Biology subjects

Lemke, B.

Publications and source records attributed to Lemke, B..

4 recordsLinked to original sources

bioRxiv2025

Parkin-dependent ubiquitination of TAX1BP1 directs efficient autophagic removal of defective mitochondria

In stressed cells, the recessive Parkinson disease (PD) associated gene products PINK1 and parkin mediate the autophagic removal of damaged mitochondria (mitophagy). Upon mitochondrial membrane potential disruption, PINK1 phosphorylation activates the ubiquitin ligase parkin which ubiquitinates various mitochondrial protein substrates. These feed-forward modifications on the mitochondria surface attract ubiquitin-binding autophagy receptors that target ubiquitinated mitochondria for degradation. Investigating post-translational protein modifications during this process, we detected transient ubiquitination of K549 within the third coiled-coil domain (CC3) of TAX1BP1 in HeLa cells expressing wild-type (WT) but not catalytically inactive parkin. Parkin-dependent ubiquitination did not target TAX1BP1 to proteasomal degradation but was rather indicative of a regulatory modification. In cells with the full complement of autophagy receptors, TAX1BP1 plays only a minor role in mitophagy. However, when expressed as a sole autophagy receptor, both WT and ubiquitination deficient TAX1BP1 were capable of promoting mitophagy, albeit mitochondria degradation was slightly delayed under mutant conditions. Use of the lysosomal inhibitor bafilomycin A indicated classical autophagolysosomal targeting of damaged mitochondria mediated by WT TAX1BP1. However, for the ubiquitination-deficient TAX1BP1, we observed an increased prevalence of enlarged endolysosomal vesicles carrying accumulated TAX1BP1-positive autophagosomes filled with mitochondrial material. Thus, while ubiquitination of the CC3 domain of TAX1BP1 is not essential for complete mitophagy, the lack of CC3 in TAX1BP1 reroutes the degradation flux to a less efficient endolysosmal degradative pathway. Interestingly, the PD gene product VPS35 becomes prominently engaged in this alternative mitophagy pathway.

cell biology↗

bioRxiv2025

When mitochondria fall apart: Unbalanced mitochondrial segregation triggers loss of mtDNA in the absence of mitochondrial fusion

Mitochondrial biogenesis and inheritance must be carefully regulated alongside cell division to ensure proper mitochondrial function and cell survival. The dynamics of the mitochondrial network, including fusion and fission, play a crucial role in mitochondrial inheritance by facilitating the distribution and quality control of mitochondria. In budding yeast, simultaneous inhibition of both fusion and fission leads to loss of mitochondrial DNA (mtDNA) integrity, resulting in an increased frequency of petite cells. Loss of mitochondrial fusion alone results in the complete loss of mtDNA. While the loss of mtDNA in the absence of mitochondrial fusion has been known for almost 30 years, the reason remained unclear. Here, we investigate the consequences of impaired mitochondrial fusion through depletion of the mitofusin Fzo1. We follow the emerging phenotype by live-cell imaging and the analysis of more than thirty thousand single cells across their cell cycle. Fzo1 depletion causes rapid mitochondrial fragmentation and a reduction in mitochondrial membrane potential, followed by a progressive decline of mtDNA content and cellular growth rate over several cell divisions. During division, Fzo1-depleted daughters obtain an unusually large amount of mitochondria, leaving the mother with too little. This results in a strong disbalance of mitochondrial mass in the population. Additionally, Fzo1-depleted cells lose the ability to adjust mtDNA synthesis to compensate for a low mitochondrial content. The combined effects of unequal distribution and reduced synthesis drive rapid mtDNA loss. These results show how fusion defects lead to mtDNA loss and mitochondrial dysfunction, contributing to understanding diseases linked to fusion defects.

cell biology↗

bioRxiv2025

InSituPy -- A framework for histology-guided, multi-sample analysis of single-cell spatial transcriptomics data

Single-cell spatial transcriptomics (scST) methodologies allow, in combination with histological stainings, an unprecedented view on disease progression. To comprehensively analyze scST data, bioinformatic analysis frameworks need to integrate the diverse set of data modalities and, just as importantly, enable the joint analysis of multiple datasets from clinical or experimental cohorts together with its corresponding metadata. Here, we present the InSituPy framework to comprehensively analyze single-cell spatial transcriptomic data from a multi-sample level down to the cellular and subcellular level. The framework contains analysis workflows for the integration of image data as well as pathological and biological expert knowledge. Increasing the accessibility of the data for non-bioinformaticians, the framework opens new ways of generating hypotheses, especially in the context of translational research.

bioinformatics↗

bioRxiv2023

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells

Cellular lineage histories along with their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have limited barcode diversity and poor single-cell lineage coverage, thus precluding their use in tissues composed of millions of cells. Here, we developed DARLIN, an improved Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) to enhance insertion events over 30 CRISPR target sites, stably integrated into 3 distinct genomic loci. DARLIN is inducible, has an estimated ~1018 lineage barcodes across tissues, and enables detection of usable barcodes in ~60% of profiled single cells. Using DARLIN, we examined fate priming within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage information in single cells. DARLIN will enable widespread high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

developmental biology↗