Biology subjects

Markovic, V.

Publications and source records attributed to Markovic, V..

6 recordsLinked to original sources

bioRxiv2026

Integration of SYT1 Interactomics and Dual-Localization Proteomics Links ER-PM Contacts to Lignin Deposition

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

molecular biology↗

bioRxiv2026

Feedback between PI4P signaling and ER-PM contact sites orchestrates polarized root hair growth

Eukaryotic cells are composed of different organelles that communicate with one another through direct contacts, which are necessary for a host of cellular reactions and for responding to different developmental and environmental changes. Plasma membrane (PM) forms extensive contacts with the endoplasmic reticulum (ER) at specific sites named ER-PM contact sites. These contacts play crucial functions in lipid homeostasis, Ca2+ regulation and signaling in all eukaryotes. However, the mechanisms by which plant ER-PM contact site proteins tether to the PM, as well as the dynamics of these contact sites, remain poorly understood. Here, we investigate the importance of phosphoinositides in the establishment and dynamics of ER-PM contact site proteins in plants. We found that phosphatidylinositol-4-phosphate (PI4P), rather than phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), is required for the association of ER-PM contact site proteins with the PM. Furthermore, we identified a PI4P phosphatase, SUPPRESSOR-OF-ACTIN7 (SAC7), that associates with the ER-PM contact site protein SYNAPTOTAGMIN1 (SYT1) and regulates its dynamic association with the PM. In particular, we found that in growing root hairs, a highly polarized cell type, SAC7 removes SYT1-containing contact sites at the growing tip. Consistently, optogenetic induction of ER-PM tethering reduced root hair elongation within minutes of blue light induction. Altogether, we propose a link between SAC7-mediated regulation of PI4P, dynamic ER-PM contact site establishment and polarized cell growth in plants.

Plant Biology↗

bioRxiv2025

Plasticity induction in the ventromedial prefrontal cortex during REM sleep improves fear extinction memory consolidation

Anxiety disorders (ADs) are among the most prevalent mental health conditions, yet first-line treatments often yield only moderate effectiveness. The fear conditioning paradigm is commonly used to investigate fear and extinction learning, revealing deficits in these processes and dysfunctional activity in the ventromedial prefrontal cortex (vmPFC) and amygdala in individuals with ADs. The vmPFC plays a critical role in regulating activity of the amygdala and consolidation of fear extinction memory. Notably, transcranial direct current stimulation (tDCS) has shown promise in enhancing fear extinction by modulating vmPFC activity. Additionally, rapid eye movement (REM) sleep has been suggested to be crucial for fear extinction memory consolidation. This study investigated the role the vmPFC during REM sleep in fear extinction memory consolidation. Thirty-two participants underwent a 3-day differential fear conditioning paradigm, with tDCS or sham stimulation applied during REM sleep over the vmPFC. Outcome measures included skin conductance responses (SCR) and subjective ratings of arousal, fear, and valence. Results indicate that tDCS during REM sleep enhances fear extinction memory consolidation, as measured by SCR. Furthermore, participants reported an increased subjective arousal following tDCS. These findings suggest that tDCS during REM sleep may hold potential for improving exposure-based treatments for ADs by strengthening fear extinction memory.

neuroscience↗

bioRxiv2024

Vanillin flavor in e-liquids does not induce endothelial cell dysfunction: the Replica Project

BackgroundThere are challenges that require collaboration among researchers to ensure that tobacco harm reduction strategies are evidence-based. One key challenge is evaluating the safety of flavors used in electronic cigarettes (e-cigarettes). While many flavorings are approved as food additives or deemed "generally recognized as safe" (GRAS) for ingestion, this does not guarantee their safety when inhaled. In this context, the international research group Replica replicated a study conducted by Fetterman and colleagues in 2018, investigating the effects of aerosolized vanillin - one of the most popular flavors in vaping - on vascular endothelium when vaporized by an electronic cigarette. MethodsWe used Aspire Zelos 3 e-cigarette and prepared e-liquids containing propylene glycol, vegetable glycerin and vanillin. The e-liquids were vaporized under two settings - regular (1 ohm coil using wattage control mode at 14 watts) and sub-ohm (0.3 ohm coil using temperature control mode at 200 {degrees}C) - using a vaping machine, following the standardized puffing regime, ISO20768:2018. The vapor was then collected into a trapping solution to prepare aqueous extracts for the treatment of human aortic endothelial cells. We evaluated cytotoxicity, oxidative stress, nitric oxide bioavailability, and inflammation addressing some gaps reported in the original study. ResultsWe observed some harmful effects, mostly attributable to ethanol, used to dilute vanillin in the original work by Fetterman, but no harmful effects on cell viability, their ability to produce nitric oxide, or oxidative stress from vanillin. Furthermore, no pro-inflammatory effects of vanillin were observed in terms of ICAM-1 and IL-6 gene expression. ConclusionsOur results confirm the endothelial cell dysfunction observed in the original paper, but clarify that these effects are mainly attributable to ethanol and not to vaporized vanillin. These findings suggest that vanillin could be a safer flavoring agent for e-cigarette, without causing adverse effects on the cardiovascular system.

pharmacology and toxicology↗

bioRxiv2024

Concerted Transport and Phosphorylation of Diacylglycerol at ER-PM Contacts Sites Regulates Phospholipid Dynamics During Stress

ABSTRACTA universal response of plants to environmental stresses is the activation of plasma membrane (PM) phospholipase C (PLC) that hydrolyzes phosphatidylinositol phosphate (PIP) to produce soluble inositol phosphate (IP) and diacylglycerol (DAG). DAG produced in this way can be either phosphorylated by PM diacylglycerol kinases (DGKs) to produce the second messenger phosphatidic acid (PA) or transferred to the endoplasmic reticulum (ER) by the Synaptotagmin 1 (SYT1) protein at ER-PM Contact Sites (CS). In Arabidopsis, the clearance of DAG at the PM (avoiding deleterious accumulation) by the transfer activity of SYT is essential to maintain PM stability after stress. In this study we identify that DGK1 and DGK2 form a module with SYT1 at ER-PM CS through interaction of their C1 and C2 domains respectively. Global transcriptomic and metabolomic analyses confirms that SYT1 and DGK1/DGK2 are functionally related and lipidomic analysis supports the hypothesis that DGK1 and DGK2 function at the ER by phosphorylating DAG transferred by SYT1 from the PM. DGK1 and DGK2 show structural similarity to human DGK{varepsilon}, the DGK isoform that function at ER-PM CS in the phosphoinositide (PI) cycle. Our data indicate that components of the PI cycle are conserved between animals and plants and provide a novel mechanism leading to an increase in the efficiency of the PI cycle by channeling the transport and hydrolysis of DAG at the ER-PM CS.

plant biology↗

bioRxiv2019

EXO70A2 is critical for the exocyst complex function in Arabidopsis pollen

Pollen development, pollen grain germination and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to fertilize ovules. Pollen grains undergo two major developmental switches: dehydration characterized by metabolic quiescent state, and rehydration upon pollination that leads to extraordinary metabolic and membrane trafficking activity, resulting in germination and rapid tip growth of pollen tubes. To sustain these processes, many plant housekeeping genes evolved their pollen-specific paralogs. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we describe that EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in Arabidopsis pollen, which governs the conventional secretory function of the exocyst, analogically to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination and pollen tube growth. GFP:EXO70A2 was localized similarly to other exocyst subunits to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for the EXO70A1 function in the sporophyte, indicating functional redundancy of these two closely related isoforms. Phylogenetic analysis revealed that the ancient duplication of EXO70A to two (or more) paralogs, one of which is highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in the Arabidopsis pollen required for efficient plant sexual reproduction.

plant biology↗