scholarly journals Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 730 ◽  
Author(s):  
Anna Osinka ◽  
Martyna Poprzeczko ◽  
Magdalena M. Zielinska ◽  
Hanna Fabczak ◽  
Ewa Joachimiak ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Protein Composition ◽  
Model Organisms ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Biochemical Analyses ◽  
Eukaryotic Organisms

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

scholarly journals The molecular structure of primary cilia revealed by cryo-electron tomography

2020 ◽  
Author(s):  
Petra Kiesel ◽  
Gonzalo Alvarez Viar ◽  
Nikolai Tsoy ◽  
Riccardo Maraspini ◽  
Alf Honigmann ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Primary Cilium ◽  
Actin Filaments ◽  
Primary Cilia ◽  
Electron Tomography ◽  
3D Structure ◽  
Molecular Composition ◽  
Structural Investigations ◽  
Cryo Electron Tomography ◽  
Motile Cilia

AbstractPrimary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known “9+0” arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.

scholarly journals PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
Author(s):  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Gang Fu ◽  
Mary E. Porter ◽  
Daniela Nicastro ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Structural Studies ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

scholarly journals Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum

2019 ◽  
Vol 116 (35) ◽  
pp. 17316-17322 ◽  
Author(s):  
Orly Levitan ◽  
Muyuan Chen ◽  
Xuyuan Kuang ◽  
Kuan Yu Cheong ◽  
Jennifer Jiang ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Spatial Organization ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Spatial Differentiation ◽  
Thylakoid Membranes ◽  
Marine Diatom ◽  
Red Algal ◽  
Antenna Proteins

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

scholarly journals Targeted dynein inhibition generates flagellar beating

2017 ◽  
Author(s):  
Jianfeng Lin ◽  
Daniela Nicastro
Keyword(s):  
Molecular Mechanism ◽  
Structural Changes ◽  
Direct Evidence ◽  
Electron Tomography ◽  
Asymmetric Distribution ◽  
Conformational Switching ◽  
Cryo Electron Tomography ◽  
Cilia And Flagella ◽  
Biochemical Analyses ◽  
Sea Urchin Sperm

Motile cilia and flagella are highly conserved organelles that are essential for the normal development and health of many eukaryotes including humans. To reveal the molecular mechanism of motility, we used cryo-electron tomography of active sea urchin sperm flagella to directly visualize the macromolecular complexes and their structural changes during flagellar beating. We resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. Our results provide direct evidence for the conformational switching predicted by the ‘switch-point-hypothesis’. However, they also reveal a fundamentally different mechanism of generating motility by inhibiting dyneins, rather than activating them, causing an asymmetric distribution of force and thus bending. Our high-resolution structural and biochemical analyses provide a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating in an all-or-none manner.

scholarly journals Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly

2018 ◽  
Author(s):  
Hiroshi Yamaguchi ◽  
Toshiyuki Oda ◽  
Masahide Kikkawa ◽  
Hiroyuki Takeda
Keyword(s):  
Sperm Motility ◽  
Electron Tomography ◽  
Protein Family ◽  
Abnormal Sperm ◽  
Kupffer’S Vesicle ◽  
Kupffer's Vesicle ◽  
Cryo Electron Tomography ◽  
Organ Specific ◽  
Axonemal Dynein ◽  
Motile Cilia

AbstractConstruction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1, pih1d2, ktu, and twister, and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer’s vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2020 ◽  
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Stable Populations

AbstractMotile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

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