Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

Gold Particle Analyser: Detection and quantitative assessment of electron microscopy gold probes

Gold particle probes are an essential electron microscopy tool to examine protein localisation, as well as protein trafficking. They can be introduced into living cells when conjugated to a protein that is endocytosed or to an antibody against a cell surface protein. Alternatively, gold particles can be introduced into fixed cells or tissue when conjugated to antibodies, immunoglobulin binding molecules or chemical probes applied to permeabilised samples or electron microscopy sections. Colloidal gold particles that have not been enlarged through chemical (gold or silver) enhancement are typically spherical and can be prepared in a range of specific sizes, allowing multiple proteins to be localised within a single sample. The typically homogeneous shape and size of the colloidal gold makes them ideal for computer assisted detection and analysis. Here we demonstrate a program developed to automatically identify two sizes of gold particle and perform a range of analyses that includes (i) distribution and cluster analysis; (ii) selection and analysis of gold particles allocated close to or either side of a membrane; (iii) measurement of organelle size; (iv) estimation of the number of gold particles within an aggregate and (v) the detection of chemically enhanced irregular sized and shaped gold particles. We show this easy-to-use program can greatly assist electron microscopists, to reliably and efficiently analyse gold particles within their images

Ua-zero as a uranyl acetate replacement when diagnosing primary ciliary dyskinesia by transmission electron microscopy

Primary ciliary dyskinesia (PCD) is a disorder affecting motile cilia. An early accurate diagnosis helps prevent lung damage and preserve lung function. To make a diagnostic assessment, one of the commonly used methods that allows for the examination of ciliary ultrastructure is transmission electron microscopy (TEM). This allows for a quantitative assessment of ciliary components to identify defects associated with PCD. Heavy metal staining is required to provide a contrast when imaging cilia in the TEM. One of the most commonly used stains is uranyl acetate (UA). UA can be applied to cellular material before embedding (en bloc), or to ultrathin sections of embedded samples (grid staining). UA is radioactive and, due to growing safety concerns and restrictions by government bodies, universities and hospitals, it is essential to find a suitable alternative. We show UA-zero (UAZ), when used en bloc, provides a high contrast and is a suitable replacement for UA. PCD diagnostic experts, having reviewed ciliary cross-sections stained with UAZ en bloc, are confident that the staining and PCD defects are readily detectable similar to samples that have been stained with UA

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.publishersversionpublishe

Changes in Mitochondrial Size and Morphology in the RPE and Photoreceptors of the Developing and Ageing Zebrafish

Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. Investigation of temporal gene expression of several mitochondrial-related markers indicated fission as the dominant mechanism contributing to the changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction

Disruption of CFAP418 interaction with lipids causes abnormal membrane-associated cellular processes in retinal degenerations

Syndromic ciliopathies and retinal degenerations are large heterogeneous groups of genetic diseases. CFAP418 is a causative gene of both disorders, and its protein sequence is evolutionarily conserved. However, the pathogenic mechanism caused by CFAP418 mutations is largely unknown. Here, we employed affinity purification coupled with mass spectrometry and quantitative lipidomic, proteomic, and phosphoproteomic approaches to address the molecular function of CFAP418 in mouse retinas. We showed that CFAP418 bound to lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but did not form a tight and static complex with proteins. Loss of Cfap418 led to membrane lipid imbalance and protein-membrane association alteration, which subsequently caused mitochondrial defects and membrane remodeling abnormalities in multiple vesicular trafficking pathways. Loss of Cfap418 also increased the activity of PA-binding protein kinase Cα. Our results indicate that membrane lipid imbalance is a new pathological mechanism underlying syndromic ciliopathies and retinal degenerations, which is associated with other known causative genes for these diseases, such as RAB28 and BBS genes

Zebrafish motile cilia as a model for primary ciliary dyskinesia

Funding Information: Funding: This work was a product of the Project LYSOCIL funded by the European Union Horizon 2020 research and innovation under grant agreement No 811087. It received funding from Fundação para a Ciencia e tecnologia, grant PTDC/BEX-BID/1411/2014; M.R. was funded by the fellowship PD/BD/136927/2018. P.S was funded by the fellowship SFRH/BD/111611/2015; C.B. was funded by the fellowship SFRH/BD/141034/2018; SSL was funded by FCT CEEC-IND 2018. Funding Information: Acknowledgments: The authors want to thank the Fish Facility from NMS. This work was developed with the support of the Fish Facility NMS|FCM that is part of CONGENTO, a Research Infrastructure co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e Tecnologia (Portugal) LISBOA-01-0145-FEDER-022170. A.P. wants to thank the Royal Brompton and Harefield hospital, part of the Guy’s and St Thomas’ foundation trust, London. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.publishersversionpublishe

Disruption of CFAP418 interaction with lipids causes widespread abnormal membrane-associated cellular processes in retinal degenerations

Syndromic ciliopathies and retinal degenerations are large heterogeneous groups of genetic diseases. Pathogenic variants in the CFAP418 gene may cause both disorders, and its protein sequence is evolutionarily conserved. However, the disease mechanism underlying CFAP418 mutations has not been explored. Here, we apply quantitative lipidomic, proteomic, and phosphoproteomic profiling and affinity purification coupled with mass spectrometry to address the molecular function of CFAP418 in retinas. We show that CFAP418 protein binds to lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 in mice disturbs membrane lipid homeostasis and membrane-protein association, which subsequently causes mitochondrial defects and membrane remodeling abnormalities across multiple vesicular trafficking pathways in photoreceptors, especially the endosomal sorting complexes required for transport (ESCRT) pathway. Ablation of Cfap418 also increases the activity of PA-binding protein kinase Cα in the retina. Overall, our results indicate that membrane lipid imbalance is a pathological mechanism underlying syndromic ciliopathies and retinal degenerations, which is associated with other known causative genes of these diseases

SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients