Biology subjects

Kozlov, M. M.

Publications and source records attributed to Kozlov, M. M..

9 recordsLinked to original sources

bioRxiv2026

Seipin and perilipin 3 promote distinct early steps of nascent lipid droplet biogenesis

Lipid droplets (LDs) are lipid storage organelles with critical roles in metabolism. Cells form LDs in response to various metabolic stresses (1). LD biogenesis occurs in the endoplasmic reticulum (ER) membrane and begins when triglyceride (TG) in the membrane phase separates (2-4). After nucleation, nascent LDs grow by absorbing TG from the membrane (5,6). How cells determine when TG in the ER membrane is added to existing LDs or is used to form new LDs or is not known. Here, we developed the first LD formation assay that allows the synthesis of TG in membranes, mimicking what occurs in cells. LDs arise stochastically after TG production begins. LD nucleation is affected by TG production rate and membrane phospholipid composition. Membrane-embedded LDs suppress nascent LD formation by sequestering TG and destabilizing nascent LDs by Ostwald ripening. We show that cells overcome this suppression in two ways. The ER membrane protein seipin accelerates LD nucleation where it localizes in a membrane, probably by increasing the kinetics of TG phase separation. The LD coat perilipin 3 inhibits nascent droplet dissipation by ripening. Together, our findings reveal a minimal LD formation machinery, and the mechanisms cells use to control whether TG in the ER drives new LD formation.

cell biology↗

bioRxiv2024

Mechanism of membrane curvature generation and caveola formation by flat disc-like complexes of caveolin

Membrane sculpting by caveolin and accessory proteins is crucial for caveola formation. The discovery of the disc-like structure of caveolin oligomers has challenged earlier models for membrane shaping by caveolins. The flat shape of the caveolin discs apparently contradicts the large curvature of caveolar membranes these discs generate upon their predicted embedding into the cytoplasmic membrane leaflet. Here we have provided a mechanism for this phenomenon. We proposed that the central factor behind the membrane shaping by caveolin discs is a differential interaction of the membrane lipid monolayers with each other and with the hydrophobic faces of the caveolin discs. Based on this hypothesis we demonstrated by computations that the caveolin disc insertion causes elastic stresses of tilt and splay in the membrane monolayers, which, in turn, drive membrane kinking along the disc boundaries. The resulting membrane shapes are predicted to have a faceted appearance in agreement with observations and the estimated effective curvatures of these shapes are equal to those measured for caveolae in cells. We predicted and analysed the membrane-mediated repulsive forces developing between the inserted caveolin discs and discussed the strength of the counterforces needed to concentrate caveolin discs in the membrane plane. Besides recovering the major features of caveolar formation and morphology, our model provides a new mechanistic understanding of the role of cholesterol and other lipids with similar intrinsic curvature in the caveola assembly and control of caveolar size.

biophysics↗

bioRxiv2023

Mechanism of tension propagation in cell membranes

The propagation of the membrane tension perturbations is a, potentially, essential mechanism of the mechanical signal transduction along surfaces of live cells. The efficiency of this process is determined by the propagation speed, which turned to be a hot and a controversial topic of the Cell Biophysics. In a stark contrast to the earlier results and expectations, the recent studies in several cell types revealed a wide range of the tension propagation speeds beginning from the strikingly low ones challenging the significance of the process and up to relatively high biologically relevant rates. The previously suggested models of the tension propagation have been based on assuming an unrealistic softness of the membranes for the stretching-compression deformations, which challenges the model ability to account for the observations. Here, we consider a different physics of the generation and the propagation of tension perturbations in cell membranes. We propose the tension to be controlled by an intra-cellular pressure and the propagation of the tension perturbations to be mediated by a membrane area redistribution between compartments, to which cell membranes are divided by the proteinic barriers, according to the picket-fence model. Using the established elastic features of cell membranes including their effective non-stretchability, this mechanism quantitatively accounts for the slowness of the propagation process and gives a natural explanation of the wide range of the observed propagation speeds. The model predictions are amenable to a direct experimental verification by controlled osmotic pressure variations.

biophysics↗

bioRxiv2022

Self-Assembly of Tunable Intrinsically DisorderedPeptide Amphiphiles

Intrinsically disordered peptide amphiphiles (IDPAs) present a novel class of synthetic conjugates that consist of short hydrophilic polypeptides anchored to hydrocarbon chains. These hybrid polymer-lipid block constructs spontaneously self-assemble into dispersed nanoscopic aggregates or ordered mesophases in aqueous solution due to hydrophobic interactions. Yet, the possible sequence variations and their influence on the self-assembly structures is vast and have hardly been explored. Here, we measure the nanoscopic self-assembled structures of four IDPA systems that differ by their amino acid sequence. We show that permutations in the charge pattern along the sequence remarkably alter the headgroup conformation and consequently alters the pH-triggered phase transitions between spherical, cylindrical micelles and hexagonal condensed phases. We demonstrate that even a single amino acid mutation is sufficient to tune structural transitions in the condensed IDPA mesophases, while peptide conformations remain unfolded and disordered. Furthermore, alteration of the peptide sequence can render IDPAs to become susceptible to enzymatic cleavage and induces enzymatically activated phase transitions. These results hold great potential for embedding multiple functionalities into lipid nanoparticle delivery systems by incorporating IDPAs with desired properties.

biophysics↗

bioRxiv2022

Tetraspanin 4 mediates migrasome formation via a two-stage mechanism

Migrasomes are recently discovered signalling organelles, enriched with tetraspanin proteins (TSPAN)1. They form by local swelling of retraction fibers, the cylindrical protrusions of cell membranes that form as a result of cell migration along external substrates. Migrasomes can grow up to several micrometers in diameter2, and allow cells to release contents such as chemokines at specific locations, hence, transmitting signals to surrounding cells through the relevant chemokine receptors. Recently, evidence emerged showing that migrasomes play essential roles in fundamental cellular processes such transfer of mRNA and proteins3, organ morphogenesis4, and mitochondria quality control5. Thus, understanding the mechanism of migrasome biogenesis is of outstanding importance. Previously, it was established that the molecules crucial for migrasome formation are tetraspanin proteins and cholesterol forming macrodomains in the migrasome membrane, while the physical forces driving local swelling of the retraction fibers originate from membrane tension and bending rigidity1. Yet, it remained unknown how and in which time sequence these factors are involved in migrasome nucleation, growth, and stabilization, and what are the possible intermediate stages of migrasome biogenesis.

biophysics↗

bioRxiv2022

Chiral growth of adherent filopodia

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryo-electron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished the filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament crosslinker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

cell biology↗

bioRxiv2022

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature

Multiple membrane shaping and remodelling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in high copy numbers in every cell type. Prominent examples are tetraspanin4 and CD9 that are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here we revealed that tetrasapnin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodelling processes.

biophysics↗

bioRxiv2021

Model for ring closure in ER tubular network dynamics

Tubular networks of endoplasmic reticulum (ER) are dynamic structures whose steady-state conformations are maintained by a dynamic balance between the persistent generation and vanishing of the network elements. While factors producing the ER tubules and inter-tubular junctions have been investigated, the mechanisms behind their elimination remained unknown. Here we addressed the ER ring closure, the process resulting in the tubule and junction removal through constriction of the network unit-cells into junctional knots followed by the knot remodeling into regular junctions. We considered the ring closure to be driven by the tension existing in ER membranes. We modeled, computationally, the structures of the junctional knots containing internal nanopores and analyzed their tension dependence. We predicted an effective interaction between the nanopores facilitating the knot tightening and collapse of additional network unit cells. We analyzed the process of the pore sealing through membrane fission resulting in formation of regular junctions. Considering the hemi-fission as the rate-limiting stage of the fission reaction, we evaluated the membrane tensions guarantying the spontaneous character of the pore sealing. We concluded that feasible membrane tensions explain all stages of the ER ring closure.

biophysics↗

bioRxiv2018

Forces and constraints controlling podosome assembly and disassembly

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation, and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. Inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was however independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues.

cell biology↗