Task 1

Cell membranes: phospholipids and more

In the late 1960s and early 1970s, a new model was proposed for the composition, architecture
and functioning of cellular membranes. It was called the "fluid mosaic model". The basis remained
the bilayer of glycero-phospholipids, but it provided a new explanation for presence of e.g. sterols
and proteins. Scientists also recognized that molecules in the membrane are not static, but highly
dynamic and constantly moving. This discovery (as always) gave rise to more questions than
answers and we ask ourselves, what are the exact roles and/or functions of all these membrane
components (Figure 1.1)?

Figure 1.1. Model of an animal cell’s plasma membrane (from Campbell Biology, 9th edition)

For example, sterols are abundant in the cell membranes of animals, plants, fungi, Protista, and
even in some bacteria (prokaryotes). The zoosterols, phytosterols (plant) and ergosterol (fungal),
are supposed to have similar functions in cellular membranes. The best-known sterol is cholesterol.
This is a molecule with a rather bad reputation since it was (and still is) seen as one of the main
culprits for cardiovascular disease in humans. There are many commercials advertising
supplements or “nutrient-drugs” to lower cholesterol levels in our blood. Apparently we can save
ourselves by reducing cholesterol intake and increasing sterols from plant or fungal sources (i.e.
phytosterols or ergosterol). However, many animals can produce their own cholesterol, and this
process is vital for survival. We know that these sterols are important precursors for hormones and
bile acids, but sterols in general have a vital function for the cellular membranes.

When it comes to function, proteins are very important constituents of the cell membranes. There
are different ways in which proteins can be integrated or associated with the membrane, and this
is related to a large variety of functions for membrane proteins. One of these functions is to regulate
transport of otherwise membrane-impermeable molecules across the membrane. This relies on the
precise control of a number of transport mechanisms for different types of molecules and
membrane proteins are very important.

For example:

Glucose uptake
The glucose molecule is membrane-impermeable, but is of vital importance for many cells as a
basic source of energy. For instance, in the intestines of animals, digestion of starch or cellulose
(polymers of glucose, remember the difference?) leads to the uptake of glucose residues into the
blood stream. Several membrane proteins play an important role in this process.

Action potential in neurons

A very important characteristic of plasma membranes is that they are an impassable barrier for
charged molecules and ions. This means that ion gradients can be generated over membranes.
Consequently, this results in a membrane potential, i.e. different quantity of charged molecules on
either side of the membrane.

The generation of an action potential requires tight coordination of a number of different transport
mechanisms to ensure conformity in action potentials. In Figure 1.2, you see a graphical
representation of an action potential: the membrane potential over time. Different membrane
transport processes can be attributed to each different phase of the graph (e.g. Na+ and K+
transport).

Figure 1.2. Graph of an action

potential. Plotted are the membrane
potential versus time.
[1] What is the fluid mosaic model? How is the membrane dynamic (key word!)?
[2] What are the membrane components and what are their functions? Make a table. Include
phospholipids, cholesterol, sphingolipids, proteins, glycoproteins, carbohydrates.
[3] For sterols, what is the difference between animal, plant and fungal sterols. Optional: is it true
that plant/fungal sterols can take on the function of cholesterol in mammalian membrane?
[4] What are the possible functions of membrane proteins?
[5] How are molecules transported over the membrane? What different types of membrane
transport are used?
[6] How are proteins involved in membrane transport of impermeable molecules?
[7] How is glucose transported over the intestinal cell membrane
[8] How is the action potential over a neuronal cell membrane regulated?
[1] They have to understand the dynamic and fluidic behaviour of the lipid bilayer. This is well
discussed and explained on pages 569-572. The fact that in most cases one of the two fatty acids
chains on the phospholipids contains unsaturated bonds, is important for the packing of the lipids
and thus the fluidity. (Figure 10-10 and 10-12). Figure 10-10 explains all the different types of
movement of the lipid molecules. There is information in the accompanying text.

[2] They have to be able to explain the figure in the manual of this task.
Lipid bilayer: Figures 10-1, 10-2 and 10-3. They have to know the basic build-up of the glycerol-
phospholipids, but not all the different head groups and fatty acids structures.
Phospholipids: basic structure of membrane; barrier function
Cholesterol: permeability, packing, fluidity (maybe – depends on the source)
Sphingolipids: lipid compound of the cell, also recognition of cell-cell etc.
Glycolipids: give hydrophilic layer on outside cell; sort of non-stick
[3] Structure of cholesterol and how it incorporates in the bilayer membrane, Figures 10-4 and 10-
5. On page 571 (halfway), there is an explanation of the effect of cholesterol in the membrane. The
role of the sterols in plants and fungi is identical or at least very similar. Apparently these sterols
can be integrated in mammalian membranes without the negative effects of cholesterol in the
bloodstream, (causing cardiovascular disease, other subject, not a topic of this task).

[4] Possible roles of membrane proteins:

- Cell attachment
- Transport
- Cell receptors for signal transduction (Task 9-10)
- Pores (transport)
- Channels (transport)
- And whatever sounds promising… let them guess a little

[5] Figure 11-1 is good, so that they know what molecules can freely
diffuse over the membrane and which need special “help” by means of
channels and/or pores (proteins).
Three different ones to know:
- Passive transport/diffusion, only downhill with the gradient, no energy
required, no saturation
- Facilitated diffusion, only downhill (electrochemical gradient), no
energy required, will saturate, is dependent on a limited number of
proteins in the membrane. Figure 11-6.
- Active transport, can also do uphill, against the gradient. Will consume
energy (can be ATP or H= gradient for instance) and will saturate
(dependent on pumps and transporters). Figures 11-7 and 11-8.

Maybe the students will come with coupled transport (symport and
antiport). This is good, they need it for one of the two examples (Figure
11-8).
[7] Glucose transport. Figure 11-9 and 11-11 with the figure legend. The trick is that glucose is
actively transported (against gradient, glucose in cell higher than outside) through a symport with
Na+. the concentration Na+ inside the cell is kept low by the Na-K pump (Figure 11-15, page 607-
608). This pump requires ATP, and this energy is stored in the Na+ gradient, which then is used to
transport the glucose in. So ATP is indirectly used for glucose transport.
[8] What happens during an action potential? There are ion channels involved, these are voltage
gated.

Pages 615-616 and 620-622 and further. Figures 11-29, 11-30 and 11-31.

Here the Campbell book comes in handy as well. Chapter 48, pages 1097-1099 (the students have
this book). Figure 48.11 is a nice explanation. They might spend some time on this, getting the
scheme on the board. You can take some time because the pre-discussion for Task 2 can be short.

K+ can leak from the cell via so-called leak channels. This leakage leaves behind unbalanced
negative charges (on large negatively charged biomolecules, e.g. DNA) and thus the K+ leakage
wills stop when the resting potential is reached. The text on page 615 will help here.

Task 2
Visualizing cells and subcellular structures
One of the major advances in studying cells and their structures is the development of ever-
improving microscope technology. There are several types of microscopy commonly used by cell
biologists. Each has its strengths and requires different considerations when preparing samples.
An understanding of the principles of microscopy is of vital importance for an experimental cell
biologist.

In Figure 2.1, you see examples of three major types of microscopy: brightfield, fluorescence, and
electron. Can you use these images to explain the different techniques? You should be able to
describe the following:

- What is the general set-up of the microscope? Can you draw a scheme?
- What is the cellular information you get from the technique?
- What are the requirements of sample preparation? How is optimal contrast achieved?

Figure 2.1. Microscopy images of different cells made with different microscopes. A) Brightfield microscopy, B)
Fluorescence microscopy, C) Electron microscopy.
A1: Brightfield micrograph of a plant stem stained with safranin (red) and fast green (green).
A2: Brightfield micrograph of testicular cells stained with haematoxylin (blue) and eosin (red).
B1: Mouse fibroblasts stained with phosphotyrosine antibodies (focal adhesions; green) and DAPI (blue).
B2: Mouse fibroblasts stained with phalloidin-TRITC (red) showing the cytoskeleton and DAPI (blue).
C1: SEM of yeast cells (Saccharomyces cerevisiae).
C2: TEM of a yeast cell (Saccharomyces cerevisiae).
 [1] Types of microscopy: brightfield and phase contrast microscopy, fluorescence
microscopy, electron microscopy (TEM and SEM). You can mention others, but don't go
into them.
[2] For each type of microscopy there should be several characteristics of the technique
discussed:
- What is the general set-up. Have them draw it.
- What cellular information can you get?
- What are the requirements of sample preparation. How do get contrast?
[3] From the lecture: The terms resolution and magnification should be clear.

[1-2] For brightfield and phase contrast microscopy (Figure 9-3), they
should realize that the light goes through the sample in this set-up. For
phase contrast, the light waves going through different parts of the
sample get out-of-phase or in-phase because of the different ways the
waves travel through different materials/media (Figure 9-4 + text); no
extreme detail here, it is not physics class. This means the sample has to
be thin. Also different breaking indices can lead to enhancement or
dimming of the light (in different forms of phase contrast microscopy; no
more detail required here). Samples may be stained to get more contrast
in the sample.
[1-2] For the fluorescence
microscopy (Figure 9-12 and 9-13):
the light comes from a laser or
halogen lamp (or these days, an LED)
and specific wavelengths are
selected with filters to illuminate the
sample. This light is then adsorbed
by the fluorescent compounds in the
sample. These then convert the light
that is sent out into a longer
wavelength (less energy; energy got
lost). This is then, via mirrors and
filters, let to the ocular/camera.

The sample does not have to be translucent or thin. The components in the sample that are of
interest are stained with a fluorescent component. This can be a specific compound that interacts
with proteins, lipids or enzymes. Alternatively it can be fluorescently labelled antibody (Figure 9-
17).

They do not have to go into the confocal fluorescence microscopy

[1-2] Electron microscopy (pages 554-560): TEM is Figure 9-41 (left), and SEM is Figure 9-50 (right).
The difference is that not light but a beam of electrons is used. This also means that special
cameras and detectors are used. One cannot look directly at the reflected or transmitted electrons.
.
[3] Resolution is the distance between two
points that can still be differentiated and seen as
two independent points. For EM, it is so much
smaller (such that smaller details can be seen)
because the wavelength is so much shorter.
Task 3
Cell walls: a target for toxins
Unlike cell membranes, cell walls are relatively static structures. They are especially important for
organisms that do not move or possess limited dispersal ability because their cells have to deal
with their immediate environment, and often they cannot relocate to a more favourable place.
Plants, fungi and many bacteria have therefore evolved this extra protective layer around the cell.
The cell wall not only protects cells from toxic compounds and nasty environmental conditions,
but it also maintains cell shape, e.g. when the cells are faced with hypotonic conditions or
mechanical stress.

The specific structure of the cell wall makes it an effective protective layer. For this, the cell wall
has to obey a number of “design” parameters: i) mechanical strength, ii) flexibility, iii) degradability
and possibility to extend, iv) porosity and permeability for nutrients, and v) use of “home-made”
abundant and “cheap” materials. Different components of the cell wall are responsible for the
different mechanical (or other) characteristics. The basic building blocks and precise architecture
may differ between plants, fungi, and bacteria, but the resulting cell walls have the same function.

Figure 3.1. Scheme (left) and scanning electron microscope (SEM) image (right) of a plant cell wall.

Penicillin
Organisms have also evolved to break down cell walls for food or to destroy competitors. A very
famous example is the fungus, Penicillium notatum. Alexander Fleming noticed that when his
bacterial plates got infected with this fungus, it created a zone in which the bacteria could not
grow. This observation eventually led to the isolation and characterization of the antibiotic penicillin.
It was a timely discovery since it prevented tens of thousands of deaths caused by infections of
wounds in the Second World War. It took much more research to discover how penicillin actually
worked.

Antifungal drugs
Fungi can also be the target of attack, as is the case with the synthetic drug called caspofungin.
This drug is used by Homo sapiens to combat several fungal species. In fact, humans are not very
fond of fungal infection when it involves themselves as the victim. It is fine growing on cheese, but
not on your organs! Since our impatient nature was not going to wait for the slow process of
evolution to develop fungal resistance, we employed organic chemistry. There is extra information
on EleUM.
 [1] What is a cell wall?
[2] Which organisms have cell walls?
[3] What is the composition of the cell wall?
[4] How is the cell wall built up and what is the resulting structure?
[5] What is the function of the cell wall?
- For [3-5], make sure they cover the differences between different organisms (bacteria,
plants, and fungi). Gram-positive AND gram-negative bacteria.
[6] How does penicillin work (in relation to the cell wall)?
[7] How does caspofungin work (in relation to the cell wall)?

[3-4] Plants: Pages 1081-1087.

- There is a primary cell wall of cellulose, pectin and cross linking glycans.
- Figure 19-63 is the figure in the task (but without text). They should come to something like:
Bacterial: Page 1267, Figure 23-3
- Composed of 1 or 2 bilayers and peptidoglycan shell
- Gram-positive: One bilayer and thick peptidoglycan outer shell
- Gram-negative: Two bilayers with thin peptidoglycan shell in between
- Gram-positive: Pentaglycine bridge connects tetrapeptides
- Gram-negative: Direct amide bond between tetrapeptides

The tetrapeptides linking adjacent backbone chains

contain an unusual γ-carboxyl linkage.
 The crosslink between tetrapeptides
in Gram-positive cell walls is a
pentaglycine bridge.

In Gram-negative cell walls, the linkage between

tetrapeptide segments involves a direct amide
bond.
Fungus
- Contains chitin and glucan
polymers (1-3 or 1-6 linked).
- Chitin is a polymer of N-acetyl-
glucosamine. This is a nitrogen
containing sugar, that also contains
an acetyl group. This polymer is very
tough and found in the exoskeletons
of insects and in fungal cell walls. It
makes the cell wall difficult to digest,
only when severe mechanical force
is used or when specialized enzymes
are available (chitinases: there are
acidic and basic forms that work
best at different conditions).
- There are not very many figures in the books we use, so some review articles are on EleUM.
 Task 4
Getting the orientation right: membrane proteins
Recall that proteins are synthesized by the ribosomes (Figure 4.1),
which ensure that the right amino acids are linked in the right order.
For membrane proteins, of which there are many types, it is critical
for their function (and that of the membrane itself) that the
integration into the membrane occurs properly.

After synthesis, the first challenge is insertion in the membrane

("translocation"). This is followed by transportation to the right
location in the cell ("compartmentalization" – for the next task). The
endoplastic reticulum (ER) has a central role in protein synthesis
because almost all proteins (whether destined for the exterior,
another organelle, or the ER itself) are first delivered to the ER Figure 4.1. Recall, protein synthesis.
lumen. There are several different mechanisms for getting a
protein into/over a membrane. The second challenge is that the right part of the protein has to end
up on the right side of the membrane.

One of the other major functions of the ER is the covalent addition of

oligosaccharides. For example, consider glycophorin (Figure 4.2), a
"single-pass transmembrane protein". This glycoprotein is present on
erythrocytes (red blood cells). It is very important that this
asymmetrical protein ends up in the outer membrane in the right
orientation. The glycosylated amino acids have to be exposed on the
outside, the extracellular space, because they are needed there for
their function. The glycosylation is a post-translational modification,
but does it occur before
or after membrane
insertion?

Also consider the membrane receptor

Figure 4.2. Glycophorin A
for adrenaline, a "multipass
transmembrane protein" that goes through the
membrane no less than seven times in animals (Figure
4.3). These receptors come up again in tasks 10 and 11. But how does a seven transmembrane
receptor end up with the correct orientation?

Figure 4.3. The beta-adrenergic receptor

 [1] What types of proteins are inserted into the membranes?
[2] What are the mechanisms of membrane insertion of proteins?
[2a] In general (broadly – not the focus of the task)
[2b] In the ER (in detail – the focus of the task)
[3] How do proteins get the right orientation?
[3a] Single-pass
[3b] Multipass
[4] How does the ER add oligosaccharides?
[5] How does the glycophorin get glycosylated? (its integration should be covered in [3a]
[6] How do seven transmembrane proteins get into the membrane in the right way? (overlap
with 3b)
[7] Optional: what are the functions of glycophorin and adrenaline receptors?
[8] Teaser: Can the transmembrane domains be predicted from the amino acid sequence?
(not required, but you can ask then at the end of the learning goal formulation).

[2] This is all explained excellently in the book on pages 669-685.

[2a] Know the general picture of co-translational translocation and post-translational translocation.
[2b] Figure 12.35 and 12.37 (below) explain how proteins are inserted into the ER.

[3a] Know the start-transfer and stop-transfer system (12-42) and the role of surrounding positive
charges (12-43) in determining the initial orientation. And even though we are talking about the ER
now, it's nice to know that this orientation is preserved even if transported elsewhere.
[3b] For multiple pass proteins this is shown in Figures 12-44 and 12-45. The trick is that the amino
acid sequence can contain several start and stop-transfer sequences. The other thing to remember
is that the protein can move away sideways out of the translocator. So the transmembrane domain
is pushed to the side, while the rest of the protein is produced by the ribosome still attached to the
ER or pushed through. This is well visible in Figure 12-39 (although this is about the initiation, but
it shows the translocator is not a solid pore; there is an opening to the side) and fig 12.42, 12.44.
[4] Glycosylation occurs in the ER lumen. Figure 12-47 gives a clear picture. The special lipid
dolichol carries a precursor oligosaccharide (composition not important) that is transferred to the
side chain of an asparagine residue, so it is an N-linked glycosylation. The glycosylation has a role
in folding and in the maturation of the protein. Also it is important for the correct transportation
from the Golgi to the target membrane (lysosomal proteins always have a mannose-6-phosphate
residue in the oligosaccharide chain).

Going further… The scheme in Figure 13-29 already says something on the maturation of
glycosylated proteins in the Golgi. This is important for the function and the targeting.

[5] Follow the principles of [4], but you can make it specific if the students want.
Schematic diagram of the interaction of Glycophorin A with Band 3 (B3). The diagram shows a
suggested topology for B3 with 12 membrane spanning segments and the proposed site of
interaction of GPA occurring with B3 between transmembrane domains 8 and 9. The close up of
the B3 and GPA interaction site shows the location of Wrb antigen residues on B3 and on GPA, the
approximate location of B3 Courcouronnes in TM 8 and also G701D in the bottom of TM 9.
Task 5
Endo- and exocytosis
For cells to survive, they have to take up materials/compounds from the environment via
invagination of the plasma membrane in a process is called endocytosis. There are multiple types
of endocytosis and different pathways that define them. Of course, cells also have to secrete waste,
and in the case of multicellular organisms, they also secrete a wide range of molecules to be used
by the other cells. This process of secretion is called exocytosis, which also has different types
and mechanisms. You may know something about exocytosis in the context of neurobiology – but
that's not the focus point here.

For all of this to work, there are highly specific mechanisms of cargo packaging, transport, and
delivery. These transport vesicles are constantly budding off one membrane and fusing to another.
It is of critical importance that transport of materials and vesicles is faultless. For example,
lysosomal enzymes being sent to the mitochondria would have terrible consequences for the cell!

Figure 5.1. A "road-map" of the secretory and endocytic pathways. Green: endocytic, blue: retrieval
pathways, red: secretory.
 [1] What are the types of endocytosis?
[2] How does endocytosis work?
[3] What are the types of exocytosis?
[4] How does exocytosis work?
[5] Other than the above, how do transport vesicles pack cargo, transport and delivery
cargo?
[6] How is the system "faultless"?
[7] How do lysosomal enzymes get transported?

[1] - Endocytosis: Uptake of

material by an invagination of the
plasma membrane and its
internalization in a membrane
bounded vesicle. There is "normal”
endocytosis and receptor-
mediated endocytosis, in which
specific membrane receptors bind
the material and concentrate in the
“pit” formed just before
invagination and internalization.
- Transcytosis: transport of vesicles
with material from one side of the
cell to another side. This to transport material through a cell layer.
- Phagocytosis: Process by which (unwanted) cells, debris, and other bulky particulate material is
endocytosed. Prominent in carnivorous cells such as Amoeba Proteus, and vertebrate
macrophages and neutrophils.
- Pinocytosis: Literally, “cell drinking”. Soluble materials are continually taken up from the
environment in small vesicles and moved into endosomes along with the membrane bound
molecules.
[2] How does endocytosis work? Starts on page 730.
The students should know the general ideas in 13-47. Endocytic
vesicles fuse near the cell periphery with an early endosome (the
primary sorting station). Then late endosomes move on
microtubules (a topic of the next task) to the cell interior. The TGN
provides a continuous supply of newly synthesized lysosomal
proteins throughout this process. Figure 13-61b can be used as a
teaser for the next tasks. They're about to learn about actin.

Cover clathrin-mediated endocytosis.

Figure 13-8 and 13-13. The assembly of the clathrin coat introduces curvature into the membrane,
which leads in turn to the formation of a coated bud (called a coated pit if it's in the plasma
membrane, like for endocytosis). The adaptor proteins bind both clathrin triskelions and
membrane-bound cargo receptors. This makes it selective. Membrane-bending and fission
proteins (e.g. dynamin GTPase) are recruited to the neck of the budding vesicle. Note: severing the
vesicle costs GTPase. Afterwards, the coat is rapidly lost and the vesicle goes on as an early
endosome fusing to form late endosomes and being targeted to the target. Often this is the
lysosome for the degradation of the material ingested. The pH is already decreasing in the
endosomal compartments, which decreases the affinity of the receptor for the cargo, so the
receptors release the cargo before the endosomes fuse with the lysosomes. This way the receptors
can be recycled to the plasma membrane for another use.
Also look at: Figures 13-48, 13-50, 13-52, 13-56, 13-58.

Receptor-mediated endocytosis.
The LDL (low-density lipoprotein)
receptor is a well-studied example.
Note that the endosomes don't all go
to the lysosome. Ask the students:
where do the endosomes end up?
Most at the lysosome but also many
at the plasma membrane (recycling
or transcytosis). This also means that the endocytotic vesicles have V-snare proteins on their
surface. This to make the fusion with their target membranes possible (these will have the T-snare).
The rab proteins are important determinants for what vesicles go where, and the Rab5A is
associated with the clathrin coated vesicles coming from endocytosis.
[3] For exocytosis, the information is on page 741-750. Especially figures 13.62, 13,63, 13.64,
13.65, 13.70 (empty vesicles), and 13.68 (exocytosis in synapse).

How are vesicles targeted to the plasma membrane?

The secret must lie in the cargo and the coating around the vesicle. The exact composition of the
lipids, the precise phosphatidyl-inostitol types present in the membrane of the vesicle, are typical
for secretory vesicles. PI(4)P remains associated with the regulated exocytotic vesicles, while they
are dephosphorylated for constitutive exocytosis. The proteins that are involved are of course again
the V-snare and T-snares (on the plasma membrane). Furthermore, the exact Rab proteins are
important. This is given in table 13-1 (page 706). For secretory vesicles, Rab3A is associated with
these vesicles.
[5] About vesicle fusion: I discussed this
extensively in the lecture. The figures in
the book are: Figures 13-16, 13-19, 13-20.
The figure legends are very good and
contain a good summary of the whole
story. Also, not forgetting the role of the
Rab proteins (table 13.1) for the specificity
of the fusion.

Page 47 of 82
[6] Figure 13.11 shows that the different vesicles involved in different forms
of endocytosis and exocytosis have different lipid composition. This is also
of importance for the different proteins that cover the vesicles. They are
specific binders of the different phosphatidylinositol lipids.

At some point, make sure that the students know a little about the different
vesicles going in between the plasma membrane and the endosomes. Also
the transport of vesicles between the ER and Golgi.
From ER à Golgi: COP II and from Golgi à ER COP I vesicles. The exact
composition of the COP vesicles is not required, only that they are different
(Figure 13-14/15). The Rab proteins also play an important role, these are
in Table 13.1. Also again figure 13-11, with the different phosphatidyl-
inositols (PIPs) is important.
[5] Lysosomal proteins are modified with a mannose-6-phosphate group. This is recognized by the
mannose-6-P receptor and packaged in vesicles targeted for the lysosome. The coat around the
vesicles (important for the budding of the lysosomal transport vesicles) is composed of clathrin.
This disassembles again when the vesicle is budded from the Golgi. For the recycling of the
mannose-6-P receptors a retromer vesicles is made, that contains a special retromer coat. Figure
13.45.
.
Task 6
Keeping the cell in shape and flexible
The shape of the cell is mainly determined and maintained by the cytoskeleton. There are different
types of fibrous molecules that together make up the cytoskeleton. The three different fibre
components are ordered according to their diameter. So we have thin, intermediate, and thick
filaments in the cell. Each has a different composition, architecture, function and molecular
mechanism. In addition to determining cell shape, the cytoskeleton is important for other, more
active functions. Changes of shape during cell movement are clearly dependent on well-regulated
changes in the cytoskeleton. The cases below will introduce you into the magical world of
cytoskeletal structures and their dynamics.

Listeria monocytogenes
Please study Figure 6.1 and the movies on EleUM and explain the underlying molecular
mechanism.

Figure 6.1. Listeria infection in a cell (left) and an extract of xenopus egg cells (right). There is an interesting role for actin
in the survival and infection of this bacteria.

Freezing dividing cells in their tracks

Vinblastine is a drug that inhibits
microtubule polymerization and is used to
treat some forms of cancer. But wait… the
molecule/drug paclitaxel (a.k.a. taxol)
strongly stabilizes microtubules and is also
used as an anti-cancer drug. What is the
explanation for this paradox?

Figure 6.2. Chemical structures of vinblastine (left) and paclitaxel

(right).
 Composition, architecture, function and molecular mechanism of:
[1] - Thin filaments
[2] - Intermediate filaments
[3] - Thick filaments
[4] How do the dynamics of the cytoskeleton get regulated?
[5] How is Listeria/Shigella propelled through the cell? How is actin involved in this?
[6] How do these drugs affect dynamics of the microtubules (as important for cancer)?

The actin filaments consist of a parallel helix of two intertwined actin polymer chains. These are
then organized in thicker bundles that are cross-linked by proteins to stabilize these bundles of
actin protofilaments. This organization increases the thermal stability of the F-actin.
[2] Intermediate filaments:
These filaments are composed of a number of different proteins, that form filaments by interacting
with each other forming protofilaments. These are then later organized in thicker filaments. The
intermediate filaments are not as dynamic as actin or microtubules. They are composed of proteins
that have a helical structure, so that
intertwining of protein molecules is
relatively straightforward. The
interaction of many protofilaments
aligning carefully, maximizing
intermolecular forces, makes these
filaments look like ropes. In table
16.1 (page 891) the different
intermediate filament proteins are
listed. Keratins are well known, but
also lamins (nuclear envelope) and
vimentin are often encountered. The
function of the intermediate filaments
is more structural. They for instance
have an important function in
keeping cellular integrity and keeping
organelles on their place. Also for axonal structure and growth, intermediate filaments (called
neurofilaments) are of critical importance.
Figure 16-67 and panel 16-2 (pages 944-945) are useful.
[3] Microtubules consist of polymers of (globular) tubulin subunits. The tubulin subunit consists of
two subunits: α-tubulin and β-tubulin, both have a binding-site for GTP. Only the GTP in the β-
subunit can be hydrolyzed to GDP (the GTP in the α-subunit is never hydrolyzed). A microtubule
consists of long chains
(protofilaments) of these
αβ-units (linked head to
tail), which are organized
in a hollow bundle (Figure
16-42). There are 13
protofilaments organized
in one microtubule (hollow
fibre) and the interactions
are between identical
tubulins (so α-subunits
only interact with
neighbouring α-subunits,
and the same goes for the
β-subunits that only
contact β-subunits).

Microtubules are formed from microtubule-organizing-centers (MTOC), for example in the

centrioles within the cell. These always lie somewhere close to the nucleus. This means that the
minus-end of the microtubules are in the cell centre, while the plus ends stick out towards the cell
periphery, to the plasma membrane.

Panel 16-1 and 16-2 as well as Figures 16-42 and 16-44 (pages 926-929).
[4] More on the regulation of the cytoskeleton (at least actin and microtubules):
In panel 16.3 and 16.4 (pages 905 and 933) overviews are given on what the different microtubule
and actin binding proteins are and what they do. The different names of the proteins and their
respective functions do not have to be known by the students. However, they should know that
proteins are very important in the dynamic nature of the cytoskeleton: so there are proteins involved
in the polymerization of the filaments, but also in the cross-linking (stability and anchoring), and in
the degradation. This is very important and the students should at least have studied panels 16.3
and 16.4 to appreciate the role of the actin- and microtubule-binding proteins

[5] Let the students read. It is on pages around pages 1286-1289 of the book and Figures 23-28
and 23-29. Also they should have a look at the movies on EleUM. The pathogenic bacteria recruit
actin monomers for actin polymerization at their cell wall. The growing F-actin chain propels them
forward inside the cell. The bacterial ARP complex is very similar to the normal ARP complex used
for F-actin initiation. This leads to a growing F-actin chain that is of course also degraded at the
minus end. This is then the reason for the comet-like structures observed after the bacteria inside
the cell. They can also get information from the web. Panel 16.3 (page 904) is useful here, showing
the ARP 2/3 complex and its function.
In table 16-1 (page 904) the effect of some drugs on
the microtubules and F-actin is given.
Paclitaxel/Taxol stabilizes microtubules and freezes
them in the polymerized state, preventing dynamic
instability.
Vinblastine prevents the polymerization of the
microtubules, so after catastrophes there is no
restoration of microtubules. This also means that
mitotic simples can never form and thus the cells
will not grow and even die.

Task 7
Cell motility: travelling cells
Did you know that your body is full of crawling cells? There are many reasons for a cell to move.
For instance, many single-celled organisms are dependent on movement to avoid toxic
environments and predators, and to chase food. In this course, we focus on the type of movement
called cell motility, which is defined as changes in cell location and limited movements of parts of
the cell. There are several forms of cell motility, almost all of which can be placed in two main
categories:

1) Actin based cell crawling. Actin filaments are used to rapidly change cell shape and to create
traction forces directed to the substratum the cells crawl on. It is a complex and highly regulated
way of cell locomotion that can be seen in Figure 7.1 and in movies online. The cell extends in the
direction of the movement, and simultaneously retracts at the trailing edge.

Figure 7.1. SEM (left) and fluorescently labelled (right) crawling cells.

2) Flagellar- and cilia-dependent movement. Specifically:

- Bacterial flagellum: Many bacterial species use a limited number of rotating flagella to propel
themselves. These flagellar motors are very similar to the ATP synthase found in mitochondria and
chloroplasts and this is reflected in their energy source. A special way of coordinating the flagella
is needed to obtain movement in the right direction.
- Flagella of human spermatocytes or single-celled flagellates: These flagella beat in a coordinated
fashion. They a completely different structure and mode of action than the bacterial flagellum.
- Ciliary motility: There is a group of Protista called the ciliates. An example of a ciliate is
Paramecium, show in the figure below. Ciliates have a large number of relatively short cilia on the
membrane that provide the coordinated movement these organisms need to survive.

Figure 7.2. Flagellar movement of a bacterium (left) and spermatocyte (middle). Ciliary
movement of a Paramecium (right).
 [1] How does actin based motility work?
[2] How do bacterial flagellum work?
[3] What is the energy source of bacterial flagellum?
[4] How do the eukaryotic flagellum and cilia work? they're
( similar)

[1] Page 951-960. Figures 16.75, 16.77, 16.78, 16.80, 16,82.

The trick is that in the leading edge of the cell (in the direction it is going) the cell sticks out a
protrusion filled with F-actin. This is newly synthesized actin, while a little bit behind this new F-
actin, the network is disassembled. In this disassembly there is an important role for the protein
cofilin, which catalyzes the breakage of actin filaments and thus the disintegration of the actin
network. The freed ARP complexes can then be used to get new actin synthesized just under the
plasma membrane.
[3] The energy comes from a proton gradient (Figure 14-35). This can be achieved by some
photosynthetic processes, or by a pump actually pumping H+ ions into the intermembrane space.
The H+ ions then flow back through the stator and rotor proteins, creating the movement. In some
cases, a Na+ gradient is used. Also then the Na+ gradient is generated by an ATP driven pump.
The motility of bacteria that use a flagellar motor costs energy.
Task 8
Mitochondria and chloroplasts: powering life
Cells constantly need energy to perform their vital functions,
such as reproduction, metabolism, synthesis and maintenance
of complex molecules and cell organelles, cell growth, signalling
and response to extracellular signals, directed cell movement,
intracellular transport and cytoskeletal dynamics.

Chemical energy is stored in the high energy bonds of energy

carrier molecules. The most familiar and most used is adenosine triphosphate (ATP). Cells have an
unquenchable thirst for energy. An ~70 kg human with a daily intake of energy (food) of 2800 kCal
will end up with 5860 kJ as ATP (50% efficiency), which at 50 kJ/mol means 117 moles of ATP are
synthesized every day. The molecular weight is 551 g/mol, so this ~65 kg of ATP!

Eukaryotes have two ways to produce ATP:

1) From catabolism of organic molecules (e.g. carbohydrates and fats). This takes place in the
cytoplasm and mitochondria. Recall the steps leading up to the production of an electron carrier
(e.g. NADH) from your previous studies. NADH goes "in" and then a series of membrane structures
and proteins perform specialized functions leading to ATP synthesis.

2) From the capture of light by chloroplasts. There are many similarities to what happens in the
mitochondria, but also many differences.

Knowledge of the ATP and energy producing process is important to understand how the cell
powers itself.

Figure 8.1. Electron transport in mitochondria (left) and chloroplasts (middle). Protons drive ATP synthesis (right).
Tip: This is not a biochemistry course, so the students do not need to go into all the steps of
glycolysis, Krebs cycle, and Calvin cycle. They should know that these are all basically redox
reactions. And they should know "what goes in, what comes out" for these.

[1] Refresher: first steps for polysaccharides and lipids?

How does oxidative phosphorylation work (mitochondria)?
[2] - Electron transport chain
[3] - ATP synthesis
How does photosynthesis work (chloroplasts)?
[4] - Electron transport chain
[5] - ATP synthesis
[3] The ATP synthase has some similarities to the bacterial flagellar motor, from task 7.
The proton motive force generates the required energy. The
students should be aware that the build-up proton gradient is what
drives the synthesis of ATP by rotating the enzyme. Also, as with
most enzymes, the reaction is reversible, so H+ can be pumped
into the intermembrane space under the hydrolysis of ATP
(providing the energy).
How are the electrons produced in the first place? For how the electrons are generated in the first
place, they have to know the principle of this. Also look at 14.45 and the text around.

The low energy electrons needed to compensate for the electrons taken away by plastoquinone,
H2O is used. O2 is generated here, and the produced protons are used to form the proton gradient
since the H2O splitting takes place in the stroma (outside of the thylakoid) and the cytochrome
complex pumps the protons into the thylakoid. It is useful that the H+ has been produced nearby.
See also figures 14-48 and 14-50.
This figure is also useful for keeping track of things:
Task 9
Cellular data flow
Cells continuously monitors signals in their environment by
specific receptors that are present in the plasma
membrane, the cytosol and the nucleus. There are many
types of signals, including pH, steroid hormones, and
proteins such as growth factors. When a signal is
membrane-permeable, there are intracellular receptors.
But most signals cannot pass through the plasma
membrane, so their receptors are able to transduce
signals from the outside to the inside of the cell. There are
three different classes of these cell-surface receptors. The
subsequent signalling pathways can illicit all kinds of
changes in cell behaviour (growth, death, motility,
differentiation, etc.). Generally, the signal-receptor
interaction is based on molecular complementarity, so it is
highly specific. And extracellular signals can act over short or long distances in the organism.

Signalling pathways may be highly specialized, but they are based on a relatively limited number
of common themes. Indeed, despite the vast number of receptors and molecules involved in
signalling (with so many acronyms!), there are only a few basic principles of how to process a signal
into a biological response. Let's look at one of the classes of cell-surface receptors: the G protein-
coupled receptors (GPCRs). These are
responsible for our sense of sight, small and
taste. And they happen to be seven
transmembrane spanning proteins
(remember those?).

Can you fill in the blanks with this

information? Epinephrine (adrenaline) and
vasopressin are two hormones that are also
used as drugs. Epinephrine has two
receptors: α (causing vasoconstriction) and
β (causing vasodilation). Vasopressin binds
to the V1 (causing vasoconstriction) and V2
(causing antidiuresis in the kidney)
receptors. There are different G proteins
involved, Gi to inhibit downstream adenyl cyclase (which converts ATP to cAMP) and Gs to
increase it. Over in the kidney, the Gq variant of the G protein signals phospholipase C, which
catalyzes PIP2 to create IP3 and DAG.
 [1] What different types of signals are known?
[2] What are the intracellular receptors and how to they work?
What are the three main types of cell-surface receptors and how do they work?
[3] - GPCR (most detail)
[4] - Ligand-gated
[5] - Enzyme-coupled (** skip the detail, it's for the next task)
[6] How can signals act over short or long distances?
[7] (From 9.2) What are second messengers? And which other molecules are 2nd messengers?
[8] Check the answers of Figure 9.2.
Figure 15-65. Nuclear receptors. An inactive
nuclear receptor is typically bound to an
inhibitory protein. When the ligand binds, it
causes to receptor to clamp shut and the
inhibitory protein dissociates. Then a
coactivator binds to the transcription factor-
activating domains and the gene is
transcribed. Not too much detail here.

[3-pre] A summary of the three types of transmembrane receptors: GPCR, ligand-gated and
enzyme-coupled.

The general scheme for signal

transduction is given in
several figures in the book.
Figures 15-1, 15-6 and 15-10.
Ligand binds to receptors, the
signal is relayed from to the
intracellular space, and a
response is generated. The
different signals that the cell
receives are integrated,
amplified, and spread over a
wide number of targets inside
the cell. This can lead to
different cellular responses,
as shown in Figures 15-4, 15-
5, 15-13.
[3] G protein-coupled receptors. Will be extensively in the
lecture. Also a good figure is: 15.21-15.23, 15.27, 15.29. The
students should understand the following: the binding of ligand
to the GPCR will lead to a conformational change of the
receptor. This will induce the activation of the G protein
(heterotrimeric G protein, the alpha subunit is the one that binds
the G-nucleotide), and the alpha subunit will release GDP and
bind a new molecule of GTP. The alpha will now be able to
activate effector enzymes. The beta-gamma subunit, can also
activate effectors, like K+ channels. The activation of effector
enzyme by the alpha subunit will proceed as long as it is bound
to GTP. The hydrolysis of GTP to GDP will inactivate the whole
system and result in association of the alpha-GDP unit with the
beta-gamma subunits. The G protein is now ready for new
activation. The G-alpha subunit has intrinsic GTPase activity,
which determines how long the alpha will be active for. The GDP
exchange for GTP is normally very slow, only the interaction with
a ligand-bound receptor accelerates this nucleotide exchange
and activated the G protein. In Table 15.3 is given which G
protein subunit interacts with what effector enzyme.

Talk about the different G proteins (stimulatory and inhibitory).

Cover the PLA/cAMP paths (Figure 15-27) and IP3/DAG (Figure 15-29)
Table 15-3 does not need to be
covered in detail, but the concept of
the different family members should
be known. Use the example from the
task.
[5] The detail in Figure 15-6 is sufficient for now. This will be the focus of the next task.

[6] The different types of signalling are given in Figure 15-2 also add autocrine signalling, the cell
produces a signal, which is secreted and then binds to the cell-surface receptor of the own cell.

[7] Second messengers are small intracellular signal molecules that are formed or released for
action in response to an extracellular signal and helps to relay the signal within the cell. Examples
are cAMP, cGMP, IP3, Ca2+, DAG, PIPx, etc. The second messenger response is almost always a
transient response. See here page 819-820. Also there is a table that depicts the limited number
of G-proteins (table 15.3 page 846 which may help you in the discussion (as an option)
Task 10
The dual role of insulin
Insulin is mostly known as the hormone lacking in patients with diabetes. Insulin binds to its
receptor and causes a response from the cell to increase glucose uptake. In this way blood glucose
levels are kept under tight control. In this task we will not discuss diabetes, but will learn about
insulin as a hormone and its signalling pathway.

Figure 10.1. (A) First commercial vial of U-10 insulin (early 1920s), (B) the molecular structure of insulin, a peptide dimer
bound together by disulphide bridges, (C) representation of a typical growth factor receptor (EGF: epidermal growth
factor) and the insulin receptor.
 [1] What is the insulin receptor?
[2] How do RTKs work?
[3] With which intracellular pathways do insulin receptors interact?
[4] How do insulin receptors actually stimulate glucose uptake?
[5] How do insulin receptors cause growth?à connection to glucose uptake or unrelated?
[6] How does the insulin receptor have the dual role? What is it caused by?
[7] Fill out the scheme

[1] The insulin receptor is a single transmembrane receptor (a receptor tyrosine kinase). Its structure
is a little more complex since it is heterotetrameric, so it does not have to dimerize upon ligand
binding (see Table 15-4 page 850 and Figure 15-43 page 851)

Figure 15-44. RTKs are activated by dimerization. Upon binding of the ligand, there is a dimerization
of receptor molecules and the two units autophosphorylate each other on tyrosine residues. This
leads to the appearance of tyrosine phosphate groups, which act as a scaffold for a number of
proteins that get translocated and activated close to the plasma membrane. One could say that
these receptors act as scaffolds only, since they have only themselves as a substrate.
Figure 15-46. Proteins with SH2 domains bind
to phosphorylated tyrosines.

[5] Figure 15-47. RTK activates Ras. First Grb2 recognizes a specific phosphorylated tyrosine on
the activated receptor by means of an SH2
domain and it recruits Sos by means of two
SH3 domains. Sos stimulates the inactive
Ras protein to replace its GDP with GTP,
which is what activates Ras to relay the
signal downstream.
[5] Figure 15-49. The MAP kinase module
(three components) is activated by Ras.
First MAP kinase kinase kinase (Raf) is
recruited by Ras to the plasma membrane and is activated. Raf then activates the MAP kinase
kinase (Erk), which in turn phosphorylates a variety of downstream proteins (other kinases and
transcription regulators in the nucleus).
 [5] Figure 15-53. Another RTK-based pathway
stimulates cell survival and growth. This is through
PI 3-kinase signalling. The activated RTK recruits
and activates PI 3-kinase, which produces
PI(3,4,5)P3, which serves as a docking site for Akt
and PDK1 (two serine/threonine kineases with PH
domains) and brings them close to the plasma
membrane. Akt is phosphorylated on a serine by a
kinase (usually mTOR), which alters Akt
conformation so it can be phosphorylated on a
threonine by PDK1. Now the activated Akt
dissociates from the plasma membrane and
phosphorylates other proteins (e.g. Bad, which
normally holds apoptosis-inhibitory proteins, so
once it is activated, these proteins are released and
can inhibit apoptosis).
[6] Figure 15-55. This shows the crosstalk of
pathways activated by GPCRs, RTKs, or both.
The students should recognize a lot of this after
tasks 9 and 10.