Biology subjects

Radulovic, M.

Publications and source records attributed to Radulovic, M..

5 recordsLinked to original sources

bioRxiv2026

Structure and function of the DFCP1 membrane barrier responsible for ER microdomain organization in lysosome repair

The endoplasmic reticulum (ER) forms specialized microdomains that organize autophagy, lipid droplet formation, and the repair of damaged lysosomes. The PI3P-binding protein DFCP1/ZFYVE1 marks these microdomains, raising the question as to whether and how one protein could define such a subcompartment. Here we determine the structure of full-length human DFCP1 bound to membranes by cryo-electron tomography and single-particle cryo-electron microscopy. DFCP1 assembles into a helical coat, built from tetramers that stack around a constricted membrane tube. Its ATPase domains form the organizing core of the helix, while its ER-targeting domains insert deeply into the outer leaflet, and its FYVE domains grip PI3P. Coarse-grained molecular dynamics simulations show that the coat does not simply follow the curvature of the membrane but acts as a barrier to lipid diffusion, almost completely blocking the movement of its cognate lipid PI3P past the filament and concentrating it beneath the coat. Point mutations that break the assembly, membrane-insertion or PI3P-binding interfaces each impair lysosomal repair in cells, and the assembly is also required for the focal recruitment of the lipid transporter VPS13C. We propose that DFCP1 collars fence off a specialized ER microdomain, so that locally made lipids can reach sites of repair rather than escaping into bulk ER.

cell biology↗

bioRxiv2025

Alizarin red perturbs skeletal patterning and biomineralization via Catalase inhibition

Alizarin Red S (AZ) is an anthraquinone dye that is commonly used in histological studies and textiles. Exposure to AZ results in morphological perturbations in several species, including rats, frogs, and dogs; however, the mechanisms by which AZ has these effects is largely unexplored, and little is known about its effect on development. We have previously shown that AZ is teratogenic to sea urchin larvae, and that AZ was the only calcium-binding mineralization marker among five tested that perturbed skeletal patterning. Here, we further characterize these defects and demonstrate that embryos exposed to AZ have abnormal skeletal element rotation, branching, and bending. Immunostains and polychrome labeling reveal delayed migration of primary mesenchyme cells and initiation of biomineralization. Although gross ectodermal dorsal-ventral specification, ciliary band restriction, and neuronal specification occur normally in the majority of AZ-treated embryos, we find abnormal patterning and connectivity of the serotonergic neurons. Temporal transcriptomics comparisons confirm delayed development and implicate changes in neuron-related GO terms with AZ treatment. Particle imaging velocimetry experiments show that ciliary beating normally directs fluid flow into the larval mouth, while AZ treatment perturbs the normal pattern of vortices and redirects flow away from the mouth. These changes in fluid flow have functional consequences for larval feeding behaviors in AZ-treated embryos. Finally, we show that the effects of AZ on skeletal patterning are largely due to its inhibition of catalase and subsequent elevation of reactive oxygen species. Specifically, catalase knockdown and transient hydrogen peroxide treatment are each sufficient to phenocopy the hallmark AZ-mediated skeletal patterning defects. This study is the first to define the teratogenic consequences of AZ exposure and show how its effects on catalase activity impact development, skeletal patterning, and biomineralization. Summary StatementAlizarin Red exposure produces teratogenic effects on skeletal and nervous systems primarily via catalase inhibition.

developmental biology↗

bioRxiv2024

CMTM4 is an adhesion modulator that regulates skeletal patterning and primary mesenchyme cell migration in sea urchin embryos

MARVEL proteins, including those of the CMTM gene family, are multi-pass transmembrane proteins that play important roles in vesicular trafficking and cell migration; however, little is understood about their role in development, and their role in skeletal patterning is unexplored. CMTM4 is the only CMTM family member found in the developmental transcriptome of the sea urchin Lytechinus variegatus. Here, we validate that LvCMTM4 is a transmembrane protein and show that perturbation of CMTM4 expression via zygotic morpholino or mRNA injection perturbs skeletal patterning, resulting in loss of secondary skeletal elements and rotational defects. We also demonstrate that normal levels of CMTM4 are required for normal PMC migration and filopodial organization, and that these effects are not due to gross mis-specification of the ectoderm. Finally, we show that CMTM4 is sufficient to mediate PMC cell-cell adhesion. Taken together, these data suggest that CMTM4 controls PMC migration and biomineralization via adhesive regulation during sea urchin skeletogenesis. This is the first discovery of a functionally required adhesive gene in this skeletal patterning system. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/616569v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b5564borg.highwire.dtl.DTLVardef@1347616org.highwire.dtl.DTLVardef@134d970org.highwire.dtl.DTLVardef@af863a_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Lysosome repair by ER-mediated cholesterol transfer

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by formation of contacts with the endoplasmic reticulum (ER), which depend on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

cell biology↗

bioRxiv2018

ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT) machinery are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX. Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens.

cell biology↗