Biology subjects

Boettcher, R.

Publications and source records attributed to Boettcher, R..

3 recordsLinked to original sources

bioRxiv2026

ILK binding to β1 Integrin is indirectly mediated by Kindlin-2

Integrin-linked kinase (ILK) and kindlin-2 (K2) are key components of focal adhesions (FAs) that regulate cell-matrix adhesion and integrin signaling. Both proteins directly bind each other, but how they influence each others localization to FAs and binding to integrins remains a subject of ongoing debate. Here, we establish a sensitive workflow to study protein-protein interactions in cells by combining methods from biochemistry, cell biology and super-resolution microscopy. Together with an analytical framework this approach allowed us to distinguish direct from indirect molecular interactions and construct detailed interaction networks. Disrupting the ILK-K2 interaction reduced ILK localization to FAs and compromised integrin function, whereas K2 recruitment was unaffected. Our interdisciplinary approach also revealed that ILK does not directly bind {beta}1-integrin cytosolic domains in vitro and in cells. Instead, ILK was recruited to integrins exclusively through a K2-dependent mechanism, primarily via K2 bridging ILK and {beta}1 integrins. These findings define the hierarchical relationship between ILK and K2 in FAs and highlight the essential role of K2-mediated ILK recruitment for integrin adhesion and signaling. Significance StatementHow cells anchor to their environment is a fundamental question in biology. Integrins provide such a connection by bridging the extracellular matrix and the cytoskeleton. A central regulator of the integrin machine is Integrin-linked kinase (ILK). How ILK is recruited to {beta}1 integrins is hotly debated since its discovery more than 30 years ago. By integrating cell biology and biochemistry with super-resolution DNA-PAINT microscopy and a novel spatial analysis framework, we demonstrate that ILK does not bind directly to integrin cytoplasmic tails. Instead, we found that ILK is recruited by kindlin-2 (K2) to active, adhesion plaque-resident integrins. This work resolves a long-standing controversy in cell biology and establishes a versatile workflow for distinguishing direct from indirect protein-protein interactions in situ.

cell biology↗

bioRxiv2025

Sequential PIDD1 auto-processing is essential for ploidy control in liver and heart

Polyploidization refers to the balanced increase in gene copy number and is a feature of specialized cells in different mammalian tissues, including the liver and the heart. During organogenesis, hepatocytes and cardiomyocytes undergo scheduled polyploidization events to increase their cellular or nuclear DNA content. This is thought to improve cellular output and enable for rapid genetic adaptation in response to stress. Yet, excessive increases in ploidy can also be disadvantageous and increase the risk of genome instability. Hence, a dedicated machinery, the PIDDosome multi-protein complex, has evolved to prevent exacerbated increases in DNA content. Using targeted mutagenesis in mice, we show that the PIDDosome controls hepatocyte ploidy in a cell-autonomous manner and that sequential and quantitative auto-processing of PIDD1 is key for accurate control of ploidy in postnatal development of liver and heart. Stoichiometric imbalances in bioactive PIDD1-fragments impair p53-dependent and independent cell cycle arrest responses during organogenesis, as well as caspase-2-dependent apoptosis caused by centrosome amplification. Strikingly, targeted mutagenesis of the caspase cleavage motif in the critical E3-ligase controlling p53 protein levels, Mdm2, impairs ploidy control in hepatocytes, but not in cardiomyocytes, indicative of the existence of alternative caspase-2 substrates that help to restrict ploidy in the heart.

cell biology↗

bioRxiv2021

Splicing stimulates antisense transcription by RNA polymerase II at DNA double-strand breaks in Drosophila cells

DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.

molecular biology↗