bioRxiv Science⌕ Search

Biology subjects

ten Have, S.

Publications and source records attributed to ten Have, S..

4 recordsLinked to original sources

The Spontaneous Evolution of Biology

We have demonstrated the potential of amino acids to polymerise into peptides, proteins, and form cell-like structures in the absence of cellular machinery, including nucleic acids, lipids or sugars. Not only has cell-free protein replication been observed, but evidence of protein templating strongly suggests protein mediated replication. We believe this is the first experimental demonstration of the link between the Miller-Urey experiment which produced amino acids from elemental starting material, and cell-like structures. Life, by definition, is the condition that distinguishes animals and plants from inorganic matter, including the capacity for growth, reproduction, functional activity, and continual change preceding death. Here we have characterised peptides which form reproducibly, into structures with longevity and which subsequently catalyse the polymerisation of free amino acids into copies of themselves. The proteomic analysis of these samples over time also enables evolution of peptide sequences to be seen and quantified. This evolution of both complex structure and functionally active proteins may potentially demonstrate a credible path to the beginnings of life, which we call the Spontaneous Evolution of Biology (SEB) Theory. One Sentence SummaryWe have shown how peptides and proteins can be made from amino acids in an aqueous media without cells, lipids or nucleic acids, duplicating themselves and forming complex structures which resemble cells.

biochemistry↗

SILAC-based quantitative proteomic analysis of Drosophila gastrula stage embryos mutant for fibroblast growth factor signaling

The application of quantitative proteomics in model organisms has been successful in determining changes in the proteome under distinct physiological conditions. Quantitative mass spectrometry-based proteomic analyses in combination with genetics provide powerful tools in developmental cell signaling research. Drosophila melanogaster is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine global changes in the proteome upon depletion of the Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signaling in Drosophila embryos at early gastrulation stages. We present a robust, single generation SILAC (stable isotope labeling with amino acids in cell culture) protocol for labeling proteins in early embryos and for selection of homozygously mutant embryos at pre-gastrula stages using an independent genetic marker. Our analyses detected quantitative changes in the global proteome of htl mutant embryos during gastrulation. We identified distinct classes of down-regulated and up-regulated proteins and network analyses indicates functionally related groups of proteins in each class. These data suggest that FGF signaling in the early embryo affects global changes in the abundance of metabolic, nucleoplasmic, cytoskeletal and endomembrane transport proteins.

developmental biology↗

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

genetics↗

The Ndc80 complex targets Bod1 to human mitotic kinetochores

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of PP2A-B56. This phosphatase controls the amount of phosphorylation of mitotic kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. The PP2A-B56 regulatory protein Bod1 directly localises to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localisation of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1s binding and inhibition of PP2A-B56, peaks in prometaphase, when PP2A-B56 localisation to kinetochores is highest. Kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Additionally, Bod1 depletion leads to a defect in the phosphorylation kinetics of Ndc80 at serine 55 (S55) within its N-terminus. Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80 phosphorylation and the stability of the attachments of microtubules to kinetochores.

cell biology↗