scholarly journals Composition and function of the C1b/C1f region in the ciliary central apparatus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ewa Joachimiak ◽  
Anna Osinka ◽  
Hanan Farahat ◽  
Bianka Świderska ◽  
Ewa Sitkiewicz ◽  
...  
Keyword(s):  
Tetrahymena Thermophila ◽  
Vortex Motion ◽  
Specific Protein ◽  
Complex Cell ◽  
Wild Type ◽  
Cell Organelles ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
Central Apparatus ◽  
And Function

AbstractMotile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.

scholarly journals Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body ofSalmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3029-3036 ◽  
Author(s):  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basal Body ◽  
Strong Interactions ◽  
Wild Type ◽  
Body Protein ◽  
C Terminus ◽  
Genes Encoding ◽  
And Function ◽  
Better Than

ABSTRACT FliE is a flagellar basal body protein of Salmonellawhose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parentalfliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

scholarly journals Periodic actin structures in neuronal axons are required to maintain microtubules

2016 ◽  
Author(s):  
Yue Qu ◽  
Ines Hahn ◽  
Stephen Webb ◽  
Simon P. Pearce ◽  
Andreas Prokop
Keyword(s):  
Super Resolution ◽  
Close Correlation ◽  
Membrane Skeleton ◽  
Cortical Actin ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
Neuronal Processes ◽  
And Function ◽  
Super Resolution Microscopy ◽  
Actin Rings

SummaryAxons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration.Significance statementAxons are cable-like neuronal processes that are up to a meter long in humans. These delicate structures often need to be maintained for an organism’s lifetime, i.e. up to a century in humans. Unsurprisingly, we gradually lose about 50% of axons as we age. Bundles of microtubules form the structural backbones and highways for life-sustaining transport within axons, and maintenance of these bundles is essential for axonal longevity. However, the mechanisms which actively maintain axonal microtubules are poorly understood. Here we identify cortical actin as an important factor maintaining microtubule polymerisation in axons. This finding provides potential explanations for the previously identified, but unexplained, links between mutations in genes encoding cortical actin regulators and neurodegeneration.

scholarly journals The Kinocilia of Cochlear Hair Cells: Structures, Functions, and Diseases

2021 ◽  
Vol 9 ◽  
Author(s):  
Difei Wang ◽  
Jun Zhou
Keyword(s):  
Auditory System ◽  
Hair Cells ◽  
Primary Cilia ◽  
Structure And Function ◽  
Biochemical Reactions ◽  
Cochlear Hair Cells ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
And Function ◽  
The Brain

Primary cilia are evolutionarily conserved and highly specialized organelles that protrude from cell membranes. Mutations in genes encoding ciliary proteins can cause structural and functional ciliary defects and consequently multiple diseases, collectively termed ciliopathies. The mammalian auditory system is responsible for perceiving external sound stimuli that are ultimately processed in the brain through a series of physical and biochemical reactions. Here we review the structure and function of the specialized primary cilia of hair cells, termed kinocilia, found in the mammalian auditory system. We also discuss areas that might prove amenable for therapeutic management of auditory ciliopathies.

scholarly journals Thioredoxins o1 and h2 Show Different Subcellular Localizations and Redox-Active Functions, but Cooperatively Affect NADPH Redox Poise and Photosynthetic Performance in Fluctuating Light

2021 ◽  
Author(s):  
Liang-Yu Hou ◽  
Martin Lehmann ◽  
Peter Geigenberger
Keyword(s):  
Photosynthetic Efficiency ◽  
Reverse Genetics ◽  
Genotypic Differences ◽  
Cell Fractionation ◽  
Wild Type ◽  
Cell Organelles ◽  
Redox States ◽  
Fluctuating Light ◽  
And Function ◽  
Glutathione Redox

Arabidopsis contains eight different h-type thioredoxins (Trx) being distributed in different cell organelles. Although Trx h2 is deemed to be localized in mitochondria, its subcellular localization and function remains a matter of debate. Here, Trx h2 localization and function were investigated using cell fractionation studies and reverse genetics. Differential centrifugation and immunodetection showed the Trx h2 protein to be distributed to the microsomal fraction rather than to mitochondrial preparations. To analyze whether Trx h2 has different roles than mitochondrial Trxs, Arabidopsis mutants lacking Trx h2 were compared with mutants deficient in mitochondrial Trx o1 and double mutants with joint deficiencies in both Trxs. Under constant medium light, trxh2 grew as the wild type, while trxo1 and trxo1h2 mutants showed impaired growth. This was accompanied by differences in the metabolite profiles. The trxo1 and trxo1h2 mutants clustered differently from the wild type during the night, revealing a decrease in ascorbate and glutathione redox states. In fluctuating light intensities, genotypic differences in growth rates were attenuated. Compared to the wild type, the fluctuating-light induced decrease in the NADPH/NADP ratio was diminished in the mutants, with the trxo1h2 double mutant showing the strongest effect. This was accompanied by an increase in the photosynthetic efficiency of the trxo1h2 mutant, specifically in the high light phases of fluctuating light. Conclusively, these results support the view that Trxs o1 and h2 are localized in different subcellular compartments, and have different effects on ascorbate and glutathione redox states and growth in medium light, but cooperatively affect NADP(H) redox state and photosynthetic efficiency in fluctuating light.

scholarly journals Gene expression profiles of Spo11−/− mouse testes with spermatocytes arrested in meiotic prophase I

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Natalya A Smirnova ◽  
Peter J Romanienko ◽  
Pavel P Khil ◽  
R Daniel Camerini-Otero
Keyword(s):  
Gene Expression ◽  
Meiotic Recombination ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Protein ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Before And After ◽  
Genes Encoding ◽  
Protein Components

Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11−/ −adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11−/ −and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.

scholarly journals Mutational Analysis of Cdc19p, a Schizosaccharomyces pombe MCM Protein

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1025-1041 ◽  
Author(s):  
Susan L Forsburg ◽  
Daniel A Sherman ◽  
Sabine Ottilie ◽  
J Randy Yasuda ◽  
Jeffrey A Hodson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Protein Function ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Wild Type ◽  
Genes Encoding ◽  
Mcm Proteins ◽  
And Function ◽  
Mutant Forms ◽  
Replication Initiator

The cdc19  + gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19  + and genes encoding subunits of DNA polymerase δ and the replication initiator cdc18  +. We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

scholarly journals Regulation of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum by the VisN/R-Rem cascade

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1673-1685 ◽  
Author(s):  
Dinah D. Tambalo ◽  
Kate L. Del Bel ◽  
Denise E. Bustard ◽  
Paige R. Greenwood ◽  
Audrey E. Steedman ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Rhizobium Leguminosarum ◽  
Flagellar Motility ◽  
Growth Phases ◽  
Wild Type ◽  
Free Living ◽  
Regulatory Cascade ◽  
Genes Encoding ◽  
And Function ◽  
Free Living Conditions

In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA–gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flaH. flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flaH is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem.

scholarly journals Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009388
Author(s):  
Rafał Bazan ◽  
Adam Schröfel ◽  
Ewa Joachimiak ◽  
Martyna Poprzeczko ◽  
Gaia Pigino ◽  
...  
Keyword(s):  
Protein Composition ◽  
Ciliary Beating ◽  
Radial Spoke ◽  
Beat Pattern ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Central Apparatus ◽  
And Function ◽  
Beating Frequency

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

scholarly journals The H1 Phosphorylation State Regulates Expression of CDC2 and Other Genes in Response to Starvation in Tetrahymena thermophila

2005 ◽  
Vol 25 (10) ◽  
pp. 3914-3922 ◽  
Author(s):  
Yali Dou ◽  
Xiaoyuan Song ◽  
Yifan Liu ◽  
Martin A. Gorovsky
Keyword(s):  
Gene Expression ◽  
Tetrahymena Thermophila ◽  
Subtractive Hybridization ◽  
Feedback Mechanism ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Genes Encoding ◽  
Positive Feedback Mechanism ◽  
Mutant Cells ◽  
Isogenic Strains

ABSTRACT In Tetrahymena thermophila, highly phosphorylated histone H1 of growing cells becomes partially dephosphorylated when cells are starved in preparation for conjugation. To determine the effects of H1 phosphorylation on gene expression, PCR-based subtractive hybridization was used to clone cDNAs that were differentially expressed during starvation in two otherwise-isogenic strains differing only in their H1s. H1 in A5 mutant cells lacked phosphorylation, and H1 in E5 cells mimicked constitutive H1 phosphorylation. Sequences enriched in A5 cells included genes encoding proteases. Sequences enriched in E5 cells included genes encoding cdc2 kinase and a Ser/Thr kinase. These results indicate that H1 phosphorylation plays an important role in regulating the pattern of gene expression during the starvation response and that its role in transcription regulation can be either positive or negative. Treatment of starved cells with a phosphatase inhibitor caused CDC2 gene overexpression. Expression of the E5 version of H1 in starved cells containing endogenous, wild-type H1 caused the wild-type H1 to remain highly phosphorylated. These results argue that Cdc2p is the kinase that phosphorylates Tetrahymena H1, establish a positive feedback mechanism between H1 phosphorylation and CDC2 expression, and indicate that CDC2 gene expression is regulated by an H1 phosphatase.

scholarly journals Thioredoxin h2 and o1 Show Different Subcellular Localizations and Redox-Active Functions, and Are Extrachloroplastic Factors Influencing Photosynthetic Performance in Fluctuating Light

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 705
Author(s):  
Liang-Yu Hou ◽  
Martin Lehmann ◽  
Peter Geigenberger
Keyword(s):  
Redox Balance ◽  
Photosynthetic Performance ◽  
Genotypic Differences ◽  
Cell Fractionation ◽  
Wild Type ◽  
Cell Organelles ◽  
Fluctuating Light ◽  
Redox Active ◽  
And Function

Arabidopsis contains eight different h-type thioredoxins (Trx) being distributed in different cell organelles. Although Trx h2 is deemed to be confined to mitochondria, its subcellular localization and function are discussed controversially. Here, cell fractionation studies were used to clarify this question, showing Trx h2 protein to be exclusively localized in microsomes rather than mitochondria. Furthermore, Arabidopsis trxo1, trxh2 and trxo1h2 mutants were analyzed to compare the role of Trx h2 with mitochondrial Trx o1. Under medium light, trxo1 and trxo1h2 showed impaired growth, while trxh2 was similar to wild type. In line with this, trxo1 and trxo1h2 clustered differently from wild type with respect to nocturnal metabolite profiles, revealing a decrease in ascorbate and glutathione redox states. Under fluctuating light, these genotypic differences were attenuated. Instead, the trxo1h2 double mutant showed an improved NADPH redox balance, compared to wild type, accompanied by increased photosynthetic efficiency, specifically in the high-light phases. Conclusively, Trx h2 and Trx o1 are differentially localized in microsomes and mitochondria, respectively, which is associated with different redox-active functions and effects on plant growth in constant light, while there is a joint role of both Trxs in regulating NADPH redox balance and photosynthetic performance in fluctuating light.

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