Concepts of Biology (BIOL116) - Dr. S.G. Saupe; Biology Department, College of St. Benedict/St. John's University, Collegeville, MN 56321; ssaupe@csbsju.edu; http://www.employees.csbsju.edu/ssaupe/

Membranes, Diffusion & Osmosis

From a more humanistic point of view, individuality entered the world when the first membrane fragment wrapped itself into a closed shell and separated the interior components from the rest of the universe.

Harold Morowitz
Mayonnaise and the Origin of Life (1985)

I. Making Mayonnaise
    Do you know how to make mayonnaise? Simply mix vegetable oil, vinegar, and egg yolk. But, how do they form a nice smooth mixture since everyone knows that oil and water "don't mix". The secret is in the egg yolk which contains the phospholipid lecithin. You will hopefully recall that phospholipids like lecithin are amphipathic (or amphiphilic). In other words, they love both (amphi-) water and lipids because they have a both a hydrophilic side and hydrophobic side. The hydrophobic portion of the molecule essentially forms a shell around the vegetable while the hydrophilic side points outward toward the vinegar mixture. This effectively emulsifies the oil and results in a smooth mayo.

    Yeah, but this isn't a cooking class! What does mayo have to do with cell membranes and how cells exchange materials with the environment? Simple, the cell membrane has the same basic structure as mayonnaise.

    For more details check out the dialog by Gink and Go. Among other things in the dialog you will learn that forming a membrane was a very early step in the evolution of life on the planet. Also, check out the article by Morowitz, "Mayonnaise and the Origin of Life".

II. Cell Membranes

A. Brief History of Discovery

1670's - Leeuwenhoek (the "Founder of microscopy; Dutch lens-maker) was one of the first to recognize the existence of the membrane. He concluded that "something" separated a cell from its environment. He came to the correct conclusion even though his initial observations were with plant cells and he was actually looking at plant cell walls. He eventually realized that animals had no walls, but had a boundary, too.

1899 - Solubility studies suggested that both polar and non-polar materials passed through the membrane indicating that it must be made of a combination of hydrophobic and hydrophilic compounds.

1910 - Studies of red blood cell "ghosts" showed that the membrane was made of phospholipids and proteins.

1935 - Davson and Danielli proposed the "unit membrane" concept. They suggested that the membrane was essentially a sandwich stuffed with phospholipids between two slices of protein bread. This model explained many features of membranes including the typical structure observed in TEM photographs.

1972 - Singer and Nicolson proposed the currently accepted "fluid mosaic model" for the cell membrane.

B. The Fluid Mosaic Model - according to this model, the membrane is a bilayer of phospholipids with proteins randomly inserted in and on the bilayer.

1. Phospholipid Structure

• Glycerol backbone (C3)
• Fatty acids are esterified to two of the carbon atoms.
• The fatty acids vary in length from C14 - C24
• One fatty acid is usually saturated, the other one is unsaturated (especially in plants).
• The unsaturated fatty acid is "kinked" which prevents the phospholipids from packing too tightly and hence, the membrane remains more fluid at cool temperatures. In fact, the degree of unsaturation in the fatty acids in phospholipids is inversely correlated with temperature. Another way that membranes remain reasonably fluid is by incorporating cholesterol and other sterols. These hydrophobic molecules also break up the tight aggregation of phospholipids that would occur at cool temperatures.
• The fatty acids are hydrophobic (non-polar).
• Phosphate - is esterified to the third carbon.
• A polar group is attached to the phosphate. Choline is a common polar group. The unifying features of the polar groups are: (a) they have an electrical charge and (b) are hydrophilic.
• Together, the phosphate and polar group make up the "head" of the phospholipid.
• A good symbolic representation of a phospholipid is a two-tailed "sperm" structure where the head represents the hydrophilic phosphate and polar group and each of the two tails are the hydrophobic fatty acids.
• As mentioned above, phospholipids have a polar/hydrophilic part and a non-polar/hydrophobic region. Thus, they are amphipathic (or amphiphilic).

2. Phospholipid Arrangement

• The hydrophobic tail regions associate with one another as do the hydrophilic head regions aggregate.
• Phospholipids readily forms a thin film that self-seals to form vesicles.
• Liposomes are small, artificial vesicles created from phospholipids used to deliver drugs

3. Proteins

• peripheral (found on the outer or inner surface of the membrane)
• integral (protein found in the interior of the membrane. A transmembrane protein completely spans the membrane.
• The distribution of proteins is asymmetric; or in other words, different proteins exist on the inside and outside of the membrane. This was first discovered by freeze-fracture studies.
• Proteins on the inner surface are synthesized by free ribosomes and make their way to the inner surface of the membrane.
• Proteins on the outer surface are synthesized by ribosomes associated with the ER. The protein is partially secreted into the ER lumen. The portion inside the lumen is chemically modified by adding a sugar. The protein pinches off in a vesicle which migrates to the golgi and proceeds from the cis to the trans faces of the golgi. It pinches off again and migrates to the cell membrane where the vesicle fuses with the membrane and the protein becomes part of the membrane.

4. What's in a name?

• Fluid - the model is termed fluid because an individual phospholipid or protein can move within the bilayer. Some elegant experiments in which mouse and human cells with labeled proteins were fused and the redistribution of the proteins was followed.
• Mosaic - based on the apparently random pattern of insertion of proteins in the membrane as observed in TEM photographs.

5. This model accounts for many observations that had previously been difficult to rationalize with the Davson and Danielli model:

• membrane thickness is not uniform, it varies
• the membrane is thinner than predicted if all the proteins are on the outsides (for you trivia buffs, about 8000 membranes make up the thickness of paper)
• permeability studies: relatively permeable to water, lipid soluble substances, uncharged (non-polar) particles; relatively impermeable to large polymers, ions.
• consistent with freeze fracture pictures showing that there is an asymmetric distribution of membrane components
• protein and lipid content variable

III. The Surface of the Cell Membrane: Glycocalyx and ECM
    The surface of the cell membrane may be "sugar-coated". In other words, many of the lipids and proteins on the outer surface of the membrane may have sugar units attached to them. These molecules are called glycolipids and glycoproteins, respectively. The sugar coating serves several functions including: (1) acts as a cell recognition system and (2) allows cells to make contact with one another. The sugar coating is called the glycocalyx.

    Animal cells in particular may also have a gel-like network of carbohydrates and fibrous proteins surrounding them. The proteins include collagen and fibronectin. They are anchored by special protein receptors in the membrane called integrins. This network of external material is the extracellular matrix (ECM).

IV. Functions of the Cell Membrane. Among the many functions of the membrane are:

1. regulates cellular traffic into/out of the cell (discussed below)
2. serves as a boundary to separate the internal and external environments (check out the quote at the beginning of this section)
3. energy transfer reactions in other words, many of the reactions in the chloroplast and mitochondria rely on membranes (more on this topic in a later class).
4. support – the membrane can be considered to help maintain the structure of the cell
5. metabolism (many of the proteins in the membrane are enzymes)
6. cellular recognition systems – involved in immunological responses
7. connecting cells to one another (i.e., gap junctions, desmosomes, tight junctions)
8. transport – many of the proteins in the membrane act as transporters to move materials from one side of the membrane to the other. Other proteins pump (using the energy in ATP) materials into/out of the cell (more at the end of this lecture).

V. Permeability of the membrane 
    We know that cell is surrounded by a membrane and that when the cells exchanges materials with its environment these materials obviously must pass through the membrane. Is the membrane permeable to all substances? The simple answer – NO.

    First, a brief digression: membranes are called semi-permeable meaning that they allow the passage of some materials but not others. Let's use a goofy analogy – consider the "Bouncer" at a night club. The bouncer permits some individuals to enter the club but not others. Thus, the club membrane (doorway) is semi-permeable allowing some individuals to pass through freely while denying others. The properties of the individuals diffusing into the club determine who enters. The doors are permeable to those 21 and older but impermeable to anyone younger. Just like the night club, the chemistry of the cell membrane serves as a bouncer that restricts the entry of some individuals but allows free passage of others.

    So, what passes through the membrane? Membranes are freely permeable to water, lipid soluble substances, and uncharged (non-polar) substances. It might seem odd that water freely passes through a membrane since it is hydrophilic and the membrane is hydrophobic. However, the reason water passes through so readily is because it is small enough to sneak into the cell between the phospholipids. Membranes are relatively impermeable to large polymers, ions and charged molecules.

VI. Moving molecules - Molecules can move from one place to another by:

A. Bulk Flow - this is the mass movement of molecules in response to a pressure gradient. The molecules move from hi � low pressure, thus following a pressure gradient. A good example would be a faucet. When you turn a faucet on, water comes out. This occurs because the water in the tap is under pressure relative to the air outside the faucet. A toilet is another example; high pressure in the tank/bowl but lower pressure in the sewer system. Some molecular movements rely on bulk flow which requires a mechanism to generate the pressure gradient. For example, animals have evolved a pump (i.e., heart) that is designed for the bulk flow of molecules through the circulatory system. Although important for the movement of blood and sap in plants, this is relatively unimportant in moving materials across membranes.

B. Diffusion - the net, random movement of individual molecules from one area to another. The molecules move from [hi] � [low], thus following a concentration gradient. Another way of stating this is that the molecules move from an area of high free energy (higher concentration) to one of low free energy (lower concentration). The net movement stops when a dynamic equilibrium is achieved.

Imagine opening a bottle of perfume containing volatile essential oils in a very still room. Initially, the essential oils are concentrated in a corner of the room. As the molecules move randomly, in every different direction, over time they will eventually appear throughout the room. Ultimately the essential oils will reach a point, dynamic equilibrium, at which they are evenly distributed throughout the room. At this point the molecules are still moving. They continue to move randomly in every direction. The only difference is that there is no net change in the overall distribution of the perfume in the room.

Now imagine that the room is divided by a partition with holes (which is analogous to a membrane). If we place a drop of perfume on one side of the partition and then count at intervals the number of essential oil molecules on either side of the partition and graph the results:

plot # molecules vs. time on both sides of the partition.

We will observe that the number of molecules on one side will decrease while the other will increase until they reach dynamic equilibrium. Once at equilibrium the molecules continue to move back and forth from one side of the partition to the other and hence the number of molecules on either side of the partition at any given time is simply chance. The number oscillates about the midpoint.

A caveat - although this theoretical example helps us to better understand the nature of diffusion, it is technically wrong. The molecular movement attributed to diffusion in this example is really due to air movements in the room, or convection. True examples of diffusion are hard to come by (see Vogel, 1994; Wheatley, 1993). Nevertheless, it serves our purpose to illustrate the general concept of diffusion.

C. Osmosis - this is a specialized case of diffusion; it represents the diffusion of a solvent (typically water) across a membrane.

D. Dialysis – another specialized case of diffusion; it is the diffusion of solute across a semi-permeable membrane. Example – consider a cell containing a sugar dissolved in water. If water (the solvent) moves out of the cell into the surroundings it moves osmotically; if the sugar (dialysis) moves into the surroundings, it is an example of dialysis.

VII. Factors influencing the rate of diffusion - Several factors influence the rate of diffusion. These include:

A. Concentration Gradient - as previously stated, solutes move from an area of high concentration to one of lower concentration; in other words, in response to a concentration gradient. Although this is true for most solutes, it is NOT important for water. The concentration of water (55.2 - 55.5 mol L^-1) is nearly constant under all conditions (i.e., MW = 18 g/mol, and 1000 g/liter; thus, 1000/18 = 55.5 mol/L).

Fick’s Law - is an equation that relates the rate of diffusion to the concentration gradient (C1 – C2) and resistance (r). Diffusion rate, also called flux density (J, in units of g m² s^-1) can be expressed in the simplified version of the equation as:

J = (C1 - C2) / r

The take-home-lessons from this equation are that the rate of diffusion is:

1. directly proportional to the concentration gradient. The greater the difference in concentration between two areas, the greater the rate of diffusion. Thus, when the gradient is zero, there will be no net diffusion, diffusion will only occur so long as a concentration gradient exists;
2. indirectly proportional to resistance. In other words, the greater the resistance to diffusion, the lower the rate of diffusion. Resistance refers to anything that reduces the rate of diffusion such as the partition in our perfume example. The width of the partitions is a resistance; the wider the partitions, the lower the resistance. And, the membrane is a resistance to the movement of ions and other charged substances in or out of cells; and
3. inversely proportional to distance traveled (a function of resistance). For example, some typical diffusion rates for water are 10 um - 0.1 sec; 100 um -1 sec; and 1 mm - 100 sec.

B. Molecular Speed. According to kinetic theory, particles like atoms and molecules are in always in motion at temperatures above absolute zero (0 K = -273 C). The take-home-lesson is that molecular movement is: (1) directly proportional to temperature; and (2) indirectly related to molecular weight (heavier particles move more slowly than lighter, smaller ones). At room temperature, the average velocity of a molecule is fast - about 2 km/sec (=3997 mph!).

C. Temperature - increases the rate of molecular movement, therefore, increases the rate of diffusion

D. Pressure - increases speed of molecules, therefore, increase the rate of diffusion

E. Solute effect on the chemical potential of the solvent. Solute particles decrease the free energy of a solvent. The critical factor is the number of particles, not charge or particle size. Essentially solvent molecules, such as water in a biological system, moves from a region of greater mole fraction to a region where it has a lower mole fraction. The mole fraction of solvent = # solvent molecules/ total (# solvent molecules + # solute molecules). This is particularly important in the movement of water. Water moves from an area of higher mole fraction or higher energy to an area of lower mole fraction or lower energy.

VIII. Measuring Osmosis
    Osmotic concentration – refers to the number of osmotically-active particles of solute in a system. The movement of water is from a low osmotic concentration to a high osmotic concentration. I prefer to remember that water always moves from an area of higher energy (lower solute concentration; lower osmotic concentration) to an area of lower energy (higher solute concentration; higher osmotic concentration).

    A solution with a lower osmotic concentration than another could be said to be hypo-osmotic (think hypodermis – under the sink). In other words, the hypo-osmotic solution has fewer solute particles than the other. Conversely, the solution with more solute is hyper-osmotic relative to the other – it has more solute particles. Iso-osmotic solutions have equal osmotic concentrations.

    Now, consider an osmometer which is a device used to measure osmosis. In its simplest form it is constructed of a glass tube attached to a semi-permeable membrane. The system is filled with water and then immersed in a beaker with water. At equilibrium, the height of water in the tube will be at the level of the water in the beaker. At equilibrium, for every water molecule that diffuses osmotically into the membrane another diffuses out. Now, what will happen if we put some sucrose inside of the membrane sac? There will be a net movement of water from the beaker into the membrane sac. Water will move into the sac (from high to low energy, or from low to high solute concentration, or from low to high osmotic concentration). As the water moves in, what happens to the column of water? – right, it moves up the tube. As it does, the water pressure inside the cell increases. Water stops moving in when the pressure inside the cell balances out the tendency for the water to move into the cell. We say that the membrane sac has a higher osmotic pressure because of the tendency of water to move into it. From the beaker's perspective, since it is loosing water it will have a lower osmotic pressure. Finally, we can consider this entire process from the perspective of the pressure, or tonicity, on the membrane sac. As water moves into the sac, the increased volume of water causes the pressure on the membrane to increase. Thus, the sac is said to be hypertonic relative to the water in the beaker. Again from the beaker's perspective, the water is hypotonic to the sac.

IX. A comparison of terminology  
    Let's try to make some sense of all the terms we have used. Imagine that the table below represents a system filled with water and separated by a semi-permeable membrane (dotted line) that allows water to pass from one side to the other, but not solute. Side 1, the left column, is filled with pure water and side 2 with water containing a solute like sugar. Now, study the different terms. In all cases the water moves from side 1 to side 2. (If the membrane was permeable to the solute, it would move from side 2 to side 1) 

XI. Spuds McSaupe – Potatoes and osmosis - Now, let's meet "Spuds" and study his experiment.

(click here to check it out)

As a review: Consider three beakers, one filled with water, a second with a dilute sugar solution and the third with a concentrated sugar solution. Place a potato core in each of the beakers and allow them to incubate for awhile. In the beaker filled with water we will observe that the potato core swells up and becomes more turgid. The pressure has increased inside the cells because water has moved into the potato from the solution. The pressure on the membrane is called tonicity. Thus the potato has greater tonicity (or is hypertonic) than the water in the solution. Conversely, we can envision that the tonicity of the solution is less or hypotonic.

Now consider the core in the concentrated solution. Water will move out of the potato into the solution (because it is hypo-osmotic to the solution, or has more energy than the solution). As water leaves, the core shrivels and the membrane pressure decreases (it is hypotonic relative to the solution in the beaker). In the dilute sugar solution, there will be no change in the core – it will neither gain no loose water. It is said to be isotonic.

1. Facilitated Diffusion – diffusion into/out of cell at rate more rapid than expect. Doesn't use ATP. Transport proteins, like glucose permease, are involved. These proteins in the membrane provide a channel through which the substance will pass. Transport is in the direction of the concentration gradient. Thus, it doesn’t require an additional, outside energy input.
2. Active Transport – substances moved against a concentration gradient. Protein transporters in the membrane move substances, require energy. Na/K pump a god example.

XI. Vesicle Mediated Transport: 
    In these processes, small vesicles are involved with moving materials across the membrane. There are two basic types based upon the direction of movement:

1. Endocytosis – cells engulfs particles/materials from the environment. As the process occurs, the materials are put into vesicles that pinches off from membrane. Lysosomes fuse with the vesciles to digest the newly obtained materials. Phagocytosis (larger particles, cell-feeding) and pinocytosis (small particles, cell drinking) are examples.
2. Exocytosis – vesicles in cell fuse with membrane and expel contents. Mechanism for cell wall production and membrane synthesis.

XII. Junctions:

• Desmosomes – spot weld to strengthen membranes (i.e., epidermal cells)
• Tight junction – junctions to prevent leakage between cells (i.e., lining gut)
• Gap junction – Gap junction, intercellular communication between cells. Similar to plasmodesmata in plants.

References:

• Hebrank, MR. 1997. Reduce confusion about diffusion. American Biology Teacher 59: 160.
• Morowitz, H. 1985. Mayonnaise and the origin of life. In Mayonnaise and the Origin of Life. Berkley Book, NY.
• Odom, AL. 1995. Secondary & College Biology Students’ misconceptions about diffusion & osmosis. American Biology Teacher 57: 409 - 415.
• Vogel, Steven. 1994. Dealing Honestly with diffusion. American Biology Teacher 56: 405-407.
• Wheatley, Denys. 1993. Diffusion theory in biolgy: its validity and relevance. Journal of Biological Education 27: 181-187.
• Zuckerman, JT. 1994. Problem solvers’ conceptions about osmosis. American Biology Teacher 56: 22-25.