Trinh, L. T. | bioRxiv Science

bioRxiv2025

RNF13 mediates pH- and Ca2+-dependent regulation of lysosomal positioning

Environmental factors such as extracellular pH (pHe) and nutritional status influence lysosomal localization and autophagy. However, the mechanisms by which pHe, intracellular pH (pHi), and Ca2+ levels coordinate the bidirectional transport of lysosomes remain poorly understood. In this study, we identify RNF13 as a critical regulator of lysosomal positioning through its ubiquitin- dependent degradation of ARL8B. RNF13 activity is modulated by both pHi and Ca2+ levels. Specifically, we demonstrate that Ca2+-activated apoptosis-linked gene 2 (ALG-2) promotes retrograde lysosomal transport while simultaneously increasing pHi and decreasing lysosomal pH (pHlys). Elevated pHi deprotonates RNF13 at His332, enabling its interaction with Ca2+-bound ALG-2. This interaction activates RNF13, which inhibits ARL8B-mediated anterograde lysosomal transport. Furthermore, we show that alkaline pHe elevates pHlys, activating the lysosomal Ca2+ channel TRPML3. This activation enhances RNF13 activity, driving lysosomes to adopt a perinuclear localization. Thus, conditions such as starvation or alkaline pHe, which induce ALG-2 activation and pHi elevation, facilitate RNF13- mediated ARL8B degradation. In contrast, under acidic pHi conditions, RNF13 activity remains suppressed regardless of ALG-2 activation, leading to increased ARL8B levels. Additionally, we provide evidence linking the loss of RNF13 activity to developmental and epileptic encephalopathy-73, a neurological disorder characterized by severe developmental symptoms. These findings deepen our understanding of lysosomal positioning mechanisms, highlighting the interplay between lysosomal Ca2+ release and dynamic changes in cytoplasmic and lysosomal pH.

cell biology↗

bioRxiv2023

Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

developmental biology↗