The Ism between Endothelial Cilia and Endothelial Nanotubules Is an Evolving Concept in the Genesis of the BBB

Abstract

The blood-brain barrier (BBB) is fundamental in maintaining central nervous system (CNS) homeostasis by regulating the chemical environment of the underlying brain parenchyma. Brain endothelial cells (BECs) constitute the anatomical and functional basis of the BBB. Communication between adjacent BECs is critical for establishing BBB integrity, and knowledge of its nanoscopic landscape will contribute to our understanding of how juxtaposed zones of tight-junction protein interactions between BECs are aligned. The review discusses and critiques types of nanostructures contributing to the process of BBB genesis. We further critically evaluate earlier findings in light of novel high-resolution electron microscopy descriptions of nanoscopic tubules. One such phenotypic structure is BEC cytoplasmic projections, which, early in the literature, is postulated as brain capillary endothelial cilia, and is evaluated and compared to the recently discovered nanotubules (NTs) formed in the paracellular spaces between BECs during barrier-genesis. The review attempts to elucidate a myriad of unique topographical ultrastructures that have been reported to be associated with the development of the BBB, viz., structures ranging from cilia to BEC tunneling nanotubules (TUNTs) and BEC tethering nanotubules (TENTs).

Keywords: BBB; brain endothelium; cilium; cytoplasmic projections; tethering nanotubules.

Figure 1

A micrograph illustrating filopodial “TNT” (tunnelling nanotubule) extensions between two normal rat kidney (NRK) epithelial cells (black arrow) (Right); during electrophysiological recordings (Left). Scale bar = 20 μm. V₁ and V₂ denote voltage applied to cell 1 and cell 2, and I₁ and I₂ denote the current injected into cell 1 and cell 2. These experiments indicated that filopodia electrically connect cells [37].

Figure 2

A schematic illustration of cytoplasmic protruding cilia, originating from the cell centrosome. (A) A typical primary cilium projecting from the cell surface, comprising cargo, viz., intraflagellar transport particles: kinesin-2 and cytoplasmic dyein; (B) a cross-section of a non-motile cilium, which assumes a 9 + 0 formation of microtubule doublets [16]; (C) a TEM image of tracheal epithelium and a cross-section of tracheal cilium displaying a 9 + 2 conformation. Ci denotes cilia, MV denotes microvilli and TJ denotes epithelial tight junctions. Scale bar = 8 μm [50].

Figure 3

An illustration postulates endothelial primary cilia as regulators of blood pressure through nitric oxide production, which is transferred to the smooth muscle in arterial tunica media [51].

Figure 4

Determination of “cilia” distribution on EC membranes of primordial cerebral vessels. Confocal micrographs of GFP-labeled primordial midbrain channel in transgenic line Tg (kdrl:mCherry-CAAX)^y171 and (bactin::Arll3b:GFP) to help label EC membranes. Scale bar = 40 μm [22].

Figure 5

Photomicrograph of primary cilia, denoted by blue arrows. An illustration of GFP-labeled cilial extensions within primordial midbrain channels. Scale bar = 5 μm [22].

Figure 6

Immunofluorescence (IF) illustration of EC “cilium-like structures”. The white arrow exhibits a gamma-tubulin AC3-positive tubular structure, which is postulated to be a cilium [52].

Figure 7

The cytoarchitectural organization of primordial cytoplasmic projections. (A) The formation of TENT extensions by cytoplasmic extension of α-tubulin in BEC bEnd5 cells, from the BEC leading membranous edges. The yellow arrows indicate TENT extensions, scale bar 20 μm [66]. (B) TENT formation on the apico-lateral surfaces of the BEC bEnd5 plasma membrane [31]. PS denotes the paracellular space; the stars represent cells 1, 2 and 3. The black arrows indicate areas of continuous membrane leading edges of BECs, governed by cytoplasmic projections, which develop into thin rope-like tethers. Scale bar 1000 nm.

Figure 8

Electron micrograph of actin-based membranous protrusions from leading edges of cultured mouse melanoma cells as denoted by the white rectangle [43].

Figure 9

TENT development on the apical surface of BEC bEnd5 plasma membrane surfaces. (A) Early/primordial TENT development on the plasma membrane surface of the BEC at high magnification, represented in the black perforated boxes. C* denotes the membrane of cell one, C^$ denotes cell two, the yellow arrow denotes a fully formed TENT structure extending across the paracellular space between two adjacent BECs and the black arrows denotes nanovesicle-induced TUNT structures. Scale bar = 200 nm [31]. (B) A SEM photomicrograph of proposed primary endothelial cilium at high magnification. Scale bar = 1 μm [71]. Note the similarity between the primordial TENT (perforated square) in (A) to the postulated cilium in (B).