Membrane Organization Research Articles

A Survey of Models of Cell Membranes: Toward a New Understanding of Membrane Organization.

The cell membrane, the boundary that separates living cells from their environment, has been the subject of study for over a century. The fluid-mosaic model of Singer and Nicolson in 1972 proposed the plasma membrane as a two-dimensional fluid composed of lipids and proteins. Fifty years hence, advances in biophysical and biochemical tools, particularly optical imaging techniques, have allowed for a better understanding of the physical nature, organization, and composition of cell membranes. This has been made possible by visualizing membrane heterogeneities and their dynamics and appreciating the asymmetrical arrangement of lipids in living cell membranes. Despite these advances, mechanisms underlying the local spatiotemporal organization of membrane components remain unclear. This review surveys various models of membrane organization, culminating in a new model that incorporates nonequilibrium processes and forces exerted by interactions with extramembrane elements such as the actin cytoskeleton. The proposed model provides a comprehensive understanding of membrane organization, taking into account the dynamic nature of the cell membrane and its interactions with its immediate environment.

Cleavage of VAMP2/3 Affects Oligodendrocyte Lineage Development in the Developing Mouse Spinal Cord.

In the developing and adult CNS, new oligodendrocytes (OLs) are generated from a population of cells known as oligodendrocyte precursor cells (OPCs). As they begin to differentiate, OPCs undergo a series of highly regulated changes to morphology, gene expression, and membrane organization. This stage represents a critical bottleneck in oligodendrogliogenesis, and the regulatory program that guides it is still not fully understood. Here, we show that in vivo toxin-mediated cleavage of the vesicle associated SNARE proteins VAMP2/3 in the OL lineage of both male and female mice impairs the ability of early OLs to mature into functional, myelinating OLs. In the developing mouse spinal cord, many VAMP2/3-cleaved OLs appeared to stall in the premyelinating, early OL stage, resulting in an overall loss of both myelin density and OL number. The Src kinase Fyn, a key regulator of oligodendrogliogenesis and myelination, is highly expressed among premyelinating OLs, but its expression decreases as OLs mature. We found that OLs with cleaved VAMP2/3 in the spinal cord white matter showed significantly higher expression of Fyn compared with neighboring control cells, potentially because of an extended premyelinating stage. Overall, our results show that functional VAMP2/3 in OL lineage cells is essential for proper myelin formation and plays a major role in controlling the maturation and terminal differentiation of premyelinating OLs.SIGNIFICANCE STATEMENT The production of mature oligodendrocytes (OLs) is essential for CNS myelination during development, myelin remodeling in adulthood, and remyelination following injury or in demyelinating disease. Before myelin sheath formation, newly formed OLs undergo a series of highly regulated changes during a stage of their development known as the premyelinating, or early OL stage. This stage acts as a critical checkpoint in OL development, and much is still unknown about the dynamic regulatory processes involved. In this study, we show that VAMP2/3, SNARE proteins involved in vesicular trafficking and secretion play an essential role in regulating premyelinating OL development and are required for healthy myelination in the developing mouse spinal cord.

Fusion peptide induced modification of membrane organization and dynamics: Implications in developing fusion inhibitors

An essential step in the entrance of enveloped viruses such as coronaviruses, the human immunodeficiency virus, and influenza is membrane fusion. A unique class of glycoprotein found on the viral envelope, known as a fusion protein, is responsible for catalyzing the fusion between the viral envelope and the host cell. Efforts have been made to block the fusion vis-à-vis the entry process by inactivating the fusion protein utilizing peptides and small molecules. Interestingly, the fusion proteins do not have any structural and sequence homology across viruses, and this limited the use of inhibitory peptides against a specific virus causing a paucity of immediate solutions against emerging and re-emerging viral diseases. Therefore, research has been initiated to develop broad-spectrum fusion inhibitors that can be effective to inhibit the entry of many viruses. In this review, we have examined the mechanistic details of membrane fusion and the potential significance of lipid composition. Moreover, we discussed the rationale for developing peptide-based, broad-spectrum fusion inhibitors that target membrane physical characteristics instead of fusion protein inactivation.

The Membrane Phase Transition Gives Rise to Responsive Plasma Membrane Structure and Function.

Several groups have recently reported evidence for the emergence of domains in cell plasma membranes when membrane proteins are organized by ligand binding or assembly of membrane proximal scaffolds. These domains recruit and retain components that favor the liquid-ordered phase, adding to a decades-old literature interrogating the contribution of membrane phase separation in plasma membrane organization and function. Here we propose that both past and present observations are consistent with a model in which membranes have a high compositional susceptibility, arising from their thermodynamic state in a single phase that is close to a miscibility phase transition. This rigorous framework naturally allows for both transient structure in the form of composition fluctuations and long-lived structure in the form of induced domains. In this way, the biological tuning of plasma membrane composition enables a responsive compositional landscape that facilitates and augments cellular biochemistry vital to plasma membrane functions.

Metabolic switch from fatty acid oxidation to glycolysis in knock‐in mouse model of Barth syndrome

Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease‐specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid‐driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell‐derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V. Treatment of mutant cells with AMPK activator reestablishes fatty acid‐driven OXPHOS and protects mice against cardiac dysfunction.

Ultra high-speed single-molecule fluorescence imaging.

In two articles in this issue, Fujiwara et al. developed an ultrasensitive high-speed camera capable of single-molecule fluorescence imaging at a microsecond timescale (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202110160). This major leap in detection speed enables the organization of plasma membrane and integrin-based adhesions to be probed in unprecedented detail (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202110162).

Molecular mechanism of GPCR spatial organization at the plasma membrane.

G-protein-coupled receptors (GPCRs) mediate many critical physiological processes. Their spatial organization in plasma membrane (PM) domains is believed to encode signaling specificity and efficiency. However, the existence of domains and, crucially, the mechanism of formation of such putative domains remain elusive. Here, live-cell imaging (corrected for topography-induced imaging artifacts) conclusively established the existence of PM domains for GPCRs. Paradoxically, energetic coupling to extremely shallow PM curvature (<1 µm-1) emerged as the dominant, necessary and sufficient molecular mechanism of GPCR spatiotemporal organization. Experiments with different GPCRs, H-Ras, Piezo1 and epidermal growth factor receptor, suggest that the mechanism is general, yet protein specific, and can be regulated by ligands. These findings delineate a new spatiomechanical molecular mechanism that can transduce to domain-based signaling any mechanical or chemical stimulus that affects the morphology of the PM and suggest innovative therapeutic strategies targeting cellular shape.

Raman Imaging Reveals Insights into Membrane Phase Biophysics in Cells.

Cellular membranes are essential components of all living organisms. They are composed of a complex mixture of lipids with diverse chemical structures and crucial biological functions. The dynamic and heterogeneous nature of cellular membranes presents a challenge for studying their biophysical properties and organization in vivo. Raman imaging, particularly coherent Raman scattering techniques such as stimulated Raman scattering (SRS) microscopy, have emerged as powerful tools for studying cellular membranes with high spatial and temporal resolution and minimal perturbation. In this Review, we discuss the scientific importance and technical challenges of characterizing membrane composition in cellular contexts and how the advances of Raman imaging can provide unique insights into membrane phase behavior and organization. We also highlight recent applications of Raman imaging in studying cellular membranes and implications in diseases. In particular, the discovery of phase separation and a solid-phase intracellular membrane on endoplasmic reticulum is reviewed in detail, shedding light on the biology of lipotoxicity.

Surfactin molecules with a cone-like structure promote the formation of membrane domains with negative spontaneous curvature and induce membrane invaginations

Surfactin uniquely influences lipid bilayer structure by initially inducing membrane invaginations before solubilization. In this study, we exposed DOPC giant vesicles to various surfactin concentrations at different temperatures and observed surfactin-induced membrane invaginations by using differential interference contrast and confocal laser fluorescence microscopy. These invaginations were stable at room temperature but not at higher temperatures. Surfactin molecules induce membrane nanodomains with negative spontaneous curvature and membrane invaginations despite their intrinsic conical shape and intrinsic positive curvature. Considering the experimentally observed capacity of surfactin to fluidize lipid acyl chains and induce partial dehydration of lipid headgroups, we propose that the resulting surfactin-lipid complexes exhibit a net negative spontaneous curvature. We further conducted 3D numerical Monte Carlo (MC) simulations to investigate the behaviour of vesicles containing negative curvature nanodomains within their membrane at varying temperatures. MC simulations demonstrated strong agreement with experimental results, revealing that invaginations are preferentially formed at low temperatures, while being less pronounced at elevated temperatures. Our findings go beyond the expectations of the Israelachvili molecular shape and packing concepts analysis. These concepts do not take into account the influence of specific interactions between neighboring molecules on the inherent shapes of molecules and their arrangement within curved membrane nanodomains. Our work contributes to a more comprehensive understanding of the complex factors governing vesicle morphology and membrane organization and provides insight into the role of detergent-lipid interactions in modulating vesicle morphology.

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.

The bacterial lectin LecA from P. aeruginosa alters membrane organization by dispersing ordered domains

The dynamic reorganization of plasma membrane nanodomains (lipid rafts) plays a key role in host cell infection by human pathogens. Bacteria can induce the reorganization of lipid rafts as a result of the interactions between bacterial lectins and lipid raft glycosphingolipids (GSLs). However, it is still unclear how such interactions trigger the initial stage of the infection mechanism. Here, we study the GSL globotriaosylceramide (Gb3), a key receptor involved in the cellular uptake of the Gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via a lipid zipper mechanism. We mimic the plasma membrane using supported lipid bilayers (SLBs), containing liquid-ordered and liquid-disordered lipid domains. We observe that the liquid-ordered domains in the SLBs disperse upon interaction with LecA, accompanied by a deformation of SLBs surface. Our results link the membrane reorganization triggered by LecA with the P. aeruginosa infections in host cells.

Azospirillum brasilense AerC and Tlp4b Cytoplasmic Chemoreceptors Are Promiscuous and Interact with the Two Membrane-Bound Chemotaxis Signaling Clusters Mediating Chemotaxis Responses.

Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense, chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense. We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization.

ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

BackgroundMelanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet.MethodsAn immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes.ResultsThe immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin β3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation.ConclusionHuman melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

Single-mutations at the galactose-binding site of enzymes GalK, GalU and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-D-galactose and azido-functionalized Gb3 analogs.

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson-Fabry disease and an elevated level of deacylated Gb3 (lyso-Gb3) is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1,4βGal1,4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of GalK, GalU and LgtC enzymes, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes GalK/GalU generate UDP-6-azido-6-deoxy-d-galactose (UDP-6AzGal), which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the three enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy-d-galactose-1-phosphate, UDP-6AzGal and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V and LgtC-Q187S, respectively, is 3- to 6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6AzGal with ~90 percent conversion yields and products AzGlobotriose and lyso-AzGb3 with substrate conversion of up to 70 percent. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series.

CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

Effect of diphenylalanine on model phospholipid membrane organization

The influence of peptides over the thermodynamic and mechanic stability of lipid membranes is studied. To this end, diphenylalanine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine are selected as model systems for peptide and lipid bilayers, respectively. Vesicles at different peptide-lipid ratios were prepared and characterized using different experimental methodologies. Densitometry and differential scanning calorimetry were used to determine the influence of the peptide over the melting transition properties (temperature, volume and enthalpy) for bulk multilamellar vesicles, whereas viscoelastic and nanomechanical properties of supported vesicle layers and supported lipid bilayers were studied using quartz crystal microbalance with dissipation monitoring and atomic force microscopy, respectively. The influence of the sample preparation methodology -solvents for obtaining the lipid films and hydration media- is also characterized. The analysis of the experimental data reveals that diphenylalanine affects the lipid membrane, decreasing lipid molecular cooperativity in a concentration-dependent manner and inducing an overall weakening of membrane organization.

Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.

Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques.

Profiling drug-protein interactions is critical for understanding a drug's mechanism of action and predicting the possible adverse side effects. However, to comprehensively profile drug-protein interactions remains a challenge. To address this issue, we proposed a strategy that integrates multiple mass spectrometry-based omics analysis to provided global drug-protein interactions, including physical interactions and functional interactions, with rapamycin (Rap) as a model. Chemoproteomics profiling reveals 47 Rap binding proteins including the known target protein FKBP12 with high confidence. Gen Ontology enrichment analysis suggested that the Rap binding proteins are implicated in several important cellular processes, such as DNA replication, immunity, autophagy, programmed cell death, aging, transcription modulation, vesicle-mediated transport, membrane organization, and carbohydrate and nucleobase metabolic processes. The phosphoproteomics profiling revealed 255 down-regulated and 150 up-regulated phosphoproteins responding to Rap stimulation; they mainly involve the PI3K-Akt-mTORC1 signaling axis. Untargeted metabolomic profiling revealed 22 down-regulated metabolites and 75 up-regulated metabolites responding to Rap stimulation; they are mainly associated with the synthesis processes of pyrimidine and purine. The integrative multiomics data analysis provides deep insight into the drug-protein interactions and reveals Rap's complicated mechanism of action.

Antibody binding reports spatial heterogeneities in cell membrane organization

The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.

Molecular mechanisms of cell functioning in norm and pathology

In our study, the following are modern information about the structural and functional organization of cell membranes, the internal apparatus, as well as the cell organelle. The features of the functioning of the structural and dynamic genome are shown, including the main epigenetic mechanisms of gene expression regulation. The cell pathology section describes the main pathological processes that occur in cells after reversible and irreversible damage. The mechanisms and signaling pathways of the phenomenon, markers of cellular aging are highlighted, as well as the significance of this phenomenon in normal and pathological conditions. The main mechanisms of hypoxic and free-radical necrobiosis are described. The mechanisms, signaling pathways, and significance for the norm and pathology of two types of programmed cell death, autophagy and apoptosis, are discussed in detail. The prospect of using fundamental knowledge about the molecular mechanisms of life activity to improve the treatment of the most urgent types of pathology is shown.