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Signaling proteins of a chemosensory pathway from sperm of Arbacia punctulata

ABSTRACT: Cilia and flagella serve as cellular antennae that translate sensory information into cellular responses. In sperm, a single chemoattractant molecule triggers signaling events that evoke a Ca2+ response and chemotactic steering. Key components of the chemotactic signaling pathway from sperm of the sea urchin Arbacia punctulata were investigated. Shotgun proteomics employing SDS-PAGE LC-MS/MS and MudPIT provided the proteome inventory of sperm flagella.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arbacia Punctulata

TISSUE(S): Sperm, Sperm Flagellum

SUBMITTER: Ansgar Poetsch

LAB HEAD: Ansgar Poetsch

PROVIDER: PXD015332 | Pride | 2019-12-11

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	AP_GeLCMS.pep.xml	Pepxml
	AP_GeLCMS.prot.xml	Xml
	AP_gel_1.raw	Raw
	AP_gel_10.raw	Raw
	AP_gel_11.raw	Raw

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Publications

Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation.

Trötschel Christian C Hamzeh Hussein H Alvarez Luis L Pascal René R Lavryk Fedir F Bönigk Wolfgang W Körschen Heinz G HG Müller Astrid A Poetsch Ansgar A Rennhack Andreas A Gui Long L Nicastro Daniela D Strünker Timo T Seifert Reinhard R Kaupp U Benjamin UB

The EMBO journal 20191227 4

Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca<sup>2+</sup> rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cel ...[more]

PMID: 31880004

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Signaling proteins of a chemosensory pathway from sperm of Arbacia punctulata

Project description:Cilia and flagella serve as cellular antennae that translate sensory information into cellular responses. In sperm, a single chemoattractant molecule triggers signaling events that evoke a Ca2+ response and chemotactic steering. Key components of the chemotactic signaling pathway from sperm of the sea urchin Arbacia punctulata were investigated. Targeted proteomics employed a spiked-in heavy isotope-labelled standard protein (QConCat) and in-gel protein digestion with trypsin. The standard protein together with the previously determined copy number of the receptor guanylate cyclase allowed determining the copy numbers for 19 polypeptides of this signaling pathway.

2019-12-19 | PXD015502 | panorama

Chlamydomonas reinhardtii flagella cc125 ift139-2 dhc1b-3

Project description:As part of a cryo EM study of IFT trains in Chlamydomonas flagella we used two mutants with defects in IFT139 or DHC1b. To determine what proteins were missing in the flagella of the mutants we did a proteomic analysis of the membrane matrix fraction from flagella isolated from the mutants.

2018-10-19 | PXD010699 | Pride

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localisation and dynamics

Project description:Identification of intact flagella of procyclic Trypanosoma brucei cells by LC-MS/MS after a trypsin shaving

2018-12-17 | PXD000051 | Pride

The distinctive flagellar proteome of Euglena gracilis

Project description:The eukaryotic flagellum is a prominent organelle with conserved structure and diverse functions. Here we present a proteomic study of the flagella and pellicle of Euglena gracilis, a photosynthetic and highly adaptable protist, which employs its flagella for both locomotion and environmental sensing. The biochemically distinct flagella of this euglenozoan yields 1,684 protein groups, which challenges previous estimates on the protein composition of motile flagella across the eukaryotes.

2021-07-16 | PXD024952 | Pride

Altered N-glycan composition impacts flagella mediated adhesion in Chlamydomonas reinhardtii

Project description:For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRIPSR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces as demonstrated by force spectroscopy and impairs polystyrene bead binding and transport. Notably, assembly, Intraflagellar Transport and FMG-1B transport into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact. Data deposited here include IS-CID data verifying altered N-glycan patterns in flagella of mutants analysed (Ursgal-SugarPy-flagella-IM-results.xls) as well as the N-glycoproteomic characterization of the novel CRISPRXylT1A mutant (Ursgal-SugarPy-CIRSPR-mutant-results.xls). Regarding the latter, PRM measurements verifying the knock out of XylT1A are also deposited here (Results_PRM-measurement_quantification_GT.xls). Further, label free quantification data on isolated flagella of original IM strains verifying that altered N-glycosylation does not impact flagellar protein localization can be found within this dataset (Flagella_IM-WT_ normalized_5_proteins.pdResult).

2020-12-22 | PXD018353 | Pride

Esherichia coli O157:H7 F9 fimbriae recognize plant xyloglucan and elicit a response in Arabidopsis thaliana

Project description:Fresh produce are often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (fml) fimbriae have been shown to be expressed at temperatures lower than 37°C, implying a function outwith the mammalian host. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared to flagella (H7) and another fimbrial type (Mat3), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response.

2021-02-01 | E-MTAB-9732 | biostudies-arrayexpress

Mutations in AK9 impair male fertility by altering sperm motility in mice and cattle.

Project description:Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination (AI) can exhibit a significant variation in fertility. During fertility analysis, a subfertile bull with a low pregnancy rate of 10% was identified. To fully characterize the phenotype, a range of in vitro, in vivo and molecular assays were carried out. Additionally, knockout mice were generated to investigate the function of the identified mutated gene. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to control bulls of proven fertility. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing and RNA sequencing of testis revealed a critical mutation in AK9 that affect splicing, shaping the majority of AK9 transcripts that leads to a premature termination codon and a severely truncated protein. Transgenic mice deficient in AK9 were generated, resulting in the production of immotile sperm that were unable to fertilize the oocyte. These sperm exhibited abnormalities, including a low ATP concentration. RNA-seq analysis of testis revealed differential gene expression of components of the axoneme and sperm flagella, as well as steroid metabolic processes. Sperm ultrastructural analysis showed a higher percentage of sperm with abnormal flagella. The infertility produced by AK9 mutant bull and in AK9 deficient mice indicates the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida.

2023-09-22 | GSE235546 | GEO

Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella

Project description:Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

2019-11-12 | PXD011730 | Pride

qRT-PCR of ex vivo Salmonella enterica in TLR4 minus/plus macrophage background

Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the toll-like receptor 4 (TLR-4) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a TLR-4 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains (WT and TMS) grown under 2 conditions (TLR-4 wildtype (plus) or TLR-4 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in quadruplicates, located on the same PCR plate. For each quadruplicates the median value was calculated and used as a data point for further normalization. A set of controls was selected for all data sets using the following criteria: the data points for the controls should have Cp <40 and be present in all the samples.

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qRT-PCR of ex vivo Salmonella enterica in NRAMP-1 minus/plus macrophage background

Project description:A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the NRAMP-1 (natural resistance-associated macrophage protein-1) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a NRAMP-1 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains ( WT and TMS) grown under 2 conditions (NRAMP-1 wildtype (plus) or NRAMP-1 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in duplicates, located on the same PCR plate. For each duplicate the mean value was calculated and used as a data point for further normalization. We averaged the Cp values for all the data points in a sample and then subtracted this sample average Cp from all the data points of the sample. Only 192 primer pairs from 288 on the platform GPL11111 were used.

2011-06-13 | E-GEOD-26806 | biostudies-arrayexpress

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