A conserved fertilization complex bridges sperm and egg in vertebrates.

Fertilization, the basis for sexual reproduction, culminates in the binding and fusion of sperm and egg. Although several proteins are known to be crucial for this process in vertebrates, the molecular mechanisms remain poorly understood. Using an AlphaFold-Multimer screen, we identified the protein Tmem81 as part of a conserved trimeric sperm complex with the essential fertilization factors Izumo1 and Spaca6.

Gpcpd1-GPC metabolic pathway is dysfunctional in aging and its deficiency severely perturbs glucose metabolism.

Skeletal muscle plays a central role in the regulation of systemic metabolism during lifespan. With aging, this function is perturbed, initiating multiple chronic diseases. Our knowledge of mechanisms responsible for this decline is limited.

The Fgf/Erf/NCoR1/2 repressive axis controls trophoblast cell fate.

Placental development relies on coordinated cell fate decisions governed by signalling inputs. However, little is known about how signalling cues are transformed into repressive mechanisms triggering lineage-specific transcriptional signatures. Here, we demonstrate that upon inhibition of the Fgf/Erk pathway in mouse trophoblast stem cells (TSCs), the Ets2 repressor factor (Erf) interacts with the Nuclear Receptor Co-Repressor Complex 1 and 2 (NCoR1/2) and recruits it to key trophoblast genes.

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.

Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs.

TurboID-based proteomic profiling of meiotic chromosome axes in Arabidopsis thaliana.

During meiotic prophase I, sister chromatids are arranged in a loop-base array along a proteinaceous structure, called the meiotic chromosome axis. This structure is essential for synapsis and meiotic recombination progression and hence formation of genetically diverse gametes. Proteomic studies in plants aiming to unravel the composition and regulation of meiotic axes are constrained by limited meiotic cells embedded in floral organs.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis.

AKIRIN2 controls the nuclear import of proteasomes in vertebrates.

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth.

The H3 histone chaperone NASP escorts CenH3 in Arabidopsis.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells.

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform.

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized.

A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function.

Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage.

Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers.

Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea.

Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions.

Global analysis of muscle-specific kinase signaling by quantitative phosphoproteomics.

The development of the neuromuscular synapse depends on signaling processes that involve protein phosphorylation as a crucial regulatory event. Muscle-specific kinase (MuSK) is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components are still poorly understood.

Analysis of meiotic protein complexes from Arabidopsis and Brassica using affinity-based proteomics.

The application of proteomics techniques to the study of plant meiosis has the potential to make a valuable contribution to our understanding of the molecular events underpinning meiotic processes. Here we describe the preparation of meiotic protein complexes from Arabidopsis thaliana and its close crop relative, Brassica oleracea, by co-immunoprecipitation for in-solution analysis by tandem mass spectrometry (MS/MS). Early results using these techniques have proved encouraging, enabling the identification of candidate AtASY1-interacting proteins in A.

Yeast protein phosphatase 2A-Cdc55 regulates the transcriptional response to hyperosmolarity stress by regulating Msn2 and Msn4 chromatin recruitment.

We have identified Cdc55, a regulatory B subunit of protein phosphatase 2A (PP2A), as an essential activating factor for stress gene transcription in Saccharomyces cerevisiae. The presence of PP2A-Cdc55 is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. We show that PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 during hyperosmolarity stress.

Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs.

Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells.

Weak organic acid stress triggers hyperphosphorylation of the yeast zinc-finger transcription factor War1 and dampens stress adaptation.

Exposure of Saccharomyces cerevisiae to weak organic acids such as sorbate, propionate, or benzoate rapidly induces the plasma membrane ABC transporter Pdr12, requiring the Zn(II)(2)Cys(6) zinc-finger transcription factor War1. Weak acid stress rapidly triggers War1 phosphorylation but its role for War1 function is not clear yet. Here, we provide new insights into sorbate-induced phosphorylation of War1.

QIKS--Quantitative identification of kinase substrates.

Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes.

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al.

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis.

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins.

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts.

Phosphoproteomics strategies for the functional analysis of signal transduction.

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques.