STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena

AbstractThe base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet–Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

10.1101/251694 ◽

2018 ◽

Cited By ~ 1

Author(s):

Laura Vuolo ◽

Nicola L. Stevenson ◽

Kate J. Heesom ◽

David J. Stephens

Keyword(s):

Transition Zone ◽

Cell Lines ◽

Primary Cilia ◽

Point Mutations ◽

Intraflagellar Transport ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

eLife ◽

10.7554/elife.39655 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Laura Vuolo ◽

Nicola L Stevenson ◽

Kate J Heesom ◽

David J Stephens

Keyword(s):

Transition Zone ◽

Primary Cilia ◽

Complex Function ◽

Intraflagellar Transport ◽

Loss Of Function ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

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Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans

The Journal of Cell Biology ◽

10.1083/jcb.201610070 ◽

2017 ◽

Vol 216 (6) ◽

pp. 1659-1671 ◽

Cited By ~ 26

Author(s):

Daniel Serwas ◽

Tiffany Y. Su ◽

Max Roessler ◽

Shaohe Wang ◽

Alexander Dammermann

Keyword(s):

Fluorescence Microscopy ◽

Transition Zone ◽

Electron Tomography ◽

Intraflagellar Transport ◽

Basal Bodies ◽

C Elegans ◽

Cilia Formation ◽

Syndrome Protein

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

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Spatial Design of Polymicrobial Oral Biofilm in Its Native Disease State

Journal of Dental Research ◽

10.1177/0022034520909313 ◽

2020 ◽

Vol 99 (6) ◽

pp. 597-603 ◽

Cited By ~ 6

Author(s):

D. Kim ◽

H. Koo

Keyword(s):

Spatial Organization ◽

Disease Process ◽

Genotypic Diversity ◽

Spatial Arrangement ◽

Dimensional Structure ◽

3 Dimensional ◽

Spatial Design ◽

Oral Biofilms ◽

And Function ◽

Human Infections

Biofilms are structured microbial communities adhered to surfaces that cause many human infections. The study of oral biofilms has revealed complex composition, spatial organization, and phenotypic/genotypic diversity of the resident microbiota at the various sites in the mouth. Yet, knowledge about the spatial arrangement, positioning, and function of the polymicrobial community across the intact biofilm architecture remains sparse. Using multiple length scale imaging and computational analysis, we discovered unique spatial designs comprising mixed interbacterial species and interkingdom communities within intact biofilms formed on teeth of toddlers with caries. Intriguing structural patterns ranging from intermixed communities with extensive coaggregation (including bacterial-fungal clustering) to spatially segregated species forming a multilayered architecture were found. Among them, a distinctive 3-dimensional structure exhibited densely clustered cariogenic pathogens that were surrounded by outer layers of mixed bacterial communities in juxtaposition, forming a highly ordered spatial organization. These findings are particularly relevant as we approach the postmicrobiome era whereby studying the spatial structure of the pathogen and commensal microbiota may be important for understanding the microbiome function at the infection site to coordinate the disease process in situ.

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Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies

eLife ◽

10.7554/elife.70794 ◽

2021 ◽

Vol 10 ◽

Author(s):

Francisco Díaz-Pascual ◽

Martin Lempp ◽

Kazuki Nosho ◽

Hannah Jeckel ◽

Jeanyoung K Jo ◽

...

Keyword(s):

Escherichia Coli ◽

Spatial Organization ◽

Interaction Strength ◽

Growth Dynamics ◽

Nitrogen Sources ◽

Spatial Arrangement ◽

Model Systems ◽

Biofilm Growth ◽

Carbon And Nitrogen ◽

And Function

Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine via the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, via the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms.

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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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Computational Micrograph Registration with Sieve Processes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100164660 ◽

1996 ◽

Vol 54 ◽

pp. 440-441

Author(s):

P.J. Phillips ◽

J. Huang ◽

S. M. Dunn

Keyword(s):

Planar Graph ◽

Efficient Algorithm ◽

Spatial Organization ◽

Spatial Arrangement ◽

Stereo Image ◽

Image Model ◽

Geometric Invariants ◽

Point Set

In this paper we present an efficient algorithm for automatically finding the correspondence between pairs of stereo micrographs, the key step in forming a stereo image. The computation burden in this problem is solving for the optimal mapping and transformation between the two micrographs. In this paper, we present a sieve algorithm for efficiently estimating the transformation and correspondence.In a sieve algorithm, a sequence of stages gradually reduce the number of transformations and correspondences that need to be examined, i.e., the analogy of sieving through the set of mappings with gradually finer meshes until the answer is found. The set of sieves is derived from an image model, here a planar graph that encodes the spatial organization of the features. In the sieve algorithm, the graph represents the spatial arrangement of objects in the image. The algorithm for finding the correspondence restricts its attention to the graph, with the correspondence being found by a combination of graph matchings, point set matching and geometric invariants.

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ЛАНДШАФТНАЯ ОРГАНИЗАЦИЯ БЕРЕГОВОЙ ГЕОСТРУКТУРЫ ОСТРОВА ШКОТА (ЗАЛИВ ПЕТРА ВЕЛИКОГО)

10.33833/tig.2019.65.23.006 ◽

2019 ◽

Author(s):

K.S. Ganzei ◽

V.V. Zharikov ◽

N.F. Pshenichnikova ◽

A.M. Lebedev ◽

A.G. Kiselyova ◽

...

Keyword(s):

Spatial Organization ◽

Peter The Great Bay ◽

Spatial Arrangement ◽

Water Area ◽

Marine Spatial Planning ◽

Landscape Approach ◽

Important Condition ◽

Statistical Comparison ◽

Peter The Great ◽

Comprehensive Study

Важнейшим условием достижения устойчивого развития прибрежноморского природопользования в заливе Петра Великого системы является морское пространственное планирование. Основой для этого является информация о природных комплексах территории и акватории, полученная на основе ландшафтного подхода. Ключевым районом для изучения пространственной организации ландшафтов прибрежных геоструктур стала территория острова Шкота и его подводных склонов. Для наземных ландшафтов было описано 49 наблюдательных пунктов, 4 профиля были заложены для подводных ландшафтов описано 64 наблюдательных пункта, проложено 18 профилей. Выделено 22 вида ландшафтов, из них 16 наземных, 6 подводных. Берега острова сформированы преобладанием абразивноденудационного и абразивного типов. В результате всестороннего изучения показаны особенности пространственной организации воздушных и водных природных комплексов. Особенностью исследуемой территории является экспозиция дифференциации ландшафтов между юговосточной и северозападной частями острова, обусловленная муссонной природой климата. Результаты полевых и картографических работ послужили основой для выбора зон интенсивного, умеренного и ослабленного взаимодействия наземных и подводных ландшафтов. Пространственное расположение зон взаимодействия четко иллюстрируется значительными различиями экспозиции. Результаты статистического сравнения ландшафтов суши и мелководья, окружающего остров, на основе картометрических характеристик указывают на неоднородность геоструктуры острова, обусловленную, прежде всего, сочетанием ландшафтообразующих факторов. The most important condition for achieving sustainable development of coastalmarine environmental management in Peter the Great Bay is marine spatial planning. The basis for this is information about the natural complexes of the territory and water area, obtained based on the landscape approach. The main area for studying the spatial organization of landscapes of coastal geostructures was the territory of the island of Shkota and its underwater slopes. For terrestrial landscapes, 49 observation points were described, 4 profiles were laid 64 observation points were described for underwater landscapes, 18 profiles were laid. 22 species of landscapes have been identified, of which 16 are terrestrial, 6 are underwater. The shores of the island are formed by the predominance of abrasivedenudation and abrasive types. Because of a comprehensive study, features of the spatial organization of air and aquatic natural complexes are shown. A special feature of the study area is the exposure of the differentiation of landscapes between the southeastern and northwestern parts of the island, due to the monsoon nature of the climate. The results of field and cartographic works served as the basis for selecting areas of intense, moderate and weakened interaction of land and underwater landscapes. The spatial arrangement of the interaction zones is clearly illustrated by significant differences in exposure. The results of a statistical comparison of the land and shallow water landscapes surrounding the island, based on the cartometric characteristics, indicate the heterogeneity of the islands geostructure, primarily due to the combination of landscapeforming factors.

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Petiole-Lamina Transition Zone: A Functionally Crucial but Often Overlooked Leaf Trait

Plants ◽

10.3390/plants10040774 ◽

2021 ◽

Vol 10 (4) ◽

pp. 774

Author(s):

Max Langer ◽

Thomas Speck ◽

Olga Speck

Keyword(s):

Transition Zone ◽

Three Dimensional ◽

Spatial Arrangement ◽

Body Plan ◽

The Body ◽

Vascular Bundles ◽

Cross Sectional ◽

Thin Sections ◽

Transition Zones ◽

The Cross

Although both the petiole and lamina of foliage leaves have been thoroughly studied, the transition zone between them has often been overlooked. We aimed to identify objectively measurable morphological and anatomical criteria for a generally valid definition of the petiole–lamina transition zone by comparing foliage leaves with various body plans (monocotyledons vs. dicotyledons) and spatial arrangements of petiole and lamina (two-dimensional vs. three-dimensional configurations). Cross-sectional geometry and tissue arrangement of petioles and transition zones were investigated via serial thin-sections and µCT. The changes in the cross-sectional geometries from the petiole to the transition zone and the course of the vascular bundles in the transition zone apparently depend on the spatial arrangement, while the arrangement of the vascular bundles in the petioles depends on the body plan. We found an exponential acropetal increase in the cross-sectional area and axial and polar second moments of area to be the defining characteristic of all transition zones studied, regardless of body plan or spatial arrangement. In conclusion, a variety of terms is used in the literature for describing the region between petiole and lamina. We prefer the term “petiole–lamina transition zone” to underline its three-dimensional nature and the integration of multiple gradients of geometry, shape, and size.

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TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

10.1101/2020.11.19.390005 ◽

2020 ◽

Author(s):

Jaap van Krugten ◽

Noémie Danné ◽

Erwin J.G. Peterman

Keyword(s):

Single Molecule ◽

Transmembrane Proteins ◽

Intraflagellar Transport ◽

Single Molecule Tracking ◽

C Elegans ◽

Normal Diffusion ◽

Cellular Signal ◽

Underlying Mechanisms ◽

And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

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