Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Introduction to Cell Biology
How Cells Are Studied
Studying the Chemical Environment in Living Cells

Classical Cell/15.html">Microscopy Methods provide insights into cellular architecture but do not offer detailed information about cellular chemistry. We have already discussed how Antibodies can be used to localize specific macromolecules within Cells. However, it is equally important to know the distribution and concentration of small molecules. Life can only be sustained through the rapid and precise Regulation of the concentrations of key metabolites such as ATP, glucose, and inorganic ions; the levels of these substances can vary significantly in different regions of Cells and Tissues. Moreover, since low-molecular-weight substances like cellular ATP, calcium, and hydrogen can act as intracellular messengers, it is crucial to be able to track changes in their concentrations in response to intracellular signals. In this section, we will discuss several methods borrowed from chemistry that allow us to determine the chemical conditions within living cells.

4.2.1. Nuclear magnetic Resonance (NMR) can be used to determine the chemical conditions in a population of living cells [14]

The nuclei of many atoms possess a magnetic moment; thus, like compass needles, they exhibit intrinsic magnetism. The magnetic properties of these atoms are influenced by their surrounding atoms. Nuclear magnetic resonance (NMR), which is non-destructive to living cells, allows the Determination of the Chemical Nature of a substance. If atomic nuclei with a magnetic moment are placed in a magnetic field, they align in one of several possible orientations. Each orientation is characterized by an energy level determined by the field strength and the chemical environment. When a collection of atoms in an identical chemical environment is irradiated with radio waves, the energy of these waves will be significantly absorbed if they have a highly specific frequency corresponding to the energy difference between the possible nuclear orientations in the magnetic field. This is known as the resonance frequency. A tissue sample contains atoms in various molecules and environments, and will absorb energy at different resonance frequencies. The absorption spectrum at these resonance frequencies for a given sample constitutes its NMR spectrum. Such a spectrum reflects the Structure and relative Abundance of each type of molecule containing magnetic nuclei.

In chemistry laboratories, NMR is widely used as an analytical technique to determine The structure of small molecules in solution. Advances in instrumentation have made it possible to apply NMR to The Study of biological systems. For example, the NMR signal from protons (hydrogen nuclei) is frequently used to study Proteins, Nucleic Acids, and other macromolecules in solution: interactions between different parts of a macromolecule affect the NMR spectrum, which therefore contains detailed information about Molecular structure and dynamics. Unlike X-ray crystallography, NMR does not require sample crystallization. However, to produce a meaningful NMR spectrum, molecules in solution must tumble rapidly. This explains the upper limit (about 20,000 daltons) restricting the size of macromolecules whose conformation can be effectively analyzed.

Only certain atoms have isotopes that yield a satisfactory NMR signal. To study macromolecules inside living cells, researchers commonly use the abundant isotopes 1H, ²³Na, ³¹P, ³⁹K, and the rare isotopes ¹³C and ¹⁵N. Given the crucial role that phosphorus compounds play in METABOLISM, ¹³P NMR spectroscopy is highly effective. This isotope is normally present in cellular phosphorus-containing substances. The signals it generates can be used to monitor Changes in the intracellular concentrations of compounds such as ATP and inorganic phosphate During Muscle contraction. NMR signals from the phosphorus isotope ³¹P are also useful for precisely measuring intracellular pH, because the resonance frequency of inorganic phosphate depends on its ionization state and, therefore, on the pH of the solution (Fig. 4-31).

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Fig. 4-31. ³¹P NMR spectrum recorded from a frog muscle (A - in a relaxed state, B - after 15 min of stimulated activity under anaerobic conditions, C - after 50 min of such activity). The five labeled peaks in the spectrum represent signals from P nuclei in different chemical environments: one peak corresponds to phosphocreatine, α, β, and γ to the three phosphate groups of ATP, and Pi to inorganic phosphate. In relaxed muscle, phosphocreatine is present in high concentration (A); it Functions as a reservoir of Free energy in Skeletal Muscle, as its phosphate is directly transferred to ADP to regenerate ATP hydrolyzed during muscle contraction. Consequently, in fatigued muscle, the phosphocreatine pool is depleted, and the concentration of inorganic phosphate (derived from ATP) increases accordingly. The P_i peak also shifts slightly to the left, reflecting A change in cellular pH due to the accumulation of lactic acid, a byproduct of Anaerobic Metabolism. From THE POSITION OF the Pi peak compared to a known standard, it can be calculated that in this case the pH shifted from 7.5 (A) to 6.4 (C). (Modified with permission from M.J. Dawson, D.J. Gadian, D.R. Wilkie, Nature, 274, 861-866, 1978. Copyright 1978 McMillan Magazine Ltd.)

The rare isotopes ¹³C and ¹⁵N are not normally present in cells in sufficient quantities, but they can be incorporated into specific macromolecules of biological importance. NMR can then be used to track their subsequent chemical transformation. If, for example, cells are grown in a medium containing ¹³C-labeled glucose, measuring the NMR spectrum of the sample over time allows the rates of many glucose-involving reactions to be determined. By using other ¹³C- and ¹⁵N-labeled compounds, it is in principle possible to trace the movement of carbon and nitrogen atoms through any metabolic pathway.

The main limitation of NMR is its low sensitivity. For example, to detect a compound using modern ³¹P-NMR techniques, a gram of living tissue must contain at least 0.2 mM of the compound of interest. However, many metabolites are present in living tissues at much lower concentrations. Furthermore, because acquiring a single NMR spectrum typically takes several minutes, rapid changes in cytochemical characteristics may go undetected. On the other hand, a major advantage of NMR is that it is non-destructive to living cells, making this method highly promising for cell biology.

4.2.2. Ion concentrations can be measured using intracellular electrodes [15]

To study individual cells, methods more sensitive than NMR must be used. One such method is based on an approach developed by electrophysiologists to study potential differences and currents across The Plasma Membrane. For this purpose, intracellular microelectrodes are prepared. They consist of fine Glass tubes with tip diameters measured in fractions of a micron; these micropipettes are filled with an electrically conductive solution (usually an aqueous solution of KCl). The tip of the microelectrode is inserted into the Cytoplasm through the plasma membrane, which seals around the capillary, fitting tightly against the glass so that The Cell remains relatively undamaged.

In the Study of Cellular contents, microelectrodes are used in two ways: they can measure the intracellular concentration of common ions, such as H⁺, Na⁺, K⁺, Cl^-, Ca²⁺, and Mg²⁺ ions, and they can also be used to inject molecules into cells. THE PRINCIPLE OF measuring ion concentration with a microelectrode is the same as in a pH meter. The tendency of ions to diffuse down a concentration gradient can be balanced by applying an opposing electrical field: the steeper the concentration gradient, the stronger the required electrical field. The magnitude of the electrical field needed to maintain a stable concentration gradient allows the determination of the ion concentration gradient. To determine the concentration of a specific ion, a barrier made of a material permeable only to that ion must be placed between a solution of known concentration and the solution being measured. The potential difference across this selectively permeable barrier in the absence of an external electrical field can serve as a measure of The ratio of the ion concentrations on either side of the barrier (see Section 6.4.15). In practice, the tip of the microelectrode is filled with an appropriate organic compound, creating a barrier selectively permeable to a specific ion. This microelectrode and a reference microelectrode are then inserted into the cell, as shown in Fig. 4-32.

Fig. 4-32. An ion-selective electrode can be used to measure intracellular ion concentrations. A. Experimental setup. B. Design of a K⁺-selective microelectrode. Typically, the tip of an ion-selective intracellular electrode is made of special glass or filled with a specific organic compound permeable only to certain ions. The rest of the tube is filled with an aqueous solution of the ion at a known concentration and contains a metal wire connected to one terminal of a voltmeter. Similarly, the other terminal is connected to an open-tipped glass reference microelectrode containing a standard conductive solution. Both electrodes are inserted through the plasma membrane into the cell under study. The voltage on the voltmeter corresponds to the potential difference across the selective barrier and reflects the ion concentration inside the cell (see text). Generally, larger cells are easier to impale with a microelectrode; if the cell diameter is less than 10 µm, the application of this method becomes more challenging.

Recently, microelectrode techniques have been adapted to study ion transport through specialized protein channels (also called Ion Channels) located in small patches of the plasma membrane. In this case, a glass microelectrode with a slightly thicker tip is required. Instead of penetrating the plasma membrane, it is pressed tightly yet gently against it (Fig. 4-33). This allows the recording of electrical Properties of the small membrane patch sealed to the tip of the microelectrode, which either remains attached to the cell or is isolated from it (Fig. 4-34). This method is known as "patch-clamp recording" (or patch recording). Its application has revolutionized the study of ion channels. It is the only method in cell biology that allows the function of a single protein molecule to be observed in real time; we will return to this topic in Chapter 6.

4.2.3. Rapid changes in intracellular ion concentrations can be measured using light-emitting indicators [16]

Ion-sensitive electrodes can only measure concentration at a single point within the cell. Furthermore, when ions are present at very low concentrations, such as Ca²⁺, the readings from such electrodes are often unreliable. However, monitoring changes in intracellular Ca²⁺ concentration is crucial for studying how cells respond to extracellular signals. These changes can be analyzed using light-emitting intracellular indicators. Some of these indicators are luminescent (emitting light spontaneously), while others are fluorescent (emitting light in response to excitation). For example, the luminescent protein aequorin, isolated from a marine jellyfish, emits light in the presence of Ca²⁺ and responds to changes in Ca²⁺ concentration in the range of 0.5–10 µM. If aequorin is injected into an egg which is then fertilized, the resulting change in cytoplasmic Ca²⁺ concentration can be detected by the flash of light emitted by the aequorin (Fig. 4-35). Recently, fluorescent indicators that bind tightly to Ca²⁺ have been synthesized. In their free state, they exhibit a different excitation or emission spectrum compared to their bound form. By measuring the ratio of fluorescence intensity at two different wavelengths, the relative Proportions of the free and Ca²⁺-bound indicator can be determined, allowing the concentration of free Ca²⁺ ions to be precisely calculated. Two well-known indicators of this type, quin-2 and fura-2, are used to continuously monitor changes in intracellular Ca²⁺ concentrations in different regions of cells using a fluorescence Microscope. Similar intracellular indicators have been developed to measure intracellular pH. Some of these can enter cells by diffusion, eliminating The Need for microinjection; this allows many individual cells to be observed simultaneously under a fluorescence microscope. The Development of new types of intracellular indicators, combined with modern Image Processing techniques, provides rapid and precise methods for measuring the intracellular concentrations of many small molecules.

Fig. 4-33. Micropipettes used for patch-clamp recording. A rod cell from a salamander retina is shown held by a suction pipette, while a fine-tipped glass pipette is pressed against the cell; the glass forms a tight seal with the plasma membrane and functions as a microelectrode. (From T.D. Lamb, H.R. Matthews, V. Torre, J. Physiol., 37, 315-349, 1986.)

Fig. 4-34. Four standard configurations of patch-clamp recording. The tip of the glass recording pipette is first pressed against The cell membrane to create a tight seal (top). The current flowing through the pipette in this membrane patch can be recorded while the patch remains attached to the cell (cell-attached configuration, A); the patch is detached from the cell, exposing its cytoplasmic surface (inside-out configuration, B); the membrane is ruptured by gentle suction, putting the electrode in direct contact with the cell interior (whole-cell configuration, C). The configuration shown on the right (D) allows recording of the cell's electrical properties similar to an intracellular electrode. In this case, the chemical environment inside the cell can be altered by introducing specific substances that diffuse into the cytoplasm through the relatively wide recording pipette. Configuration D arises from configuration B when the pipette is pulled away from the cell, and the membrane patch in contact with the electrode reseals. In configuration D, it is typically the extracellular, rather than the cytoplasmic, surface of the membrane that faces the electrode (compare with B).

Fig. 4-35. The luminescent protein aequorin emits light in the presence of free Ca⁺⁺ ions. Aequorin was injected into a fish egg, where it diffused through the Cytosol. The egg was then artificially fertilized and observed using an image-intensifying technique. Four photographs were taken from the side of sperm entry at 10 s intervals. A wave of Ca⁺⁺ ions, released from internal stores located just beneath the cell membrane, was observed in the cytosol. Starting from the site of sperm entry, this wave sweeps across the entire egg, as shown in the diagram on the left. (Photographs reproduced from J.C. Gilkey, L.F. Jaffe, E.B. Ridgway, G.T. Reynolds, J. Cell Biol., 76, 448-476, 1978. Copyright Rockefeller University Press.)

Fig. 4-36. Micrographs of a region of an early Drosophila embryo injected with rhodamine-labeled tubulin (tubulin is the microtubule protein). At this early stage of development, the nuclei share a common cytoplasm, so microtubules are labeled throughout the embryo. A. Microtubules in the living embryo radiate from two bright spots on either side of each interphase Nucleus; a centrosome is at the center of each spot. B. The same embryo a few minutes later, as all nuclei synchronously enter mitosis. The microtubules remain attached to the centrosomes but have reorganized to form the mitotic spindle. (Courtesy of Douglas Kel^-

4.2.4. Several methods exist for introducing membrane-impermeant molecules into cells [17]

Occasionally, There is a need to introduce membrane-impermeant molecules into cells. These may include light-emitting indicators (such as aequorin), fluorescently labeled cellular proteins, or molecules that influence cell behavior. One approach involves microinjecting molecules into cells using a glass micropipette. This is a highly effective technique, The Essence of which is as follows: a purified protein is coupled to a fluorescent tag and then injected into cells. Using an appropriate microscope, researchers can monitor The behavior of this protein during Cell Growth and Division (Fig. 436).

Although microinjection is a highly effective and widely used method, It is important to remember that each cell must be injected individually, which limits the number of cells that can be observed simultaneously. Other methods allow the simultaneous permeabilization of cell membranes in large populations of cells. This can be achieved using a powerful electrical discharge or chemical Treatment, such as a low-concentration detergent solution. The electrical discharge creates large Pores in the plasma membrane without damaging intracellular membranes. These pores remain open for several minutes or even hours, depending on the cell type and the intensity of the electrical exposure. Through these pores, even macromolecules can rapidly enter or leave the cytosol. With limited exposure, the membrane reseals in a large proportion of cells, allowing them to survive. A third method for introducing large molecules into cells involves fusing membrane-enclosed vesicles containing the desired molecules with the cell's plasma membrane. All three methods are widely used in cell biology (Fig. 4-37).

Fig. 4-37. Three methods are used for the intracellular Introduction of membrane-impermeant substances. A. The substance is introduced into the cell using a micropipette via hydraulic plunger pressure or the electrical charge of the injected molecules, causing the substance to enter The Cell as an ion current (iontophoresis). B. The cell membrane is disrupted by a short, powerful electrical discharge (2000 V/cm for 200 µs), allowing specific substances to enter the cell. C. Membrane fusion is utilized. At THE START OF the Procedure, membrane-enclosed vesicles (Liposomes) are prepared. The liposomes are then loaded with the desired substance by mixing a concentrated solution of this substance with a phospholipid suspension. Another modification of this method involves initially disrupting Erythrocyte membranes, causing them to lose their cellular contents, and subsequently placing the resulting 'erythrocyte ghosts' into a solution of the desired substance, where they are filled and their Plasma Membranes reseal. Both types of carriers (both liposomes and 'erythrocyte ghosts') can be introduced into target cells via membrane fusion mediated by specific viral proteins (synthesized by the virus to facilitate entry into cells).

Conclusion

Measuring the concentration and distribution of inorganic ions and other low-molecular-weight substances in Cells must be performed on intact living tissue. Nuclear magnetic resonance (NMR) is highly effective for this purpose. NMR is a completely non-invasive method used to measure the relative concentrations of many small molecules; unfortunately, however, its application requires a substantial Amount of sample. To determine the concentration of specific ions in individual cells or cell compartments, fluorescent indicator Dyes can be used. Glass microelectrodes are indispensable not only for measuring electrical potentials and ion flux across the plasma membrane, but also for determining the concentration of specific intracellular ions. Microelectrodes can also be used to inject membrane-impermeant molecules into cells. Alternative approaches involve temporarily permeabilizing membranes or fusing cells with membrane-enclosed vesicles containing macromolecules.