recently been described from an ultrastructural perspective, highlighting for the first time, the importance of nanotubules (NTs) in the physical alignment of adjacent BECs by utilizing high-resolution electron microscopy (HREM) [2]
Recent high-resolution scanning electron microscopy (HR-SEM) observations in our laboratory showed that the nanoscopic morphology of the BEC confluent monolayers exhibits cytoplasmic projections, which are continuous with the leading edges of the BEC membrane (See Figure 1 below)

Summary

Introduction

Academic Editor: Hyunsoo ChoThe brain microvascular endothelial cells (BECs) play a critical role as an interdependent network constituting the basic angioarchitecture of the blood-brain barrier (BBB) [1].The functionality of the BBB is closely related to the regulation of permeability and entails paracellular (PC) sealing by intercellular tight junction (TJ) protein complexes as a central feature of the barrier’s physical function. Compared to systemic endothelial cells, the apico-lateral expression of the TJs is specific, unique, and depends on the correct orientation of the BECs into the apical and basal domains [1]. The mechanics that are required for two juxtaposed BEC membranes to align in a manner that enables TJ interaction with its counterparts on adjacent cells have recently been described from an ultrastructural perspective, highlighting for the first time, the importance of nanotubules (NTs) in the physical alignment of adjacent BECs by utilizing high-resolution electron microscopy (HREM) [2]. Two types of novel NTs were discovered as a form of direct BEC–cell interaction at the apico-lateral domains of BECs and can be categorically differentiated into (i) nanovesicle (NV)-induced tunneling NTs (TUNTs) and (ii) tethering NTs (TENTs). TENTs were described as novel nanostructural cross-bridges that emerged transiently as a supplementary means of direct cell-cell communication during immortalized mouse BEC (bEnd3/bEnd5) monolayer development in vitro, which has become

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