Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1−/− cells.

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Superresolution microscopy reveals photoreceptor-specific subciliary location and function of Cep290

10.1101/2020.10.28.357806 ◽

2020 ◽

Author(s):

Valencia L. Potter ◽

Abigail R. Moye ◽

Michael A. Robichaux ◽

Theodore G. Wensel

Keyword(s):

Electron Microscopy ◽

Epithelial Cells ◽

Fluorescence Microscopy ◽

Mouse Models ◽

Primary Cilia ◽

Cultured Cells ◽

Precise Location ◽

Superresolution Microscopy ◽

Functional Roles ◽

And Function

AbstractMutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ syndromic ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used superresolution fluorescence microscopy and electron microscopy (TEM) to localize CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency. Radially, CEP290 co-localizes with the microtubule doublets and extends beyond them. Longitudinally, it is distributed throughout the length of the CC but is strictly confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, the ciliary sub-structures between microtubules and membrane, at the base of the transition zone in primary cilia of epithelial cells and throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. They did not lead to loss of the Y-shaped links but caused changes in their structures. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.

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Interpreting the pathogenicity of Joubert Syndrome missense variants in Caenorhabditis elegans

Disease Models & Mechanisms ◽

10.1242/dmm.046631 ◽

2020 ◽

pp. dmm.046631

Author(s):

Karen I. Lange ◽

Sofia Tsiropoulou ◽

Katarzyna Kucharska ◽

Oliver E. Blacque

Keyword(s):

Caenorhabditis Elegans ◽

Primary Cilia ◽

Model Organism ◽

Joubert Syndrome ◽

Compound Heterozygous ◽

Inherited Disorders ◽

Missense Variants ◽

C Elegans ◽

Variant Alleles ◽

And Function

Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have employed a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S, G155S) in B9D2/mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment, and regulates the cilium's molecular composition and sensory/signaling functions. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and TMEM216/MKS-2. Together, these data establish C. elegans as a paradigm for interpreting JBTS mutations, and provide further insight into MKS module organisation.

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Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e19-09-0499 ◽

2020 ◽

Vol 31 (7) ◽

pp. 619-639 ◽

Cited By ~ 2

Author(s):

Henrietta W. Bennett ◽

Anna-Karin Gustavsson ◽

Camille A. Bayas ◽

Petar N. Petrov ◽

Nancie Mooney ◽

...

Keyword(s):

Molecular Structure ◽

Cell Lines ◽

Single Molecule ◽

Primary Cilia ◽

Three Dimensional ◽

Fibrillar Structure ◽

Systematic Analysis ◽

Superresolution Microscopy ◽

Superresolution Imaging

Using three-dimensional single-molecule superresolution imaging and systematic analysis of knockout cell lines, we have determined the molecular structure and composition of the inversin compartment in primary cilia. INVS establishes fibrillar structures that recruit ANKS6-NEK8 complexes to sequester NPHP3 in this unique periaxonemal compartment.

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INPP5E controls ciliary localization of phospholipids and odor response in olfactory sensory neurons

Journal of Cell Science ◽

10.1242/jcs.258364 ◽

2021 ◽

pp. jcs.258364

Author(s):

Kirill Ukhanov ◽

Cedric Uytingco ◽

Warren Green ◽

Lian Zhang ◽

Stephane Schurmans ◽

...

Keyword(s):

Sensory Neurons ◽

Primary Cilia ◽

Electrical Response ◽

Joubert Syndrome ◽

Gene Replacement ◽

Inactivation Kinetics ◽

Olfactory Sensory Neurons ◽

Efficient Recovery ◽

And Function ◽

Ciliary Localization

The lipid composition of the primary cilia membrane is emerging as a critical regulator of cilia formation, maintenance, and function. Here, we show that conditional deletion of the phosphoinositide 5’-phosphatase gene, Inpp5e, causative of Joubert syndrome in terminally developed mouse olfactory sensory neurons (OSNs) led to a dramatic remodeling of ciliary phospholipids that was accompanied by marked elongation of cilia. PI(4,5)P2 normally restricted to the proximal segment redistributed to the entire length of cilia in Inpp5e knockout mice with a reduction in PI(3,4)P2 and elevation of PI(3,4,5)P3 in the dendritic knob. The redistribution of phosphoinositides impaired odor adaptation, resulting in less efficient recovery and altered inactivation kinetics of the odor-evoked electrical response and the odor-induced elevation of cytoplasmic Ca2+. Gene replacement by adenoviral expression of Inpp5e restored the ciliary localization of PI(4,5)P2 and odor response kinetics in OSNs. Our findings support the role of phosphoinositides as a modulator of the odor response and in ciliary biology of native multi-ciliated OSNs.

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Conformation of Ribosomes from Escherichia Coli

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051062 ◽

1974 ◽

Vol 32 ◽

pp. 210-211

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Fluorescent Dyes ◽

Protein Biosynthesis ◽

Three Dimensional ◽

Spatial Arrangement ◽

Dimensional Structure ◽

Complete Understanding ◽

And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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Structure and function of neural networks in the retina

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102213 ◽

1988 ◽

Vol 46 ◽

pp. 26-27

Author(s):

Peter Sterling

Keyword(s):

Ganglion Cells ◽

Three Dimensional ◽

Structure And Function ◽

Synaptic Connections ◽

Visual Axis ◽

One Dimensional ◽

Cat Retina ◽

Ultrathin Sections ◽

And Function ◽

Insight Into

The synaptic connections in cat retina that link photoreceptors to ganglion cells have been analyzed quantitatively. Our approach has been to prepare serial, ultrathin sections and photograph en montage at low magnification (˜2000X) in the electron microscope. Six series, 100-300 sections long, have been prepared over the last decade. They derive from different cats but always from the same region of retina, about one degree from the center of the visual axis. The material has been analyzed by reconstructing adjacent neurons in each array and then identifying systematically the synaptic connections between arrays. Most reconstructions were done manually by tracing the outlines of processes in successive sections onto acetate sheets aligned on a cartoonist's jig. The tracings were then digitized, stacked by computer, and printed with the hidden lines removed. The results have provided rather than the usual one-dimensional account of pathways, a three-dimensional account of circuits. From this has emerged insight into the functional architecture.

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Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098629 ◽

1981 ◽

Vol 39 ◽

pp. 424-425

Author(s):

M. Boublik ◽

N. Robakis ◽

J.S. Wall

Keyword(s):

Three Dimensional ◽

Uranyl Acetate ◽

Dimensional Structure ◽

Electron Microscopic ◽

Ribosomal Subunits ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

Scanning Transmission ◽

And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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Predicting spatial activity patterns in a neural map from a probabilistic atlas of 3-D reconstructed neurons

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140439 ◽

1995 ◽

Vol 53 ◽

pp. 812-813

Author(s):

G. Jacobs ◽

F. Theunissen

Keyword(s):

Activity Patterns ◽

Three Dimensional ◽

Sensory System ◽

Neural System ◽

Probabilistic Atlas ◽

Uniform Sample ◽

Dimensional Reconstruction ◽

The Time Domain ◽

And Function ◽

Visualization Techniques

In order to understand how the algorithms underlying neural computation are implemented within any neural system, it is necessary to understand details of the anatomy, physiology and global organization of the neurons from which the system is constructed. Information is represented in neural systems by patterns of activity that vary in both their spatial extent and in the time domain. One of the great challenges to microscopists is to devise methods for imaging these patterns of activity and to correlate them with the underlying neuroanatomy and physiology. We have addressed this problem by using a combination of three dimensional reconstruction techniques, quantitative analysis and computer visualization techniques to build a probabilistic atlas of a neural map in an insect sensory system. The principal goal of this study was to derive a quantitative representation of the map, based on a uniform sample of afferents that was of sufficient size to allow statistically meaningful analyses of the relationships between structure and function.

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Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies.

Biochemical Society Symposium ◽

10.1042/bss0720119 ◽

2005 ◽

Vol 72 ◽

pp. 119-127 ◽

Cited By ~ 19

Author(s):

Tamara Golub ◽

Caroni Pico

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Actin Cytoskeleton ◽

Lipid Rafts ◽

Patch Clustering ◽

Pkc Protein Kinase C ◽

Membrane Bound ◽

And Function ◽

Cytoskeleton Dynamics ◽

Syndrome Protein

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P2-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P2-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P2, Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P2 in a Ca2+/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P2-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.

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