Thomas Burgoyne

Methamphetamine-induced nitric oxide promotes vesicular transport in blood–brain barrier endothelial cells

Methamphetamines (METH) neurotoxicity is thought to be in part due to its ability to induce blood–brain barrier (BBB) dysfunction. Here, we investigated the effect of METH on barrier properties of cultured rat primary brain microvascular endothelial cells (BMVECs). Transendothelial flux doubled in response to METH, irrespective of the size of tracer used. At the same time, transendothelial electrical resistance was unchanged as was the ultrastructural appearance of inter-endothelial junctions and the distribution of key junction proteins, suggesting that METH promoted vesicular but not junctional transport. Indeed, METH significantly increased uptake of horseradish peroxidase into vesicular structures. METH also enhanced transendothelial migration of lymphocytes indicating that the endothelial barrier against both molecules and cells was compromised. Barrier breakdown was only observed in response to METH at low micromolar concentrations, with enhanced vesicular uptake peaking at 1 μM METH. The BMVEC response to METH also involved rapid activation of endothelial nitric oxide synthase and its inhibition abrogated METH-induced permeability and lymphocyte migration, indicating that nitric oxide was a key mediator of BBB disruption in response to METH. This study underlines the key role of nitric oxide in BBB function and describes a novel mechanism of drug-induced fluid-phase transcytosis at the BBB.

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151ts272a and mouse Ccdc151Snbl mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the ‘empty’ CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.

Differential apicobasal VEGF signaling at vascular blood-neural barriers

Summary The vascular endothelium operates in a highly polarized environment, but to date there has been little exploration of apicobasal polarization of its signaling. We show that VEGF-A, histamine, IGFBP3, and LPA trigger unequal endothelial responses when acting from the circulation or the parenchymal side at blood-neural barriers. For VEGF-A, highly polarized receptor distribution contributed to distinct signaling patterns: VEGFR2, which was found to be predominantly abluminal, mediated increased permeability via p38; in contrast, luminal VEGFR1 led to Akt activation and facilitated cytoprotection. Importantly, such differential apicobasal signaling and VEGFR distribution were found in the microvasculature of brain and retina but not lung, indicating that endothelial cells at blood-neural barriers possess specialized signaling compartments that assign different functions depending on whether an agonist is tissue or blood borne.

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Calcium signaling at ER membrane contact sites.

Communication between organelles is a necessary consequence of intracellular compartmentalization. Membrane contact sites (MCSs) are regions where the membranes of two organelles come into close apposition allowing exchange of small molecules and ions including Ca²⁺. The ER, the cells major Ca²⁺ store, forms an extensive and dynamic network of contacts with multiple organelles. Here we review established and emerging roles of ER contacts as platforms for Ca²⁺ exchange and further consider a potential role for Ca²⁺ in the regulation of MCS formation. We additionally discuss the challenges associated with the study of MCS biology and highlight advances in microscopy-based solutions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Visualization of cardiac muscle thin filaments and measurement of their lengths by electron tomography

Abstract Aims An intriguing difference between vertebrate skeletal and cardiac muscles is that the lengths of the thin filaments are constant in the former but variable in the latter. The thick filaments have constant lengths in both types of muscles. The contractile behaviour of a muscle is affected by the lengths of both types of filaments as the tension generated during contraction depends on the amount of filament overlap. To understand the behaviour of cardiac muscle, it is important to know the distribution of the thin filament lengths. The previous detailed analysis by Robinson and Winegrad used serial transverse sections to determine the lengths of the thin filaments. However, the precision, set by the 100 nm section thickness, was low. Here, we have used electron tomography to produce 3D images of rat and mouse cardiac muscles in which we can actually see individual thin filaments up to the free ends and see that these free ends have variable locations. For comparison, we also measure the thin filament lengths in skeletal muscle (frog sartorius). Methods and results Cardiac papillary muscles were obtained from a rat (Sprague–Dawley) and a mouse (C57/B6). Skeletal muscle (sartorius) was obtained from a frog (Rana pipiens). Longitudinal sections (100 nm thick) were used to produce tilt series and tomograms from which the thin filament paths were traced. Cardiac papillary muscle thin filaments in rat and mouse range from 0.94 to 1.10 µm, with a mean length of 1.04 µm and standard deviation of 0.03 µm. For frog sartorius muscle, the thin filament length was 0.94 µm with standard deviation of 0.01 µm. Conclusion Electron tomography of cardiac and skeletal muscles allows direct visualization and high precision measurement of the lengths of thin filaments.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

Significance Photoreceptors of the vertebrate retina contain specialized outer segments (OSs) where phototransduction begins. Rod OSs contain stacks of ordered membranous discs that undergo a daily renewal process essential for vision. Mechanisms underlying disc renewal are unclear. The biosynthetic machinery resides in the inner segment (IS), which is connected to the OS via a connecting cilium. Here, we use electron microscopy and tomography to show that the visual pigment, rhodopsin, traffics to the OS via the ciliary plasma membrane, which evaginates to form discs that are initially extracellularly exposed and that make novel contacts with the IS. Leading edges of adjacent evaginations then fuse to form discrete discs. Tomographic analysis leads us to propose a potential mechanism underlying the evagination process. The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that “zipper” up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

Characterizing the ultrastructure of primary ciliary dyskinesia transposition defect using electron tomography.

Primary ciliary dyskinesia is an autosomal recessive disorder affecting the motility of cilia. There are a range of ultrastructural ciliary defects that lead to associated clinical symptoms including ineffective mucus clearance, reduced lung function, infertility, and left‐right isomerism. Mutations in radial spoke head proteins are a known cause of primary ciliary dyskinesia. Ultrastructually these defects are identified by a portion of cilia lacking a central pair and transposed outer microtubular doublets. We have repeatedly observed an intermittent loss of the central pair in patients with a transposition defect. To further understand the central pair changes in these radial spoke head mutations we employ electron tomography, a high resolution electron microscope technique, to elucidate in three dimensions the ultrastructural arrangements caused by mutation of the RSPH4A gene. We thereby provide an explanation of the structures observed by conventional electron microscopy studies. We demonstrate that the central pair can be present within the cilium. In some cilia, the central pair rotates at the base of the axoneme. We propose that it is this rotation that gives rise to an intermittent appearance of the central pair when viewed under conventional electron microscopy. We discuss the potential causes and consequences of these findings.