scholarly journals Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopies

2017 ◽  
Author(s):  
Michael A. Robichaux ◽  
Valencia L. Potter ◽  
Zhixian Zhang ◽  
Feng He ◽  
Michael F. Schmid ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Electron Tomography ◽  
Super Resolution ◽  
Intraflagellar Transport ◽  
Structural Features ◽  
Animal Cells ◽  
Ciliary Membrane ◽  
Acetylated Tubulin ◽  
Sensory Cilium ◽  
Syntaxin 3

ABSTRACTPrimary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as “ciliopathies” give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca2+-binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including Arl13B, intraflagellar transport proteins, BBS5, and syntaxin-3. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS7 and BBS9 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region, but alter the distribution of syntaxin-3 clusters.

scholarly journals Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wangxi Luo ◽  
Andrew Ruba ◽  
Daisuke Takao ◽  
Ludovit P. Zweifel ◽  
Roderick Y. H. Lim ◽  
...  
Keyword(s):  
High Speed ◽  
Primary Cilia ◽  
Protein Transport ◽  
Genetic Diseases ◽  
Super Resolution ◽  
Intraflagellar Transport ◽  
Protein Diffusion ◽  
Spatiotemporal Resolution ◽  
Cytosolic Proteins ◽  
Relative Contribution

AbstractTransport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α–tubulin utilizes diffusion besides IFT.

scholarly journals Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Fan Ye ◽  
David K Breslow ◽  
Elena F Koslover ◽  
Andrew J Spakowitz ◽  
W James Nelson ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Active Transport ◽  
Single Molecule ◽  
Primary Cilia ◽  
Somatostatin Receptor ◽  
Intraflagellar Transport ◽  
Signal Propagation ◽  
Protein Diffusion ◽  
Ciliary Membrane ◽  
Signaling Receptors

The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.

scholarly journals Three-dimensional architecture of epithelial primary cilia

2019 ◽  
Vol 116 (19) ◽  
pp. 9370-9379 ◽  
Author(s):  
Shufeng Sun ◽  
Rebecca L. Fisher ◽  
Samuel S. Bowser ◽  
Brian T. Pentecost ◽  
Haixin Sui
Keyword(s):  
Primary Cilia ◽  
Structural Model ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Cell Communication ◽  
Intraflagellar Transport ◽  
Transport Processes ◽  
Fibrous Protein ◽  
Junction Protein ◽  
Major Player

We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron tomography. Our data demonstrate the architecture of primary cilia differs extensively from the commonly acknowledged 9+0 paradigm. The axoneme structure is relatively stable but gradually evolves from base to tip with a decreasing number of microtubule complexes (MtCs) and a reducing diameter. The axonemal MtCs are cross-linked by previously unrecognized fibrous protein networks. Such an architecture explains why primary cilia can elastically withstand liquid flow for mechanosensing. The nine axonemal MtCs in a cilium are found to differ significantly in length indicating intraflagellar transport processes in primary cilia may be more complicated than that reported for motile cilia. The 3D maps of microtubule doublet–singlet transitions generally display longitudinal gaps at the inner junction between the A- and B-tubules, which indicates the inner junction protein is a major player in doublet–singlet transitions. In addition, vesicles releasing from kidney primary cilia were observed in the structural maps, supporting that ciliary vesicles budding may serve as ectosomes for cell–cell communication.

scholarly journals Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Alison Leaf ◽  
Mark Von Zastrow
Keyword(s):  
Dopamine Receptors ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Dopaminergic Receptors ◽  
Guanine Nucleotide Binding Protein ◽  
Ciliary Membrane ◽  
Specific Receptors ◽  
Guanine Nucleotide Binding ◽  
Transport Complex

Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

scholarly journals Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy

2019 ◽  
Vol 116 (47) ◽  
pp. 23562-23572 ◽  
Author(s):  
Michael A. Robichaux ◽  
Valencia L. Potter ◽  
Zhixian Zhang ◽  
Feng He ◽  
Jun Liu ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Structural Features ◽  
Nanometer Scale ◽  
Bardet Biedl Syndrome ◽  
Sensory Cilium ◽  
Light Sensing ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Subtomogram Averaging ◽  
Microscopic Techniques

Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.

scholarly journals Transient accumulation and bidirectional movement of KIF13B in primary cilia

2021 ◽  
Author(s):  
Alice Dupont Juhl ◽  
Zeinab Anvarian ◽  
Julia Berges ◽  
Daniel Wustner ◽  
Lotte B Pedersen
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Primary Cilia ◽  
Cell Imaging ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Ciliary Membrane ◽  
Bidirectional Movement ◽  
And Function ◽  
Male Specific

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia

2021 ◽  
Author(s):  
Yamato Ishida ◽  
Takuya Kobayashi ◽  
Shuhei Chiba ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Protein Trafficking ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Missense Variant ◽  
Cellular Level ◽  
Compound Heterozygous ◽  
Hypomorphic Allele ◽  
Genes Encoding ◽  
Specific Proteins ◽  
Compound Heterozygous Mutations

Abstract Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140, and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

scholarly journals Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function

2021 ◽  
Vol 7 (1) ◽  
pp. eabe4310
Author(s):  
Yue Li ◽  
Adam Eshein ◽  
Ranya K.A. Virk ◽  
Aya Eid ◽  
Wenli Wu ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Super Resolution ◽  
Population Level ◽  
Average Diameter ◽  
Live Cells ◽  
Molecular Function ◽  
Label Free ◽  
Multiple Length Scales ◽  
Chromatin Packing ◽  
Analysis Platform

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

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