peroxisome-staining-mammalian-cells-using-peroxisome-specific

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization ...

University of Southampton

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes.

Bacteria and archaea deploy diverse, sophisticated defence systems to counter virus infection, yet many immunity mechanisms remain poorly understood. Here, we characterise the Kiwa defence system as a membrane-associated supercomplex that senses changes in the membrane induced by phage infection and plasmid conjugation. This supercomplex, comprising KwaA tetramers bound to KwaB dimers, as its basic repeating unit, detects structural stress via KwaA, activating KwaB, which binds ejected phage DNA through its DUF4868 domain, stalling phage DNA replication forks and thus disrupting replication and late transcription. We show that phage-encoded DNA mimic protein Gam, which inhibits RecBCD, also targets Kiwa through KwaB recognition. However, Gam binding to one defence system precludes its inhibition of the other. These findings reveal a distinct mechanism of bacterial immune coordination, where sensing of membrane disruptions and inhibitor partitioning enhance protection against phages and plasmids.

Kiwa is a bacterial membrane-embedded defence supercomplex activated by phage-induced membrane changes.

Peroxisomes carry out a diverse set of metabolic functions, including oxidation of very long-chain fatty acids, degradation of D-amino acids and hydrogen peroxide, and bile acid production. Many of these functions are upregulated on demand; therefore, cells control peroxisome abundance, and by extension peroxisome function, in response to environmental and developmental cues. The mechanisms upregulating peroxisomes in mammalian cells have remained unclear. Here, we identify a signaling regulatory network that coordinates cellular demand for peroxisomes and peroxisome abundance by regulating peroxisome proliferation and interaction with ER. We show that PKC promotes peroxisome PEX11b-dependent formation. PKC activation leads to an increase in peroxisome-ER contact site formation through inactivation of GSK3β. We show that removal of VAPA and VAPB impairs peroxisome biogenesis and PKC regulation. During neuronal differentiation, active PKC leads to a significant increase in peroxisome formation. We propose that peroxisomal regulation by transient PKC activation enables fine-tuned responses to the need for peroxisomal activity.

Protein Kinase C promotes peroxisome biogenesis and peroxisome-endoplasmic reticulum interaction.

Bacteria and archaea deploy diverse antiviral defense systems, many of which remain mechanistically uncharacterized. Here, we characterize Kiwa, a widespread two-component system composed of the transmembrane sensor KwaA and the DNA-binding effector KwaB. Cryogenic electron microscopy (cryo-EM) analysis reveals that KwaA and KwaB assemble into a large, membrane-associated supercomplex. Upon phage binding, KwaA senses infection at the membrane, leading to KwaB binding of ejected phage DNA and inhibition of replication and late transcription, without inducing host cell death. Although KwaB can bind DNA independently, its antiviral activity requires association with KwaA, suggesting spatial or conformational regulation. We show that the phage-encoded DNA-mimic protein Gam directly binds and inhibits KwaB but that co-expression with the Gam-targeted RecBCD system restores protection by Kiwa. Our findings support a model in which Kiwa coordinates membrane-associated detection of phage infection with downstream DNA binding by its effector, forming a spatially coordinated antiviral mechanism.

Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites.

Alpha-synuclein (aSyn) is an intrinsically disordered protein involved in phase separation and several age-associated neurodegenerative disorders, including Parkinson's disease. However, its function and pathological role remain elusive. Here, we modeled different aSyn assemblies in living cells by exploiting its interaction with synphilin-1 (Sph1). We developed a model that reports on gel- and solid-like inclusions through coexpression of aSyn and Sph1. Distinct morphological differences emerged between VN-aSyn + aSyn-VC and VN-Sph1 + aSyn-VC assemblies, showing unique antibody recognition, proteinase K resistance, and protein mobilities. The VN-Sph1 + aSyn-VC interaction could be manipulated to alter inclusion size and number. These inclusions also contained lysosomes and AP-1 vesicles, aligning with observations in human brain tissue. Our study offers new insight into aSyn aggregation and release, highlighting the importance of Sph1 and other aSyn-interacting proteins in synucleinopathies, which involve diverse copathologies only now beginning to be understood.

Article	Year
Fluorescent fatty acid conjugates for live cell imaging of peroxisomes. Nature communications\| PubMed ID: 38773129	2024
Kiwa is a bacterial membrane-embedded defence supercomplex activated by phage-induced membrane changes. bioRxiv : the preprint server for biology\| PubMed ID: 39896579	2025
Protein Kinase C promotes peroxisome biogenesis and peroxisome-endoplasmic reticulum interaction. The Journal of cell biology\| PubMed ID: 40689797	2025
Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites. Cell\| PubMed ID: 40730155	2025
Synphilin-1 modulates alpha-synuclein assembly, release and uptake. NPJ Parkinson's disease\| PubMed ID: 41266386	2025

Triana Amen

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