Triosephosphate isomerase deficiency (TPI Df) is an ultra-rare genetic enzymopathy. Previously, the TPIR5G allele was found to cause TPI Df when combined with a null allele. Here, we report a 1.15 Å TPIR5G crystal structure providing insight into disease pathogenesis. Previously, we conducted a high-throughput screen that identified TPI-inducing compounds, including predicted hypoxia inducible factor (HIF) inducers. We have investigated repurposing HIF activators/prolyl hydroxylase domain inhibitors (PHDIs) as TPI Df treatments. We tested the efficacy of these compounds in cells from individuals with TPI Df. Our results demonstrate that PHDIs increase TPI protein levels and TPI activity, suggesting they should be further developed for TPI Df. RNA-sequencing and reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments were performed to analyze PHDI-induced gene expression changes. We discovered that chronic PHDI treatment results in HIF1-antisense 2 (HIF1-AS2) activation, which operates as a negative feedback loop in the HIF pathway. These results demonstrate that repurposing PHDIs for TPI Df is a promising avenue of research deserving further investigation. Our results also suggest that PHDIs may also benefit dozens of other heritable disease conditions if treatment avoids HIF1-AS2 activation.

The second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) have been implicated in the regulation of flagellar motility in diverse eukaryotes, yet the specific contributions of PKA isoforms remain unclear. Moreover, the kinetoplastid PKA is insensitive to cAMP. Here, we investigated subcellular localisations of PKA subunits in Leishmania and study the flagellar waveform parameters in PKA deletion mutants. Leishmania mexicana has three PKA catalytic (LmxPKAC1, LmxPKAC2 and LmxPKAC3) and two PKA regulatory (LmxPKAR1 and LmxPKAR3) isoforms. Expansion microscopy revealed that LmxPKAR1 was localised between the paraflagellar rod and the axoneme, and LmxPKAC1, LmxPKAC2 depend on LmxPKAR1 for enrichment in the flagellum. LmxPKAC3 required LmxPKAR3 for colocalisation at the cell cortex. Deletion of LmxPKAR3 resulted in increased flagellar LmxPKAC3 signal and increased swimming speed. Conversely, deletion of LmxPKAC3 resulted in reduced flagellar beat frequencies and swimming speed. The absence of LmxPKAC1 resulted in the loss of high frequency tip-to-base waveforms, and consequently reduced swimming speed. LmxPKAC1 dissociated from cytoskeletons upon addition of guanosine, adenosine or inosine, consistent with a nucleoside-based PKA activation mechanism. These data suggest that this non-canonical PKA pathway regulates the motility of Leishmania through the modulation of flagellar waveforms.

Ca2+ homeostasis is essential for cellular functions, with regulation by store-operated Ca2+ entry (SOCE) omnipresent. Due to a lower affinity for endoplasmic reticulum (ER)-luminal Ca2+, STIM2 regulates basal cytosolic Ca2+ but also increases interaction and activation of ORAI proteins at ER-plasma membrane junctions after stimulation, whereas STIM1 requires stronger store depletion. In brain, STIM2 is highly expressed in hippocampal neurons. Here, we describe a short STIM2 splice variant, STIM2.3 (also known as STIM2G), that is present only in Old World monkeys, apes and humans, with expression mostly in brain. In contrast to other variants and despite lack of the polybasic domain, expression of STIM2.3 increased SOCE. Structure-function analysis delineated the role of the C-terminal motifs of STIM2 for Ca2+ entry as well as for basal and induced activation of the NFAT transcription factor NFATc1. STIM2.3 displayed reduced interaction with AMPK and with activated AMPK. Neuronal expression of STIM2.3, in comparison to STIM2.2, increased the size of dendritic spine heads, suggesting a specific regulatory role in spine maintenance. Regulated splicing of STIM2.3 in brain might present a rapid mechanism to increase STIM2-mediated effects on gene expression, spine morphology or spontaneous excitability, potentially facilitating an evolutionarily recent expansion of brain complexity.

Mitophagy - the selective degradation of damaged mitochondria - preserves mitochondrial quality; yet, how mitochondrial fission is coordinated with autophagy initiation remains unclear. Here, we identified the mitochondrial outer membrane protein MTFR1L as a key component of mitophagy initiation hubs after using a synthetic FKBP-FRB system to tether ULK1 kinase to mitochondria independently of damage. We found that MTFR1L was enriched at ULK1 foci together with additional fission factors, and constitutive mitochondrial targeting of MTFR1L shifted mitochondrial morphology towards fragmentation. MTFR1L depletion decreased respiratory capacity, elevated apoptosis and impaired mitophagy flux. Upon mitophagy induction, MTFR1L was phosphorylated in a ULK1 kinase-dependent manner and reciprocally modulated ULK1 activity, establishing a feedback loop. Moreover, MTFR1L was required for proper ATG13 stability. These findings position MTFR1L as a critical link between mitochondrial fission and the autophagy machinery, coordinating mitophagy initiation and cell survival.

Glycine-N-methyltransferase (Gnmt) is highly upregulated in the aging Drosophila melanogaster eye. Gnmt regulates S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) availability by catalyzing the transfer of a methyl group from SAM to glycine, producing sarcosine and SAH. Gnmt is well studied in the liver; however, little is known about the consequences of increased Gnmt in neurons. We overexpressed Gnmt in the eye and profiled diurnal rhythmic gene expression and histone methylation in photoreceptor neurons. Here, we show that eye-specific Gnmt overexpression altered rhythmic gene expression without impacting photoreceptor viability. Gnmt overexpression decreased global repressive histone methylation but had no major effect on H3K4 methylation, suggesting that methylation reactions are selectively inhibited by Gnmt and that Gnmt disrupts rhythmic gene expression independently of H3K4 methylation. Gnmt overexpression did not alter rhythmicity of core clock genes and did not impact circadian behavior. These results suggest that Gnmt plays a role in the regulation of light-dependent rhythmic gene expression in photoreceptors that does not involve the molecular clock.

In mitosis, the duplicated genome is aligned and accurately segregated between daughter nuclei. CTCF is a chromatin looping protein that localizes to the centromere in mitosis with an unknown role. We previously published data showing that CTCF constitutive knockdown causes mitotic failure, but the mechanism remained unknown. To determine the role of CTCF in mitosis, here, we used a CRISPR CTCF auxin inducible degron cell line for rapid degradation. CTCF degradation for 3 days resulted in increased failure of mitosis and decreased circularity in post-mitotic nuclei. Upon CTCF degradation, CENP-E was still recruited to the kinetochore and there was a low incidence of the polar chromosomes that occur upon CENP-E inhibition. However, immunofluorescence imaging of mitotic spindles revealed that CTCF degradation caused increased intercentromere distances and a wider and more disorganized metaphase plate, representing a disruption of key functions of the centromere. These results are similar to what is seen upon partial loss of cohesin, an established component of the centromere. Thus, we reveal that CTCF is a key maintenance factor of centromere function, successful mitosis, and post-mitotic nuclear shape.

Polytopic copper (Cu)-ATPases are central regulators of the essential micronutrient copper in all organisms. In polarized epithelia, the vertebrate homologues ATP7A and ATP7B undergo copper-induced trafficking from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively, to mediate efflux of excess copper. To probe (1) inter-domain interactions that drive trafficking and (2) the extent of divergence between homologous domains constituting Cu-ATPases, we replaced the copper-binding N-terminal (NT), nucleotide-binding (NBD) and/or C-terminal (CT) domains of ATP7B with those of ATP7A. The functionally active chimeras exhibited distinct trafficking phenotypes. Notably, the ATP7B-NT substitution led to constitutive basolateral membrane trafficking, whereas simultaneous NT-NBD substitution led to steady-state TGN localization, suggesting that interaction between the two domains, as confirmed by in vitro NT-NBD-binding studies, might be essential for TGN localization. Interestingly, reciprocal replacement of the ATP7A-NBD and -NT with that from ATP7B did not rescue membrane localization, indicating that domain compatibility is restricted, suggesting greater evolutionary divergence of ATP7B domains. Analysing orthologous Cu-ATPase domain-sequences from diverse organisms, however, revealed similar evolutionary relationships between the NT and NBD, suggesting their co-evolution. We thus correlate the copper-responsive trafficking ability of Cu-ATPases with evolutionary stringency imparted onto Cu-ATPase domains.

ABSTRACTA significant challenge in studying the biology of the Drosophila centriole is its small size. Advanced super-resolution techniques have provided valuable insights but require specialized equipment and can be difficult to implement in tissues. Expansion microscopy (ExM) offers an accessible alternative, yet its application in Drosophila centriole research has been sparse. We provide an ExM protocol for cultured S2 cells and fly tissues that reveals new insights into procentriole biology. In S2 cells we document overduplication in the form of the classic ‘rosettes’, while in spermatids we uncover an unexpected movement of the procentriole-like structure (PCL). ExM has also refined existing molecular models. In S2 cells we document the distal tip protein Cep97 as a ring, which clarifies its role in capping the growing centriole. In spermatids, we spatially segregate the inner nuclear membrane protein Spag4 and the cytoplasmic protein Yuri, leading to the new hypothesis that they play independent roles at the centriole–nucleus contact site. Finally, we show that our ExM protocol is a hypothesis generator and discovery tool applicable beyond Drosophila centrioles by imaging synaptonemal complexes in the Plodia interpunctella moth.

In the colon, the single-layered epithelium forms crypts that extend into the mucosa and are surrounded by a fibroblast network essential for extracellular matrix (ECM) production, remodeling and epithelial support. Fibroblasts are heterogeneous, but inconsistent nomenclature and lack of markers have hindered their classification. Using single-cell RNA sequencing (scRNA-seq), we identified six distinct fibroblast subpopulations in mouse colonic mucosa, each with unique molecular profiles and specialized functions. Some fibroblasts focus on ECM production and remodeling, whereas others show high contractility. Certain subsets of the fibroblasts secrete cytokines promoting epithelial differentiation or stem cell niche maintenance. Spatial mapping revealed their organization within the mucosa, and trajectory analysis suggested distinct differentiation pathways. Cell cycle scoring confirmed that fibroblasts remain largely non-proliferative under homeostasis. By integrating our dataset with published ones, we identify conserved fibroblast populations and propose a standardized nomenclature for intestinal fibroblasts. This framework enhances communication and understanding of fibroblast diversity and their roles in gut homeostasis and disease.

Lysosomes are the main digestive organelles and serve as a signaling hub linking environmental cues to cellular metabolism. Through these functions, lysosomes play a crucial role in maintaining cellular and organismal homeostasis. However, how lysosomal homeostasis itself is maintained is not well understood. Lysosomes are frequently damaged by a variety of substances, including crystals, silica, lipids, bacteria, toxins, amyloid proteins and reactive oxygen species. When lysosomes are damaged, their acidic contents leak out, leading to oxidative stress, inflammation and cell death. Damaged lysosomes are thus harmful to cells, and to restore lysosomal function after damage, cells have developed several defense mechanisms, collectively called the lysosomal damage response (or endo-lysosomal damage response). Recent studies have shown that this response is composed of three main pathways depending on the degree and duration of damage - repair, removal of the damaged lysosomes, and lysosomal biogenesis and regeneration. Growing evidence suggest that the failure and/or dysregulation of this response is implicated in aging and several diseases, including neurodegenerative diseases and kidney disease. In light of the rapid growth of this field, this Review summarizes our current knowledge of the lysosomal damage response, its significance in aging and diseases, and future perspectives.