NISHIMURA, T. | Search bioRxiv

bioRxiv2025

Rational engineering of lipid-binding probes via high-throughput protein-lipid interaction screening

Lipid-binding domains, originally isolated from natural proteins, are useful tools essential for analyzing membrane lipids in cells, and their applications are varied. Yet, there is no general strategy for engineering lipid-binding domains. Here, we present a robust method for monitoring protein-lipid interactions, named the Cell surface Liposome Binding (CLiB) assay. Using this technique, we isolated high-affinity lipid-binding domains and nanobodies that preferentially bind to phosphatidylinositol phosphates. Furthermore, by combining the CLiB assay with next-generation sequencing, allowing the analysis of more than 10,000 clones in parallel, we identified novel variants with enhanced binding to phosphatidylinositol phosphates and uncovered a common structural feature: a positively charged pocket necessary for binding, formed by the three loop regions. This study opens a new avenue for the rational design and generation of lipid-binding probes on demand.

cell biology↗

bioRxiv2023

DNA methylation is linked to the monoallelic expression of MRP3, a diatom mating type determining gene

Diatoms are unicellular microalgae widely distributed in aquatic ecosystems. In diatoms, sexual reproduction is needed to counteract cell miniaturization imposed by the rigid silica shell, and only small cells, below a species-specific size threshold, are competent for sex. We performed a genome-wide Enzymatic Methyl-seq analysis in the heterothallic diatom Pseudo-nitzschia multistriata comparing cells of different size and opposite mating type (MT) to investigate potential epigenetic controls in life cycle transitions. We found an imprinting-like pattern of methylation at the sex locus: alleles of the gene responsible for the specification of the MT+, MRP3, are hypermethylated in MT- and differentially methylated in MT+, with transcription occurring only on the MT+ hypomethylated variant. The methylation pattern is overall stable over the P. multistriata life cycle. Absence of methylation in MRP3 is necessary for its expression but not sufficient, since large non-sexual cells have the same methylation profile of small sexual cells but do not express MRP3, suggesting that additional controls are involved in the mechanism of sex determination. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/561864v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ea2482org.highwire.dtl.DTLVardef@2d553org.highwire.dtl.DTLVardef@350aaorg.highwire.dtl.DTLVardef@1a4f2f6_HPS_FORMAT_FIGEXP M_FIG C_FIG The methylation profile of the mating type determining gene MRP3 of the diatom Pseudo-nitzschia multistriata is different between opposite mating types. Lack of methylation is necessary but not sufficient for MRP3 expression in the sexually competent MT+ strains. HighlightsO_LI- The mating type locus is differentially methylated in MT+ and MT- C_LIO_LI- DNA methylation is linked to MRP3 expression C_LIO_LI- DNA methylation does not play a role in the acquisition of sexual competence C_LI

genetics↗