Gerhard Obermeyer

Background: Substrate curvature is an important biophysical cue regulating cell behavior through mechanotransduction. However, how three-dimensional curvature regulates osteoblast transcriptional adaptation remains poorly understood. We investigated the temporal transcriptional response of pre-osteoblastic cells to defined substrate curvature. Methods: Murine MC3T3-E1 pre-osteoblasts were cultured on three-dimensional polydimethylsiloxane capillary bridges or flat substrates for 7 and 23 days to investigate the effects of surface curvature on gene expression. A genome-wide transcriptomic approach was employed to comprehensively characterize differential gene expression, followed by a focused analysis of selected biological pathways, including osteogenic signaling, extracellular matrix (ECM) organization, cell–matrix adhesion, cytoskeletal regulation, and mechanosensitive signaling. Results: Substrate curvature induced pronounced, time-dependent transcriptional changes. At day 7, curved substrates activated immediate-early and stress-responsive genes, including Egr1, Egr2, Fos, Junb, and Nr4a1 . By day 23, the response shifted toward ECM remodeling, receptor-mediated signaling, and structural adaptation, including increased expression of Ankrd1, Ereg, Serpine1, and Plaur . Despite these changes, canonical osteogenic transcription factors, including Runx2, Cb@, Osx/Sp7, and Atf4 , showed limited alterations. In contrast, BMP, WNT, FGF, and Notch signaling, ECM components, integrins, and actin-associated genes were dynamically regulated, whereas Piezo1, Piezo2, and most septins remained comparatively stable. Conclusions: Substrate curvature regulates osteoblast transcriptional adaptation primarily through coordinated changes in ECM organization, cell–matrix interactions, cytoskeletal architecture, and signaling networks rather than reprogramming the canonical osteogenic transcriptional machinery.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H⁺ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H⁺ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H⁺ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H⁺ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H⁺ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H⁺ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H⁺ ATPase sequence suggests a different regulation compared to higher plant PM H⁺ ATPases. A higher abundance of H⁺ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Fertilization in plants relies on fast growth of pollen tubes through the style tissue towards the ovules. This polarized growth depends on influx of ions and water to increase the tube’s volume. K+ inward rectifying channels were detected in many pollen species, with one identified in Arabidopsis. Here, an Arabidopsis AKT1-like channel (LilKT1) was identified from Lilium longiflorum pollen. Complementation of K+ uptake deficient yeast mutants was only successful when the entire LilKT1 C-terminus was replaced by the AKT1 C-terminus. No signals were observed in the plasma membrane (PM) of pollen tubes after expression of fluorescence-tagged LilKT1 nor were any LilKT1-derived peptides detectable in the pollen PM by mass spectrometry analysis. In contrast, fluorescent LilKT1 partly co-localized with the lily PM H+ ATPase LilHA2 in the PM of tobacco leaf cells, but exhibited a punctual fluorescence pattern and also sub-plasma membrane localization. Thus, incorporation of LilKT1 into the pollen PM seems tighter controlled than in other cells with still unknown trafficking signals in LilKT1’s C-terminus, resulting in channel densities below detection limits. This highly controlled incorporation might have physiological reasons: an uncontrolled number of K+ inward channels in the pollen PM will give an increased water influx due to the raising cytosolic K+ concentration, and finally, causing the tube to burst.

Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.