BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 4. MOLECULAR ORGANIZATION AND BIOLOGICAL FUNCTIONS OF MEMBRANES

4.4. Membrane Fusion during Exocytosis and Endocytosis

The discussed mechanisms of passive, primary, and secondary Active Transport are incapable of transporting macromolecules. A key feature of both exocytosis and endocytosis is the translocation of macromolecules that are secreted or engulfed within membrane vesicles. These vesicles can fuse only with specific membranes, ensuring targeted Transport of substances. The types and cytological aspects of exocytosis and endocytosis are covered in the "Cytology" course; therefore, we will focus on the biochemical and physicochemical mechanisms of membrane fusion. These processes are crucial because they occur during every instance of exocytosis and endocytosis, Cell and mitochondrial division, Fertilization, multinucleated Cell Formation, differentiation, and infection by enveloped Viruses. Artificially induced membrane fusion is widely used in various biotechnological and biomedical Applications. Since exocytosis and endocytosis are closely related processes, let us consider the fusion mechanism using mast Cells as an example. These cells and their secretory granules (containing histamine, heparin, and serotonin) are so large that the entire course of exocytosis can be captured using an Electron microscope.

As shown in the diagram (Fig. 4.20), in the initial state, secretory granules are located far from The Plasma Membrane. In response to an external stimulus, the granule comes into direct contact with the plasma membrane. In the contact zone, the plasma membrane invaginates toward the granule membrane, forming a dimple with a highly curved tip. The area of this contact is ~10 nm. It is precisely here that the fusion pore is formed, enabling the release of the secret from the granule into the extracellular space; thus, fusion is the merging of membranes with The formation of a pore. The walls of these pores are formed by highly curved lipid bilayers. Protein macromolecular structures containing Actin are located around the pore. It is assumed that these structures facilitate dimple formation.

The pore opens (forms) abruptly, with a radius of 0.2 nm. It flickers open and closed, and at a certain point, its diameter begins to increase rapidly. During this time, the surface area of the granule membrane increases significantly due to The transfer of ~ 10⁵ lipid molecules from the plasma membrane, meaning that the pore walls themselves are lined with Lipids. The driving force behind this lipid flow is the difference in surface tension between the plasma and granular membranes. The primary role in generating local tension belongs to Proteins, often referred to as membrane fusion proteins.

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Fig. 4.20. Diagrams of exocytosis processes (A), fusion of two flat BLMs (B), and stalk formation (C).

A: 1 - secretory granule, 2 - contact between the granule membrane and the plasma membrane, 3 - plasma membrane, 4 - dimple

(PM invagination toward the granule), 5 - fusion proteins, 6 - fusion pore. B - sequence of intermediate stages of fusion of two BLMs (1-4; explanation in the text). C - diagram of stalk formation and development (a-d; explanation in the text)

In studies of flat bilayer lipid membranes, it was possible to determine the distance between membranes at which their fusion occurs. The approach of two such membranes to each other (due to a hydrostatic pressure gradient) leads to a gradual thinning of the Water film between them until an equilibrium plane-parallel contact is established (Fig. 4.20; 2). This state is metastable, meaning that after a certain period, a spontaneous transition from state 2 to state 3 occurs. At this stage, the two bilayers merge into one. This process is called "hemifusion": the membranes merge through the fusion of only the outer monolayers, while the aqueous volumes remain separated. State 3 is quite stable, and further fusion processes occur only under the action of fusion factors, which include an electric field. Under METABOLISM/18.html">The Influence of such a field, membrane fusion is completed (state 4), meaning that both the membranes and the solutions are now merged.

Fig. 4.21. Diagram of low-density lipoprotein (LDL) endocytosis:

1 - LDL receptor in the plasma membrane (corresponds to the fusion proteins in Fig. 5.20); 2 - LDL; 3 - endocytic vesicle (clathrin coat of the vesicle is not shown); 4 - fusion with an endosome (invagination of the endosome toward the vesicle corresponds to the dimple in Fig. 5.20); 5 - endosome; 6 - peripheral endosome with a dissociated LDL receptor (dissociation is caused by the acidic environment); 7 - perinuclear endosome, which fuses with a transport vesicle (8) from the Golgi apparatus (* - enzyme), forming an endolysosome (9); 10 - transport vesicle detached from the peripheral endosome, containing the LDL receptor;

11 - invagination (dimple) of the plasma membrane

The hemifusion state is energetically favorable because the transition from state 2 to state 3 (Fig. 4.20) reduces the total surface area of the system; a single bilayer is obtained instead of two. Under the influence of thermal fluctuations, local bending deformations occur, resulting in perturbations (bulges) that expose the hydrophobic tails of lipid molecules to the solution. The attraction between such hydrophobic sites on adjacent membranes (Fig. 4.20, B, a) leads to the formation of a bridge (b, c) between them, termed a stalk. The latter is characterized by high energy because The Lipid Bilayer forming it is highly curved. Stalk expansion, accompanied by the fusion of distal monolayers (opposite to the monolayers where the stalk forms), leads to the formation of a contact bilayer (Fig. 4.20, d).

Thus, the fusion of Introduction/36.html">Biological Membranes, including during exocytosis and endocytosis (Fig. 4.20), is a multi-step process involving proteins and lipids. Proteins (primarily receptors) ensure local contact between the two fusing membranes. Fusion proteins generate additional tension (or bending moment) in the contacting bilayers, the rupture of which leads to the formation of a fusion pore.

Solubilization and reconstitution of membranes. Membrane research is based on two main methodological approaches: dismantling the membrane into its components and subsequent complete (or partial) restoration of its Structure. These two approaches are termed membrane solubilization and reconstitution.

The first attempts at membrane solubilization were made in the 1960s. Methods of mechanical disruption, ultrasonication, and The Use of organic Solvents were employed, which are now considered inadequate. Only after the introduction of detergents into membrane research practice was it possible to uncover the key secrets of membrane Structure and function.

Detergents (Latin: detergere - to wash, clean) are Surfactants that form stable colloidal solutions in water and adsorb at the water-air and water-oil interfaces, due to the amphiphilic nature of their molecules. In water, these substances form micelles containing up to a hundred molecules oriented such that their nonpolar groups form an inner Hydrophobic core, while their hydrophilic polar groups contact water molecules. It is precisely due to the presence of this hydrophobic core that detergent micelles are able to solubilize membranes, bringing substances that are practically insoluble in water into solution.

Several hundred different detergents of two classes are known (Table 4.5): ionic and non-ionic. Ionic detergents are divided into cationic, anionic, and zwitterionic (the latter possess both positive and negative charges, making the molecule electrically neutral overall). The main parameters characterizing the ability of detergents to form micelles are the critical micelle concentration (CMC) and the aggregation number. CMC is the lowest concentration of a detergent at which micelles begin to form. The aggregation number indicates how many molecules make up a single micelle. Detergents most commonly used for membrane solubilization are characterized by high CMC values (10^-4-10^-2 mol/L) and belong to "mild" detergents, which do not disrupt The activity of membrane Enzymes or cause significant Denaturation of Membrane Proteins.

Table 4.5

Examples of detergents most commonly used for membrane solubilization and reconstitution

Membrane proteins and lipids are also amphiphilic molecules. However, their CMC is 10«¹⁰ -10^-9 mol/L; they do not dissolve in water but aggregate instead. Therefore, in the presence of detergents and Membrane Lipids, a "conflict" arises: the detergent "wants" to adopt a micellar configuration, while the lipid "wants" to maintain its bilayer packing. When the detergent concentration is low, it only creates defects in the lipid packing, forming pores. As the detergent concentration increases, the defect in the lipid bilayer is enhanced. When the detergent concentration in the membrane reaches the CMC, the membrane is disrupted, forming mixed lipid-detergent and protein-detergent micelles, which are essentially a solution of membrane components in an excess of detergent (Fig. 4.22). This is the moment of solubilization. In the solubilized state, membrane proteins can easily be separated from the bulk of lipids, allowing the subsequent isolation of membrane proteins. For this purpose, methods such as Gel filtration, Chromatography, and Electrophoresis in an aqueous buffer are used.

Fig. 4.22. Membrane solubilization by detergents:

1 - membrane, 2 - detergent addition; 3 - partial membrane fragmentation; 4 - addition of detergent at a concentration above the CMC; 6 - detergent micelles. The addition of detergent leads to membrane disruption, forming mixed detergent-lipid (5) and detergent-protein (7) micelles. Leftward arrows indicate a decrease in detergent concentration, leading to membrane reconstitution

Membrane reconstitution is carried out using the same principles as solubilization. If the detergent concentration in mixed micelles is decreased, the micellar aggregates are converted into membranes (Fig. 4.22). Four methods of detergent removal are most commonly used: dialysis, gel filtration, dilution of the solubilizate, and detergent adsorption on hydrophobic polymers. Since detergent molecules are relatively small (Table 4.6), they freely pass through the pores of the dialysis membrane, and their concentration decreases. This method is suitable for detergents with a high CMC (sodium cholate). Non-ionic detergents such as Triton X-100 are not completely removed by dialysis; moreover, this method is too time-consuming. During gel filtration, the solubilizate is passed through an inert gel with a pore size large enough to accommodate detergent molecules but small compared to membrane particles, which pass freely between the gel beads. An even faster way to reconstitute a membrane is by rapid dilution. To do this, the solubilizate is diluted with a large volume of medium (without detergent); the detergent content in the micelles drops sharply, and the membrane is reconstituted.

Using these and other methods, almost all membrane proteins have been isolated, which has made it possible to elucidate the MOLECULAR MECHANISMS OF their functioning and intermolecular interactions in membranes, as well as to reconstitute the Functions of Cellular energy supply, metabolite transport, reception, and signal Transduction. Understanding the principles of reconstitution helps clarify how membrane biogenesis occurs and how to design membrane systems for Practical Applications, such as targeted drug delivery.

Thus, cell membranes are complex supramolecular structures. Their molecular complexes form an ordered liquid-crystalline mosaic, which provides membranes with their biological Specificity. By interacting with stimuli of various natures, membranes transduce their energy into the energy of biological cell excitation. This initiates the processes that ensure the spatiotemporal Organization of biochemical, physicochemical, and physical processes within the extremely confined volume of The Cell. Therefore, the Molecular organization of cell membranes remains one of the key areas of research in modern biochemistry.