Biology subjects

Dimke, H.

Publications and source records attributed to Dimke, H..

3 recordsLinked to original sources

bioRxiv2021

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV- Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

molecular biology↗

bioRxiv2021

Differential activation of parathyroid and renal Ca2+-sensing receptors underlies the renal phenotype in autosomal dominant hypocalcemia 1

BackgroundParathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of CaSRs in kidneys and intestine attenuates local transepithelial Ca2+ transport. In patients with autosomal dominant hypocalcemia 1 (ADH1) due to activating CASR mutations, treatment of symptomatic hypocalcemia can be complicated by treatment-induced hypercalciuria, resulting in nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body are activated by increased extracellular Ca2+ concentrations, it is not understood why some ADH1 patients have reduced PTH, but not hypercalciuria at presentation, despite CaSR expression in both kidney and parathyroid. MethodsActivation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells. ResultsMice with a gain-of-function mutation in Casr are hypocalcemic with reduced plasma PTH levels. However, renal CaSRs are not activated as indicated by normal urinary calcium handling and unaltered renal Cldn14 expression. Activation of renal CaSRs only occurred after increasing plasma Ca2+ levels. Similarly, calcimimetic administration to wildtype mice induced hypocalcemia without activating renal CaSRs. Moreover, significant hypercalciuria was not observed in an ADH1 patient until blood Ca2+ was normalized. In vitro experiments suggest that increased CaSR levels in the parathyroid relative to the kidney contribute tissue-specific CaSR activation thresholds. ConclusionHere we delineate tissue-specific CaSR activation thresholds, where parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even with overactivating mutations. These findings may aid in the management of ADH1 patients.

physiology↗

bioRxiv2018

A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity

Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. The E. coli protein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. A randomly mutated BirA library was used to identify novel variants. Bacteria displaying peptide sequences from 13 out of 14 tested proteins were strongly enriched after 3-5 selection rounds. Moreover, a clone selected for biotinylation of a C-terminal peptide from red-fluorescent protein TagRFP showed site-specific biotinylation. Thus, active BirA variants with novel specificity are effectively isolated with our bacterial display system and provides a basis for the development of BirA variants for site-selective biotinylation.

bioengineering↗