Biology subjects

Thiele, L.

Publications and source records attributed to Thiele, L..

3 recordsLinked to original sources

bioRxiv2026

Arteriolar GMP signaling revealed by FRET-based real-time measurement in vivo

cGMP evokes arteriolar vasorelaxation and is generated in smooth muscle by the soluble guanylyl cyclase (sGC) stimulated by endothelial NO. Phosphodiesterases (PDEs) degrade cGMP and may contribute in diameter regulation. We examined arteriolar cGMP levels in real-time in vivo upon NO and acetylcholine (ACh) and effects of PDE inhibition. Changes in cGMP were measured by FRET using the indicator protein cGi500. Arterioles in the cremaster muscle of anesthetized mice expressing cGi500 were exposed for imaging. They were stimulated with various NO donors (DEA/NO, SNP, SNAP) and ACh with or without PDE inhibition (non-specific: IBMX; PDE-5 specific: sildenafil). Furthermore, dilations induced by ACh and SNP were studied in sGC-deficient mice. DEA/NO and SNAP led to a fast, concentration-dependent rise of fluorescence ratio of cGi500 (by 4.0% at 10{micro}M), that declined on removal slowly. These ratio changes indicated cGMP increases as verified by inhibition of the sGC using ODQ. PDE inhibition increased cGMP levels slowly despite NO synthase inhibition. However, it did not amplify NO-induced ratio increases, but the decline after drug removal was decelerated. Interestingly, ACh did not modify cGMP levels and dilations were not impaired in sGC-deficient mice. FRET-based imaging allows reliable real-time assessment of arteriolar cGMP levels in vivo. PDE activity does not limit the amplitude, but the duration of cGMP signaling after stimulation. Furthermore, we conclude that ACh does not release endothelial NO in murine arterioles. Preliminary experiments demonstrate that simultaneous measurement of cGMP and diameter is feasible using mice with expression of cGi500 in smooth muscle cells.

physiology↗

bioRxiv2025

Peroxisome protein import deficiency causes heart failure in mouse and human

Peroxisomes are ubiquitous cellular organelles with potentially vital roles in lipid and reactive oxygen metabolism. The metabolic demands of the heart are substantial; however, the contribution of peroxisomes to cardiac development, health, and their role in heart failure (HF) remain largely unexplored. We developed and examined a mouse and an engineered human myocardium (EHM) model with a deficiency in cardiac peroxisome biogenesis to investigate the role of peroxisomes in cardiac function and pathology. In the EHM, loss of peroxisome protein import and subsequent peroxisomal metabolic impairment trigger mitochondrial damage and compromise cellular respiration and energy production. Peroxisome dysfunction results in incoherent electrical conduction, defective Ca2+-handling, and ultimately presentation of a HF phenotype with pathological force generation. These phenotypes are mirrored in an orthogonal murine model system with defective cardiac peroxisome biogenesis. Preload-dependent deficits in force generation due to insufficient energy supply are eventually fatal. Thus, peroxisomes play an important role in sustaining normal heart operations. Vice versa, peroxisome maintenance is compromised in pressure overload-induced HF, establishing peroxisomes as potential modulators of pathology and targets of therapy.

cell biology↗

bioRxiv2018

A Systematic Approach to Identify Recycling Endocytic Cargo Depending on the GARP Complex

Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. In a previous paper we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Frohlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.

cell biology↗