Biology subjects

Kovtun, O.

Publications and source records attributed to Kovtun, O..

9 recordsLinked to original sources

bioRxiv2026

ATF4 Coordinates Transcriptomic and Structural Adaptations in Aging Muscle

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

cell biology↗

bioRxiv2026

Microvascular architecture and dynamics of the choroid plexus brain barrier

The choroid plexus is a specialized blood-cerebrospinal fluid barrier that supports cerebrospinal fluid production, immune surveillance, and molecular exchange between the circulation and the central nervous system, yet its vascular bed remains poorly understood. Here, we combine whole-tissue clearing and light-sheet imaging, single-nucleus transcriptomic reanalysis, and live calcium imaging of intact murine explants to define the structural and functional organization of choroid plexus endothelial networks across development. Three-dimensional imaging and reconstruction highlights a dense, epithelial-ensheathed vascular plexus that is continuous with the broader cerebrovasculature and organized into anatomically distinct inflow and ventricular margin regions. Transcriptomic analysis identified developmentally stratified endothelial subtypes, with embryonic populations enriched for proliferative, motor, and membrane remodeling programs and adult and aged populations enriched for adhesion, extracellular matrix, transport, and mechanosensory pathways. Endothelial subsets across stages expressed genes linked to flow sensing and calcium-dependent mechanotransduction, including Piezo1, Piezo2, and Trpv4. Consistent with these signatures, intact explants exhibited spontaneous, spatially graded calcium oscillations, and pharmacologic activation by Piezo1, triggered robust network-wide calcium responses in embryonic and adult tissue with distinct temporal dynamics. Piezo1 activation also promoted stabilization of PECAM1-associated endothelial adhesion under ex vivo flow conditions. Together, these findings establish the choroid plexus endothelium as a structurally specialized, developmentally dynamic, and mechanosensitive vascular network and provide a framework for investigating endothelial contributions to blood-cerebrospinal fluid barrier function in health and disease.

neuroscience↗

bioRxiv2026

Stress-Encoded Mitochondrial Plasticity: ATF4 Control of Mega-Mitochondria and Nanotunnel Communication

Mitochondrial structural plasticity is a critical adaptive response to cellular stress, yet the transcriptional networks governing the formation of specialized mitochondrial architectures remain poorly defined. Here, we identified and demonstrated that activating transcription factor 4 (ATF4), the master regulator of the integrated stress response, directly regulates mitochondrial morphological remodeling through a novel ATF4-NRF1/Nrf2-MFN2 signaling axis. Using serial block-face scanning electron microscopy and three-dimensional reconstruction in Drosophila flight muscle, primary myotubes, and human skeletal muscle, we show that overexpression of ATF4 promotes significant mitochondrial elongation, increased cristae concentration, enhanced mitochondrial-endoplasmic reticulum contact site (MERC) formation, and the initiation of Mitochondrial Nanotunnels. In contrast, loss of ATF4 results in mitochondrial fragmentation and impaired aerobic capacity. Chromatin immunoprecipitation sequencing reveals direct ATF4 binding at the promoters of the genes encoding NRF1 and Nrf2, which in turn regulate MFN2 expression. Small-molecule inhibition studies further establish that activation of this hierarchical pathway is both necessary and sufficient for stress-induced mitochondrial structural adaptation. Together, these findings position ATF4 as a master regulator of mitochondrial architectural plasticity, providing a direct mechanistic link between cellular stress signaling and organelle remodeling.

cell biology↗

bioRxiv2025

The role of MICOS in modulating mitochondrial dynamics and structural changes in vulnerable regions of Alzheimer's Disease

Mitochondrial contact site and cristae organizing system (MICOS) complexes are critical for maintaining the mitochondrial architecture, cristae integrity, and organelle communication in neurons. MICOS disruption has been implicated in neurodegenerative disorders, including Alzheimers disease (AD), yet the spatiotemporal dynamics of MICOS-associated neuronal alterations during aging remain unclear. Using three-dimensional reconstructions of hypothalamic and cortical neurons, we observed age-dependent fragmentation of mitochondrial cristae, reduced intermitochondrial connectivity, and compartment-specific changes in mitochondrial size and morphology. Notably, these structural deficits were most pronounced in neurons vulnerable to AD-related pathology, suggesting a mechanistic link between MICOS disruption and the early mitochondrial dysfunction observed in patients with AD. Our findings indicate that the loss of MICOS integrity is a progressive feature of neuronal aging, contributing to impaired bioenergetics and reduced resilience to metabolic stress and potentially facilitating neurodegenerative processes. MICOS disruption reduced neuronal firing and synaptic responsiveness, with miclxin treatment decreasing mitochondrial connectivity and inducing cristae disorganization. These changes link MICOS structural deficits directly to impaired neuronal excitability, highlighting vulnerability to AD-related neurodegeneration. These results underscore the importance of MICOS as a critical determinant of neuronal mitochondrial health and as a potential target for interventions aimed at mitigating AD-related mitochondrial dysfunction.

neuroscience↗

bioRxiv2024

Molecular basis for the assembly of the Vps5-Vps17 SNX-BAR proteins with Retromer

Retromer mediates endosomal retrieval of transmembrane proteins in all eukaryotes and was first discovered in yeast in complex with the Vps5 and Vps17 sorting nexins (SNXs). Cryoelectron tomography (cryoET) studies of Retromer-Vps5 revealed a pseudo-helical coat on membrane tubules where dimers of the Vps26 subunit bind Vps5 membrane-proximal domains. However, the Vps29 subunit is also required for Vps5-Vps17 association despite being far from the membrane. Here, we show that Vps5 binds both Vps29 and Vps35 subunits through its unstructured N-terminal domain. A Pro-Leu (PL) motif in Vps5 binds Vps29 and is required for association with Retromer on membrane tubules in vitro, and for the proper recycling of the Vps10 cargo in Saccharomyces cerevisiae. CryoET of Retromer tubules with Vps5-Vps17 heterodimers show a similar architecture to the coat with Vps5-Vps5 homodimers, however, the spatial relationship between Retromer units is highly restricted, likely due to more limited orientations for docking. These results provide new mechanistic insights into how Retromer and SNX-BAR association has evolved across species.

cell biology↗

bioRxiv2022

FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation. 125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

cell biology↗

bioRxiv2020

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signalling, immunity, neuroprotection and virus infection. To function in different trafficking routes, retromer core (VPS26/VPS29/VPS35) assembles with a range of sorting nexins to generate tubular carriers and incorporate assorted cargoes. We elucidate the structural basis of membrane remodelling and coupled cargo recognition by assembling metazoan and fungal retromer core trimers on cargo-containing membranes with sorting nexin adaptor SNX3 and determining their structures using cryo-electron tomography. Assembly leads to formation of tubular carriers in the absence of canonical membrane curvature drivers. Interfaces in the retromer coat provide a structural explanation for Parkinsons disease-linked mutations. We demonstrate that retromer core trimer forms an invariant, evolutionarily-conserved scaffold that can incorporate different auxiliary membrane adaptors by changing its mode of membrane recruitment, so modulating membrane bending and cargo incorporation and thereby allowing retromer to traffic assorted cargoes along different cellular transport routes.

molecular biology↗

bioRxiv2020

Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cells plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 {beta}2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

molecular biology↗

bioRxiv2019

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions (DR1-3) that flank positively charged -helical regions (HR1 and HR2). Here we show that the three DR domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between DR and HR regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1 (CAV1), and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and CAV1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

cell biology↗