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IOP Concise Physics

Introduction to Cellular Biophysics, Volume 1Membrane transport mechanisms

Armin Kargol

Chapter 1

Introduction to cells

1.1 Cell structure and chemistryAll living organisms are made of cells. Some are single-cell organisms where all lifefunctions are performed by that one cell, while in multicellular organisms the cellsmay be specialized for one particular function only. Groups of cells sharing a similarfunction form organs and the entire organism depends on all of its organs workingtogether. While it has been possible to isolate certain types of cells and grow themin vitro, normally the cells of multicellular organisms cannot survive outside theorganism, and a failure of one type of cell, or one organ, may lead to the death of theentire organism.

A cell is a small compartment isolated from its environment by a cell membraneand filled with concentrated solutions of various chemicals. When cells are studiedby different sciences, they are seen differently by biologists, chemists, or physicists.Biologists concentrate their attention on cell structure and function. What do cellsconsists of? Where are the organelles? What function does each organelle fulfil?From a chemists’ point of view, a cell is a complex chemical reaction chamberwhere various molecules are synthesized or degraded. The main question is howthese, sometimes very complicated, chains of reactions are controlled. Finally,from a physics standpoint, one of the main questions is the physical movement ofall these molecules between organelles within the cell, as well as their exchangewith the extracellular medium. The aim of this book is to look into the basicphysical phenomena occurring in cells. These physical transport processes facili-tate chemical reactions in the cell and that in turn leads to the biological functionsnecessary for the cell to satisfy its role in the mother organism. Ultimately, thegoals of every cell are to stay alive and to fulfil its function as a part of a largerorgan or organism. The first volume of this book is an inventory of physicaltransport processes occurring in cells while the second volume will be a closer lookat how complex biological and physiological cell phenomena result from these verybasic physical processes.

doi:10.1088/2053-2571/aaf86dch1 1-1 ª Morgan & Claypool Publishers 2019

1.1.1 The biology of a cell

Cells are surrounded by the cell membrane—a phospholipid bilayer whose role is toisolate the inside of the cell from the extracellular medium and to facilitate a controlledexchange of various substances between the cell interior and the environment. Cells areusually shown as compact objects with oval shapes. Their size can vary greatly. Typicalcells in multicellular organisms have dimensions of the order of 10 μm, but some can beas large as 1 mm. The exceptions are neurons which have complex shapes with multipledendrites and a single axon protruding from the cell soma. The lengths of some of theseprocesses can reach macroscopic dimensions, e.g. some human neurons have axons upto 3 feet long, and giant squid axons can have diameters of up to 1 mm.

Biology recognizes two types of cells: prokaryotic and eukaryotic. The former areall single-cell organisms. Their internal structure is simple—a single compartmentcontaining the intracellular fluid, the cytosol, surrounded by a plasma membrane.Their DNA is concentrated in a central region but it is not separated from the rest ofthe cytosol by any distinctive barrier. In addition to the plasma membrane,prokaryotes have a cell wall made of complexes of proteins and oligosaccharides.Its role is to protect the cell and provide some rigidity.

All plant and animal cells, including some unicellular fungi (e.g. yeast, mold) areeukaryotes. They differ from prokaryotes in having specific organelles where variouschemical and physiological processes are physically located. The organelles are boundedby internal phospholipid membranes which separate them from the rest of thecytoplasm. Here are the most important organelles and their role in the cell (figure 1.1):

• A cell nucleus surrounded by a double membrane contains all geneticmaterial.

• Glycoproteins and lipids are synthesized in endoplasmic reticula.• The Golgi apparatus is responsible for packaging proteins into Golgi vesicleswhich are then transported to their destination.

• Fatty acids and amino acids are degraded in peroxisomes.• Lysosomes, found in animal cells only, degrade worn out and foreign material.• Chloroplasts, found in certain plant cells only, are the location for photosynthesis.• The cytoskeleton is formed by interlinking filaments and tubules spanning thecytoplasm.

1.1.2 Some molecules involved

A cell contains a complex solution of various molecules differing greatly in size andin function. Some are structural, providing barriers separating various cellularcompartments (organelles) or rigidity, others participate in energy harvesting fromextracellular sources or energy transfer within a cell, yet others are for signaling orcontrol of various processes, etc. In order to understand the complexity of chemicalreactions occurring in cells we need to first review the types of molecules involved.

Phospholipids consist of two long fatty acyl groups (the tails) linked with a small,highly hydrophilic group (the head). They are characterized as amphipathic, that issome parts (the head) are hydrophilic (i.e. attracted to water) and other (the tails) arehydrophobic (i.e. tend to avoid contact with water) (figure 1.2).

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Due to this amphipathic nature, the behavior of phospholipids in aqueoussuspensions is rather interesting. They tend to aggregate in such a way that theirheads are exposed to water and their tails are shielded from direct contact withwater. They spontaneously form structures known as micelles, liposomes, or bilayers(figure 1.3).

This self-organization of phospholipids is an area of active research in biophysics,however, it is outside the scope of this book. Some of these structures have foundpractical applications. For instance, liposomes are used for cells transfection, i.e. theinsertion of DNA into cells. In one commercially used transfection method, asuspension of phospholipids and DNA in water is mixed and incubated. Theincubation process allows for a spontaneous formation of liposomes, which containin their cavity some of the dissolved DNA. When such liposomes are spread over acell culture, they fuse with the cell membranes, releasing their contents (including theDNA) into the cell. Another example of these phospholipid structures are bilayers,

Hydrophilic head Hydrophobic tails

Figure 1.2. A schematic drawing of a phospholipid molecule.

Figure 1.1. A typical animal cell, including a nucleus and some of the organelles (Reprinted by permissionfrom Springer Nature, from Friedman 2008).

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which serve as membranes separating cells from their environment and definingvarious organelles inside the cell. They are used in scientific research as artificialbilayers spanning a specially prepared support. It is worth noticing that thephospholipid bilayer is an example of a two-dimensional (2D) liquid. What thatterm means is that while bilayers form well-defined surfaces defining the boundariesof the cells, within these surfaces the molecules can move around, just like they do inordinary 3D liquids, e.g. like water molecules in a beaker of water. We will discussthese properties of cell membranes in the next section.

Proteins are single, unbranched chains of amino acids. The structure of a typicalamino acid is well known in biochemistry, and consists of amino and carboxylgroups, a hydrogen atom, and a side chain, all linked by a central carbon atom, as infigure 1.4.

While this basic structure is conserved among the different amino acids, theydiffer in their type of side chain. There are 20 types of amino acids commonly foundin proteins, and they may differ in their size, shape, electric charge, hydrophobicity,reactivity, etc. The different types of amino acids are labeled using either a one-letteror a three-letter code. For example, the amino acid lysine is denoted by K or Lys, thearginine is R or Arg, and so on. Amino acids of different types can bind together inan arbitrary order and form a long linear chain called a polypeptide, while the termprotein is usually reserved for a polypeptide with its 3D structure.

Figure 1.3. Typical self-assembled phospholipid structures: a micelle, a liposome, and a bilayer.

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When a polypeptide chain is synthesized, parts of it interact with other parts andwith the environment, ultimately turning this long linear molecule into a compact3D shape. This process is called protein folding and it is also a subject of intenseresearch that goes outside the scope of this book. In order to understand proteinstructure and shape, we distinguish four levels. The primary structure is the sequenceof amino acids in the polypeptide chain. It is usually presented as a chain of letters,each indicating the type of amino acid present at that particular location in thechain. The process of identifying the primary structure is known as proteinsequencing and can now be done by commercial labs. The secondary structuredenotes the local shapes in polypeptide chains, stabilized by chemical bonds. Thetwo most commonly identified secondary structures are α-helices and β-sheets. Theformer are formed when the chain turns into a helix and neighboring turns in thathelix bind to each other. The latter structures involve the chain folding on itself andthe two parts binding side-by-side, one in a reverse order. The tertiary structure isprobably the most important as it describes the overall 3D conformation of theentire molecule, showing where different α-helices and β-sheets are located and howthey position themselves within the molecule. The basic dogma of molecular biologyis that the primary structure determines the tertiary structure, which in turn decidesthe protein function. Our main goal is to understand the relation between the tertiarystructure and functions, as this would allow researchers to custom-design proteinsfor specific purposes. It is worth noting that the protein function often requiresdynamic behavior, i.e. changes in the 3D conformation that modify the molecule’sproperties. In the case of, e.g., ion channels such conformational changes bring thechannel from a closed state to an open one and vice versa. These changes will bediscussed in subsequent chapters. Finally, in the case of multimeric proteins,consisting of more than one polypeptide chain (multiple subunits), we also talkabout the quaternary structure of proteins to characterize the relative positions ofsubunits.

Proteins are crucial to the survival of all living cells as they are used in virtually allliving processes. To mention only some of these, proteins act as enzymes andantibodies, as structural elements in cells, and as transport mechanisms in cell

C

H

C H2N

R

OH

O

central carbon atom

carboxyl group

amino group

side chain

hydrogen atom

Figure 1.4. A structure of an amino acid. Among different amino acids the conserved parts are the centralcarbon atom (C), the carboxyl (COOH) and amino (H2N) groups, and the hydrogen atom (H). The differenceis in the type of side chain (R).

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membranes. They are involved in cell motility and in cell signaling, e.g. as receptors.We will discuss some of the specific proteins involved in membrane transport in laterchapters.

Nucleic acids. There are two types of nucleic acids: DNA—deoxyribonucleic acid,and RNA—ribonucleic acid. Both are linear polymers of nucleotides. A nucleotide isa molecule consisting of a five-carbon sugar—pentose (ribose or deoxyribose—hencethe names, RNA and DNA, respectively), an organic base molecule, and a number(1, 2, or 3) of phosphate groups (figure 1.5).

In DNA and RNA chains there are five different nucleotides with bases: cytosine (C),thymine (T), uracil (U), adenine (A), and guanine (G). Of the five only A, G, C, and Tare found in DNA, while only A, G, C, and U are found in RNA.

The basic role of DNA and RNA is to store and transfer genetic information usedto synthesize proteins. In other words, the sequences of nucleotides in DNA or RNAchains form codes for the protein primary structure, i.e. the sequence of amino acids.However, since there are 20 amino acids in proteins, while only four nucleotides inDNA or RNA, that coding cannot be 1:1. In fact it cannot be 2:1 since there are only16 different two-nucleotide sequences. The correspondence between nucleotides andamino acids is 3:1, i.e. each amino acid in a protein is represented by a sequence ofthree nucleotides in DNA or RNA. Since there are 64 such three-nucleotidesequences, there is a redundancy and the same amino acid can be coded by differentnucleotide sequences. For instance, the amino acid arginine (Arg) can be coded asAGA, AGG, CGA, CGC, CGG, or CGT.

The DNA and RNA molecules play a somewhat different role in proteinsynthesis. The processes involved are fairly complex but, simplifying things a little,we can describe DNA as used mainly for storage of genetic information, while RNAis used mainly for transfer. Typically, from stored DNA the cell generates a strand ofRNA by a process called transcription, and that RNA is used to synthesize proteinsin the so-called translation process.

Pentose

Nitrogenous base

Phosphate group

Figure 1.5. A schematic picture of a nucleotide. A sugar pentose (ribose or 2-deoxyribose) is linked with 1–3phosphate groups and a nitrogenous base. DNA chains are formed from nucleotides with deoxyribose and oneof the four bases: adenine, guanine, cytosine, thymine, while RNA chains consist of nucleotides with riboseand one of the following bases: adenine, guanine, cytosine, and uracil.

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AMP, ADP, ATP. Among all nucleotides a special role is played by adenosinetriphosphate ATP, and its ‘cousins’ adenosine diphosphate ADP and monophos-phate AMP. As nucleotides, they contain the base adenine and they differ in thenumber of phosphate groups they attach. Of the three the ATP is the most importantand well known as it is used to store and release energy in cells. The chemicalreaction describing this process can be summarized as

↔ + +−ATP ADP HPO energy.42

This reaction proceeding left-to-right is known as hydrolysis of ATP, while right-to-left the reaction is called phosphorylation of ADP. ADP phosphorylation requiresenergy from some other source, e.g. metabolism or photosynthesis, while ATPhydrolysis releases energy at the site of the reaction. By moving ATP moleculeswithin the cell, the energy can be transported from the site where it is harvested, e.g.from sunlight by photosynthesis, to a location where the energy is utilized.

Nutrients. Under this term we understand various molecules that are used inmetabolic processes, e.g. glucose or salts.

Waste products are the result of various, e.g. metabolic, chemical processes takingplace in the cell. An important example is urea, which must be removed from the cellto avoid poisoning.

Ions are the smallest chemical species present inside and outside the cell, since theyare, well, ions. The main physiologically relevant ions are K+, Na+, Ca2+, Mg2+, andCl−. Despite their chemical simplicity, ions are present in relatively large concen-trations, hence they are key factors in any diffusive or osmotic process in the cell. Forinstance, they are required to maintain the cell membrane potential and cellosmolarity, and play a crucial role in electrical signaling.

1.1.3 The subject of cellular biophysics

Whether it is a unicellular organism or a cell in a larger organ or organism, all cellsshare basic functions. These are:

• Maintaining internal composition and volume in response to changingextracellular conditions (homeostasis).

• Transporting nutrients in and waste products out of the cell.• Performing the cell’s physiological function, e.g. as a part of an organ ororganism, for the benefit of the whole. There is a multitude of specific cellularfunctions, including:

○ Electrical signaling (nerve cells).○ Mechanical excitation (muscle cells).○ Transport (e.g. enterocytes, erythrocytes).○ Secretion and absorption, etc.

The two main questions about cellular functions we ask from the point of view ofbiophysics are:

• What transport and control mechanisms does the cell use to survive in achanging environment and to function as intended?

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• What physical processes are at the root of biologically and physiologicallyimportant cellular functions?

We attempt to give at least a partial answer to these question in this book and in thecompanion volume 2. We need to emphasize, however, that the subject of cellularbiophysics is larger than this and some topics will not be discussed here. Forinstance, we will omit the topics of cell motility, i.e. their ability to move throughaqueous media, or molecular motors.

1.2 Properties of cell membranes1.2.1 Composition of cell membranes

The cell membrane and the membranes of organelles in eukaryotic cells arecomposed mainly of lipids. As we mentioned in section 1.1.2, one of the stableconformations of an ensemble of lipids in an aqueous environment is a lipid bilayer.Cell membranes are just such bilayers, typically 7.5–10 nm in thickness.

The most important lipids in membranes are phospholipids, described in section1.1.2. In a bilayer formation the hydrophilic heads of these amphipathic moleculesare in contact with the aqueous phase on either side of the membrane. Another classof lipids are steroids, in particular cholesterol, found in large amounts in themembranes of mammalian and plant cells. Cholesterol is absent from membranesof prokaryotic cells.

While lipids constitute 25%–75% of membrane mass, depending on the type ofcell, most of the rest consists of membrane proteins. We can categorize thesemembrane proteins based on their location within the membrane as:

• Peripheral proteins, which are relatively loosely attached to the face of thebilayer. They are water-soluble and easy to extract.

• Integral proteins, which are embedded deeply in the membrane, oftenspanning the entire thickness of the membrane. They are typically notwater-soluble but may be extracted using detergents.

or based on their function within the membrane as:• Structural proteins, which provide a degree of stability to the membrane oradhesion in multicellular structures.

• Transport proteins, which participate in transport of various other moleculesto and from the cells.

• Receptor proteins, which detect and bind certain extracellular or internalsubstances, initiating various cellular responses through this molecularsignaling mechanism.

The remaining, up to 5%, of the cell membrane mass is made up of hydrocarbons.They can be bound to membrane proteins, forming glycoproteins or to lipids asglycolipids. They increase the hydrophilic character of the lipids and proteins andhave a stabilizing effect on the membrane as a whole.

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1.2.2 The membrane as a dynamical structure

Self-assembly of a bilayer from phospholipids and proteins is studied usingthermodynamics and statistical physics methods, as well as various numericaltechniques (molecular dynamics, Monte Carlo methods, etc). One importantproperty of the phospholipid bilayer is that it behaves like a 2D liquid. This hasbeen demonstrated experimentally, e.g. in an experiment in which two cellscontaining different types of membrane proteins are fused together (Lodish et al2016). One of the proteins can be detected by fluorescent antibodies. Immediatelyafter fusing, one half of the composite cell shows the presence of fluorescent proteins,the other does not. After a sufficiently long time the fluorescent molecules diffusearound and cover the entire surface of the cell.

Certain membrane properties show some degree of temperature dependence. Insufficiently low temperatures the lipids’ hydrophobic tails are maximally extended(this is known as the ‘trans’ configurations) and the membrane has maximumthickness and a gel-like consistency. As the temperature is increased, a phasetransition to a fluid-like state occurs. Another factor influencing membrane fluidityis the cholesterol content. There is evidence of lipid rafts, i.e. areas of increasedmembrane thickness and rigidity, forming with the cholesterol content in them being3–5 times greater than in the surrounding membrane.

Finally, a phospholipid bilayer membrane which separates two fluids, theextracellular and cytosolic fluids, differing in their composition, is usually asym-metric. Integral proteins often have a specific orientation and the exoplasmic andcytosolic ends of these molecules have different properties. The concentrations ofcertain peripheral proteins (e.g. receptors) on both sides of the membrane may bedifferent. Also, the lipid concentrations of both leaflets in the bilayer can bedifferent. While in artificial bilayers lipids were never observed to flip-flop to theother leaflet, in natural membranes they can occasionally do so with the help ofspecialized membrane proteins—flippases.

1.3 Membrane transport processes and their significance to cellfunctions

The biochemical and biophysical picture of a cell that emerges from these sections isthat of a small ‘chemical factory’ where various reactions take place and the reagents aswell as products of these reactions are moved to different organelles or exchanged withthe extracellular medium. While the nature of these reactions is studied by biochemists,the transport of various molecules is the subject of cell biophysics. In addition tocharacterizing these transport processes, in this book we also aim at understanding howthey are coupled together and how they determine the observed physiological functions.

In general, transport processes can be separated into two classes, passive andactive. Passive processes result from random thermal motions of molecules. Activeones require energy input from some source, which is typically the ATP hydrolysis.The following transport processes will be discussed in detail in subsequent chapters,including the explanation of all the terminology used:

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• Diffusion through a lipid bilayer: while the bilayer forms an impermeablebarrier to most molecules, some substances, such as oxygen and carbondioxide, are soluble in lipids and, due to the fluid-like nature of the bilayer,can diffuse through it.

• Water channels: also known as aquaporins, these are protein-lined pores incell membranes. They facilitate diffusive flows of substances such as ureadown their concentration gradients or osmotic flows of water from less tomore concentrated solutions.

• Ion channels: also pores in cell membranes formed by proteins. They are ion-specific and usually gated (i.e. opened or closed) by some external gatingvariable, such as voltage, ligands, mechanical stress, light, etc. Ion channeltransport is driven by electrochemical potential gradients.

• Carrier-mediated transport: carriers are macromolecules (proteins) that bindsolute molecules on one side of the membrane, then the complex drifts or flip-flops to the other side, where the solute is released. This transport mechanismis reserved for larger solutes, such as sugars, amino acids, mononucleotides,phosphates, etc. From a functional standpoint there are different varieties oftransporters:

○ Uniport: transports one molecule at a time.○ Symport: transports two different solutes in the same direction.○ Antiport: also transports two different solute molecules but in opposite

directions.• Pumps: are similar to carriers in the mechanics of how they transport solutemolecules, mainly ions, but they require energy input, typically from ATPhydrolysis, to ‘pump’ these molecules against their electrochemical potentialgradient. They also come in uniport/symport/antiport varieties, and of coursethey are examples of active transport mechanisms. Some of the most commonexamples are:

○ Sodium–potassium pump: an antiport moving three sodium ions out ofthe cell for two potassium ions entering the cell.

○ Calcium pump: a uniport used to remove intracellular calcium.○ Sodium–calcium exchanger: really a hybrid mechanism between carriers

and pumps since it uses energy stored in the Na+ concentration gradientacross the membrane to fuel Ca2+ pumping against its gradient.

• Endo- and exocytosis: based on forming vesicles which fuse with the cellmembrane and release their contents. This mechanism is used to transportvery large molecules.

1.4 Experimental methods for membrane transportAll these transport mechanisms require different experimental methods to determinetheir properties. For instance, chemical methods involve direct measurements of theamounts of permeating solutes by periodically collecting liquid samples andanalyzing them with standard chemical techniques such as mass spectrometry orchromatography. We also use radioactively labeled solutes, radioactive tracers, in a

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manner similar to chemical methods. Fluid samples are collected and evaluated forthe amounts of radioactive tracers in them. Both methods are time-consuming andgive poor time resolution of the results. Optical methods rely on changes in theoptical properties of solutions with concentration, such as light absorption,fluorescence, the presence of dyes, or interference patterns. Cell volume changescan also be used to evaluate water transport rates in membranes. Finally, theelectrical methods rely on the presence of ions in physiological fluids and the voltagechanges and currents due to these ions. The most commonly used methods aremeasurements of electrical potentials in membranes and the patch clampingtechnique, in which the measurement of ionic currents in membranes providesinformation on ion flows. All these methods will be discussed in more detail asnecessary in the following chapters, and we refer the interested reader to the book byWeiss (1996).

ReferencesFriedman M 2008 Principles and Models of Biological Transport (Berlin: Springer)Lodish H et al 2016 Molecular Cell Biology (London: Macmillan)Weiss T 1996 Cellular Biophysics (Cambridge, MA: MIT Press)

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