Background
Odds are you have learned about diffusion and osmosis a few times before—but that doesn’t mean we shouldn’t revisit these topics. It turns out there is an extraordinary amount of cellular energy devoted to regulating these two processes. Cells are literally filled with protein-based highways that allow for the movement of molecules packaged up in vesicles (membrane bubbles), far more filled than any picture you’ve seen lets you imagine. Given this simple fact, let’s look at these processes a bit more.

Diffusion
Physics tells us a lot of what we need to know in this case. Following laws of thermodynamics, everything moves until we reach a particular temperature, absolute zero, at which point…other things happen. This movement is at random, thanks to some physics ideas, and we know that there is definitely an energy component.

Sometimes that energy component is easy to see – let’s add electricity during electrophoresis – but sometimes it’s not as obvious. There is energy associated with concentrations, or how much stuff you pack into a space. We can measure concentrations with various units you learned in chemistry. Sometimes the energy source is from a cellular molecule, such as ATP, which needs to undergo a chemical reaction in order to be accessed. There could be pressure gradients (gradient: difference), too. That’s wind! All of these processes involve a concept called free energy, which is the only driver of reactions. We just say “ok” and move on.

Image 1: Fundamental differences between passive and active transport

Part of diffusion is we go from states of order to chaos naturally. This is where we get the concept of concentration driving diffusion (though not perfectly accurate): a bunch of things in one location is order. But what happens? They move away and spread out. That’s chaos.

Image 2: Distinguishing between net diffusion and equilibrium

Moving into and out of cells
Recall cells are membrane bubbles that maintain their internal environment (saltwater) differently than the outer saltwater. The membrane is rather important and worthy of our analysis.

Our membrane – all cell membranes, actually – are composed of a phospholipid bilayer, with the outsides of this bilayer being attracted to water, and the middle layer being pushed away by water (hydrophobic is actually a misnomer of how the interactions occur). What does this do? We have a sort of bubble formed, where an insanely thin layer of fatty acids separate the saltwater environments. That’s how your cells are formed. That’s how all cells are formed. Is your mind blown yet?! Without that layer of fatty acids, no life as we know it.

Image 3: Diagrammatic view of the plasma membrane with a fluid-mosaic model approach

Some molecules are capable of getting close enough to this fatty layer and can slip through. Those are nonpolar molecules, typically fats that were capable of being transported in water with help, but also gases. These move according to their own diffusion gradients, and thus follow what is known as passive diffusion. We don’t really get much say in controlling these except by sequestering the molecules with other molecules. (Tada, now you know how we move oxygen and carbon dioxide in the lungs and circulation!)

Image 4: Fundamental differences between ionic/polar and nonpolar diffusion through the membrane

If you don’t turn out to be one of these molecules, you’re going to need assistance. For that, proteins (hey…a tie-in from last lab!) are necessary. These protein transporters are found stuck inside of the phospholipid bilayer and would be known as an integral protein. These transporters would be divided into two camps: facilitator proteins and pump proteins. Let’s look at each one.

Facilitator proteins serve to simply allow the movement of molecules along their free-energy gradients. These can be open all the time (“channel” proteins), or they can have an open-close switch (“gate” proteins). A famous example of a channel protein is called an aquaporin, which you could probably guess what it lets move: water.

Image 5: The aquaporin

Gate proteins typically can be opened or closed through the manipulation of their environment. How does this happen? As you recall, protein’s ability to fold depends on its environment – if we change the environment, we can change its structure (such as opening or closing a gate!). We can do this by changing the charges near the gate, or by adding a chemical that can force the gate open or closed. Easy.

Image 6: (a) Closed sodium- and potassium- ion channels, (b) Open potassium-ion channel

Pump proteins force the movement of molecules against their free-energy gradient. This is done by coupling the fight against free-energy (such as moving Na+ from low to high concentrations) with the breakdown of an energy-storage molecule like ATP (full name: adenosine triphosphate). Perhaps the most famous example of a pump protein is the Na+-K+ ATPase, or “sodium-potassium pump.” It’s diagrammed out below, but it runs like this:

1. Sodium ions bind to the protein (they have affinity for some sites);
2. ATP is hydrolyzed – breaks – to ADP and a phosphate that remains attached to the protein;
3. The break and addition of the phosphate forces the protein to change its shape and release sodium ions to the other side of the membrane (why do you think the sodium ions leave?);
4. Potassium ions now bind to the protein, where there is affinity;
5. The phosphate is cleaved off, forcing the protein to change its shape; and
6. The potassium ions leave onto the other side of the membrane (why?), and we repeat.

Image 7: The sodium-potassium pump, the most famous example of active transport

Diffusion across membranes can happen for any molecule. Sometimes we need more than just a protein to transport these molecules, which is when bulk transport is utilized. Inside the cell, we can rely on diffusion…but it’s really difficult. Cells are massive compared to their internal parts, so we can speed up diffusion by attaching molecules to transporters within the cell. You can look up vesicular transport yourself – it’s pretty amazing to watch. (Have you seen the video produced by Harvard about what goes on inside of a cell? No? Watch it!)

Osmosis
Everything can diffuse. When we deal with water, we call it osmosis. Knowledge does not transfer by osmosis, although it is – in a sense – moved by diffusion. () There are a few differences between osmosis and regular, run-of-the-mill diffusion:

1. Osmosis requires a semi-permeable membrane (hey…cells have that!);
2. Osmosis goes from high water potential to low water potential;
3. Water potential is dictated by pressure, temperature, the dissolved nature of solutes, the concentrations of solutes, location, etc.

Osmosis is such a big deal that there could be courses devoted just to it. However, we don’t have the time. But you know quite a bit about it, as you know how to dehydrate foods (add salt – draws out water – osmosis!), or force water to become purer (“reverse osmosis,” which isn’t a thing, but that’s what people call it), or why you shouldn’t go drinking salt water.

In Class
We are going to walk through four simulations today found online. They are

• Diffusion factors
• Diffusion across a semipermeable barrier
• Diffusion across semipermeable barriers
• Diffusion across a membrane

Diffusion factors
This is a PhET simulation, so it’s not necessarily as pretty as you’re used to with the previous set of simulations but they allow you to have a lot of control over the simulations. What we can see is how four different factors affect the rates of diffusion. These four factors are

• number of particles (concentration),
• mass,
• radius (size), and
• temperature.

The temperature is recorded in Kelvin (K), which is a number 273 more than the temperature in Celsius. That is, a room temperature of 22 C is the same as 295 K. Describe the resulting diffusion pattern when each of the following patterns are set. Make sure you set a “particle flow rate,” so you can see arrows that show where the net diffusion rate is by just modifying the blue particle. You must remove the barrier to see the results.

50 particles, 28 amu, 125 pm, 300 K
100 particles, 28 amu, 125 pm, 300 K
50 particles, 32 amu, 125 pm, 300 K
50 particles, 14 amu, 125 pm, 300 K

Now repeat but by modifying both the red and blue particles. Generate two distinct situations and describe the results. List your conditions for both types of particles in the table.

Blue Particle Red
Particle
Description of Results

Diffusion across a semipermeable barrier
This simulation is very similar to the first simulation, with the exception that there is no “the barrier is present, then it is gone” situation. You can adjust the pore size, as noted below, to the five positions shown. Using the pause function, record the number of green and blue particles in the left and right chambers.

Image 8: Settings for pore size adjustments

Complete the data table with your counts with the pore size position the particles at each location and time. I would recommend writing the number of green particles first, using a slash, then writing the number of blue particles (i.e., “5 / 2”).

Position Left chamber Right chamber
0 s 5 s 10 s 15 s 20 s 0 s 5 s 10 s 15 s 20 s
1
2
3
4
5

How would you best visualize these data? Why do you make that recommendation?

Generate your visualization of the data, as you recommended above, into the text below.

Diffusion across semipermeable barriers
You will notice this set up is very similar to the previous simulation, with the exception being there are two barriers not just one. Repeat the same set of experiments as above, but describe the results in the three chambers rather than make a quantitative observation. (You may include numbers if you wish, however.)

Position Left chamber Middle chamber Right chamber
1
2
3
4
5

Diffusion across a membrane
All of these simulations, so far, have been nice in that they are simple. But now let’s turn to how a true semipermeable membrane, such as the phospholipid bilayer, would serve with nonpolar molecules in their diffusion. This simulation looks at CO2 and O2, both are nonpolar gas molecules.

The initial levels are set three non-exact levels: none (N), low (L) and high (H); you can see relative concentration levels to the right of the simulation screen. There is no timer, so you would need to use your phone to serve as a timer for this simulation.

Record the relative concentrations (100%, 80%, 50%, etc.) of each of four setups below after letting the simulation run for 30 seconds. You may set the O2 and CO2 levels inside and outside of the cell be either N, L or H—but please record what conditions you selected.

Trial Initial (N/L/H) Final (%)
Description
O2 (out) O2 (in) CO2 (out) CO2 (in) O2 (out) O2 (in) CO2 (out) CO2 (in)
1
2
3
4

Follow-up questions
Complete the following questions regarding your experiences from this lab.
1. You just completed four simulations that manipulated various factors that would affect diffusion rates. What factors seemed to decrease the time to reach an equilibrium (where all chambers were approximately “equal” in concentrations)? Justify using your data!

2. In water, CO2 diffuses approximately 0.0016 mm2/s. The plasma membrane is, at most, 10 nm thick. Knowing that 1 mm = 103 m = 106 nm and assuming that CO2 diffusion rate could be approximated to be linear, how long would it take for CO2 to diffuse through the cell membrane? (Dimensional/unit analysis skills from chemistry are useful now!)

3. Eukaryotic cells in the human body average around 20 m in diameter – should the cell rely on diffusion alone for transport within the cell? Justify your response. You may assume essential nutrients to have a mass near that of CO2.

4. COPD, or chronic obstructive pulmonary disorder, is a collection of disorders that affect the respiratory apparatuses of the body. One of them, emphysema, reduces the amount of cell membrane within the lungs. How would this affect diffusion rates?

5. You were working with a recently discovered species of ants and interested in their cellular biology. Among the novel discoveries you make you notice they have a rather unique transmembrane protein that seems to be involved with regulating the movement of ions into and/or out of the cell. How could you determine if it is a pump protein or a channel/gate protein?