MG5607, Cell Biology

Instructor:
Paul K. Herman
842 Riffe Building
Office hours: Wed 4-5 PM
E-mail: herman.81@osu.edu

Mid-Term Exams (In class):

Mon. Sept. 29th (25%)
Mon. Nov. 12th (25%)

Final Exam:
Mon. Dec. 11th (50%)
Same room; from 4:00 - 5:45 PM
 Molecular Genetics 5607, Cell Biology (14)
Date Topics Covered Reading Assignment
Aug. 27 (W) Herman Overview/ Membrane Structure Chapter 10
Aug. 29 (F) Herman Membrane Transport Chapter 11
Sept. 1 (M) ******* No Class - Labor Day
Sept. 3 (W) Herman Membrane Transport
Sept. 5 (F) Herman Membrane Transport
Sept. 8 (M) Herman Protein Import Chapter 12
Sept. 10 (W) Herman Import into Mitochondria
Sept. 12 (F) Herman Import into ER
Sept. 15 (M) Herman Vesicular traffic: Coated vesicles Chapter 13
Sept. 17 (W) Herman Vesicular traffic
Sept. 19 (F) Herman Protein folding & degradation Chapter 6
Sept. 22 (M) Herman Cell Signaling Chapter 15
Sept. 24 (W) Herman Cell Signaling
Sept. 26 (F) Herman Review
Sept. 29 (M) Herman MIDTERM EXAM-1
Oct. 1 (W) Herman Cell Signaling
Oct. 3 (F) Herman Cell Signaling
Oct. 6 (M) Fisk How Cells are Studied Chapter 9
Oct. 8 (W) Fisk How Cells are Studied
Oct. 10 (F) Herman Cell Signaling
Oct. 13 (M) Herman Apoptosis Chapter 18
Oct. 15 (W) Herman Cancer Chapter 20
Oct. 17 (F) Herman Cancer
Required Text: Alberts et al., 5th Edition
Molecular Biology of the Cell

Reading Assignment I:
Ch. 12 Compartmentalization (pp. 695 - 699)
Ch. 10 Membrane Structure (pp. 617 - 640)
Ch. 11 Membrane Transport (pp. 651 - 683)
Ch. 12 Protein Sorting (pp. 699-704, 713-745)
Ch. 13 Vesicular Traffic (pp. 749 - 766)
Ch. 6 Protein Folding (pp. 387-398)
Ch. 15 Cell Signaling (pp. 879-937)
Ch. 18 Apoptosis (TBA)
Ch. 20 Cancer (TBA)
Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-2 Molecular Biology of the Cell (© Garland Science 2008)
 Cell Compartmentalization

* Eukaryotic cells are subdivided into a set of functionally-

distinct, membrane-enclosed compartments
* Internal compartments comprise ~50% of cell volume
* Plasma membrane contains ~5% of total cell membrane
* Organelles are nonrandomly distributed within the cell
 Questions that we will be addressing:
* What are the major organelles and their functions?
* How are the individual compartments formed? How are
the relevant proteins delivered to the correct target
organelle?
* Why and how did the eukaryotic cell become
compartmentalized?
* How do small and large molecules pass through the
membrane barrier?
* How do cells communicate with each other across their
respective plasma membranes?
* How are extracellular signals recognized & processed by
the cell?
 Cell Compartments
Nucleus:
- contains DNA genome
- site of DNA and RNA synthesis

Cytoplasm:
- comprises ~50% of cell volume
- site of protein synthesis and intermediary metabolism

Endoplasmic Reticulum (ER):

- made up of smooth and rough ER
- site of entry into the secretion pathway
- major site of lipid biosynthesis
- comprises > 50% of total cell membrane

Golgi Complex:
- made up of stacks of disc-like compartments
- an intermediate compartment of the secretion pathway
 Cell Compartments (cont’d)
Mitochondria:
- responsible for most of the ATP production in eukaryotic cells
- enclosed within a double membrane

Lysosomes:
- contains a variety of digestive or degradative enzymes
- responsible for degradation of cellular macromolecules, endocytosed
material and defunct organelles (autophagy)

Endosomes:
- a series of compartments that serve as an intermediate destination for
endocytosed material

Peroxisomes:
- vesicular compartments that carry out a variety of oxidative reactions
Evolution of eukaryotic cells
* The precursor to first eukaryotic cells likely resembled bacterial cells.
* Membrane-dependent functions were carried out by plasma
membrane proteins.
* The proliferation of internal membranes may have come about, in part,
as a response to an increase in cell size.
* The evolution of internal membranes likely occurred concomitantly
with the specialization of membrane function.

The cell size problem:

As cells became larger, the surface-to-volume ratio decreases. As a
consequence, the plasma membrane would no longer be sufficient to
carry out those membrane reactions essential for cell viability.

The Solution?
Increase membrane area by accumulating specific membranes within
the interior of the cell.
 Possible origin of the ER and nucleus

Figure 12-4a Molecular Biology of the Cell (© Garland Science 2008)

 Possible origins of the mitochondria

Figure 12-4b Molecular Biology of the Cell (© Garland Science 2008)

 Membrane Structure
* Lipid molecules in biological membranes are arranged as
continuous double layer, known as a "bilayer".
* All lipids in cell membranes are amphipathic (amphiphilic).

* Phospholipids are the most abundant cellular lipids.

* Phospholipids possess a polar head group and two

hydrophobic hydrocarbon tails.
* Lipid bilayers are two-dimensional fluids; proteins and
lipids both exhibit rapid lateral diffusion.
* Fluidity of a lipid bilayer depends on its composition.
 The Lipid Bilayer

Part A shows an electron micrograph of a plasma membrane (of human RBC) seen in cross section.
Parts B & C show two- and three-dimensional views of a cell membrane.

Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008)

 The parts of a phospholipid molecule:

Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008)

 Packing arrangements of lipid molecules in an
aqueous environment

Figure 10-7 Molecular Biology of the Cell (© Garland Science 2008)

 The spontaneous closure of a
phospholipid bilayer to form a
sealed compartment.

Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008)

 Liposomes

100 nm
 Phospholipid mobility:

Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)

Lipid rafts are transient domains within a bilayer that form as a
result of the physical properties of some lipid molecules

Lateral phase separation in artificial lipid

bilayers (Fig. 10-13)

Particular proteins can be concentrated

within lipid rafts (Fig. 10-14)
 The structure of cholesterol

Figure 10-4 Molecular Biology of the Cell (© Garland Science 2008)

 Schematic drawing of a cholesterol molecule interacting with two phospholipid
molecules in one monolayer of a lipid bilayer.
The presence of cholesterol tends to decrease the permeability of the bilayer.

Figure 10-5 Molecular Biology of the Cell (© Garland Science 2008)

 The four major phospholipids (A-D) in mammalian plasma membranes

Figure 10-3 Molecular Biology of the Cell (© Garland Science 2008)

 Membrane Proteins
* Most of the functions of the membrane bilayer are
performed by associated proteins.
* Proteins associate with membrane bilayers in several
different ways.
* Transmembrane proteins have amino acid stretches
embedded within the lipid bilayer.
* The membrane-spanning regions tend to be -helical,
especially in single-pass transmembrane proteins.
* In multipass transmembrane proteins, the TMDs may be
arranged as a -sheet in the form of a closed barrel.
* Membrane proteins may diffuse laterally within the plane
of the lipid bilayer.
 1. Single a helix.
2. Multiple a helices.
3. b-barrel (rolled-up b sheet).
4. Anchored with an a helix that partitions into one bilayer leaflet.

Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)

 5, 6. Anchored by a lipid attached to the protein.
7, 8. Attached via noncovalent interactions with other proteins;
peripheral membrane proteins.

Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)

 A segment of a trans-
membrane polypeptide
chain crossing the lipid
bilayer as an  helix.

Figure 10-21 Molecular Biology of the Cell (© Garland Science 2008)

 Using hydropathy plots to predict TMDs

Figure 10-22 Molecular Biology of the Cell (© Garland Science 2008)

Figure 10-27
 The carbohydrate layer on the cell surface

Figure 10-28a Molecular Biology of the Cell (© Garland Science 2008)

 Steps in the folding of a multipass membrane protein

Figure 10-25 Molecular Biology of the Cell (© Garland Science 2008)

Solubilizing membrane proteins with a mild detergent

Figure 10-30
Chapter 11

• Membrane Transport of
Small Molecules and the
Electrical Properties of
Membranes
 Membrane Transport (Ch. 11)
* Diffusion across a membrane bilayer is determined by the size
and polarity of the solute molecule.
* The hydrophobic core of the lipid bilayer serves a barrier to
polar molecules.
* The barrier function of membranes allows cells to maintain
different concentrations of solutes in different compartments.
* However, cells must be able to transport specific water-soluble
solutes across the membrane bilayer.
* Transport across membranes is carried out by specialized
transmembrane proteins, known as membrane transport
proteins.
* Cells can also transport larger molecules, such as proteins,
across the membrane bilayer.
(Moles/sec/cm2 )

Figure 11-1/2
 IN (mM) OUT (mM)

Na+ 5 - 15 145
K+ 140 5
Ca2+ 10-4 1-2
Cl- 5 - 10 110

Table 11-1
 Membrane Transport Proteins

- Multiple pass transmembrane proteins

- Exhibit solute specificity

- Two major classes:

1) Transporter proteins
- Also called carriers or permeases
- Bind to solute and undergo a series of
conformation changes that result in the
transfer of bound solute across bilayer

2) Channel proteins
- Form a hydrophilic pore; do not bind solute
- Faster rate of transfer
1) Passive Transport 2) Active Transport
- Also called "Facilitated - Transfer solutes against their
Diffusion" electrochemical gradient
- Solutes are transferred - Transport is coupled to an
passively across membrane expenditure of energy; e.g. ATP
- Mediated by both carrier & hydrolysis
channel proteins - Mediated by carrier proteins

Figure 11-4a
Forces driving passive transport:

For uncharged molecules = concentration

gradient

For charged = concentration + membrane

?
molecules gradient potential + + + + + +
- - - - - - - -
= electrochemical gradient
Figure 11-4b Molecular Biology of the Cell (© Garland Science 2008)
 Three ways of driving active transport.

Figure 11-7 Molecular Biology of the Cell (© Garland Science 2008)

 Types of Carrier Proteins

Uniporters
Transport a single solute
from one side of the
membrane to another.

Coupled Transporters
Transfer of one solute
depends on the simul-
taneous transport of
a second solute.

Two types of coupled transport reactions, symport and antiport.

Symporters transfer second solute in the same direction as the first.
Antiporters transfer second solute in the opposite direction as the first.
 A model of how a conformational change in a carrier protein could
mediate the passive transport of a solute.

Figure 11-5 Molecular Biology of the Cell (© Garland Science 2008)

 Using the Na+ gradient to drive glucose uptake

Note: Na+ and glucose bind

cooperatively

Figure 11-9 Molecular Biology of the Cell (© Garland Science 2008)

An asymmetric distribution of carrier proteins underlies
transcellular transport in epithelial cells

Figure 11-11
 Three types of ATP-driven pumps

Figure 11-12 Molecular Biology of the Cell (© Garland Science 2008)

 Plasma Membrane Na+/K+ Pump
* Present in the plasma membranes of almost all animal cells.
* Functions as an antiporter; Na+ is pumped out and K+ is pumped
in.
* Na+ gradient is used to regulate cell volume and to drive the
transport of sugars and amino acids into the cell.
* Almost 33% of the energy required by a typical animal cell is
used to drive this pump.
* ATP hydrolysis is required for Na+/K+ pump action.
* This pump can also be run in reverse resulting in the synthesis of
ATP.
* Pump is electrogenic, as it pumps 3 Na+ out for every 2 K+
pumped in.
 Three types of ATP-driven pumps

Figure 11-12 Molecular Biology of the Cell (© Garland Science 2008)

Figure 11-14 Molecular Biology of the Cell (© Garland Science 2008)
Na+-K + Pump
 A model of the
pumping cycle of
Figure 11-15
the Na+-K+ pump
Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008)
Osmolarity problem?
If the cell does nothing to control osmolarity, the concentration of
solutes will be higher inside than outside.

- +
- + The concentration of water will
- + - be higher outside the cell
+ - + resulting in a constant influx of
- + + water that could lead to cell lysis.
- Panel 11-1

Solutions?
1. Control intracellular osmolarity by actively pumping out inorganic
ions, such as Na+ (animal cells, bacteria).
2. Prevent swelling by possession of a rigid cell wall. At equilibrium, the
internal turgor pressure forces out as much water as is entering due
to osmolarity differences (plant and yeast cells).
3. Extrude water from special contractile vacuoles (protozoa).
 Responses of human RBC to changes in the osmolarity of the
extracellular fluid.

Figure 11-16 Molecular Biology of the Cell (© Garland Science 2008)

ABC Transporters

Figure 3-78a MBoC Ed. 5

 OUT

IN

Multi-drug (MDR)
resistance proteins
Figure 11-17b Molecular Biology of the Cell (© Garland Science 2008)
 Drug resistance in cancer patients

The over-expression of
multidrug resistance
transporter proteins, like
MDR1 and MRP1, results in
a resistance to chemo-
therapeutic agents in many
cancer patients.
 Some ABC transporters act as ion channels

Cystic fibrosis (CF) is caused by

mutations that inactivate an ABC
transporter that functions as a Cl-
ion channel in the plasma
membrane.
In this case, ATP binding and
hydrolysis regulates the opening &
closing of the channel pore.
 Ion Channels
* Form hydrophilic pores across membranes that allow for passage
of specific ions.
* Display ion-selectivity.
* More efficient than carrier proteins; a single channel can carry
>100 million ions per second, a rate 105 times greater than that
observed with carrier proteins.
* Mediate passive transport only.
* Transport is regulated by "gates" that open in response to specific
stimuli.
 Three types of ion channels

Figure 11-21 Molecular Biology of the Cell (© Garland Science 2008)

 A schematic representation of an ion channel

How is solute/ion selectivity achieved?

Figure 11-20 Molecular Biology of the Cell (© Garland Science 2008)

 Ion specificity of channel proteins

Figure 11-24

Figure 11-23a

Selectivity filter of bacterial K+ channel

K+
Na+
 K+ Leak Channels & Membrane Potential
* The membrane potential in animal cells is due primarily to K+ leak
channels and the K+ gradient across the plasma membrane.

* K+ leak channels are slightly open even in an unstimulated state.

* The leakage of K+ ions through the leak channels leaves the

plasma membrane with a relative negative charge.

* The movement of a relatively small number of ions can have a

large effect on membrane potential. The ions giving rise to
membrane potential lie in a thin (<1 nm) surface layer next to the
membrane.

* Therefore, almost any change in membrane permeability has an

effect on membrane potential. This is the key principle relating
the electric excitability of cells to the activity of ion channels.
The ionic basis of membrane potential
+

Figure 11-22
 Nerve Impulses (Action Potentials)
* Neurons utilize ion channels for receiving, conducting and
transmitting signals.
* The signal carried by neurons is always the same; a change in the
electrical potential across the neuronal plasma membrane.
* Communication occurs because an electrical disturbance in one part
of the cell spreads to other parts.
* Signals over a specific threshold result in a self-amplifying response
that travels rapidly down the neuron (an action potential).

* Membrane potential arises as a result of an electrical charge

difference between the two sides of a membrane.
* In general, the inside of an animal cell plasma membrane tends to
be negative relative to the outside. This results in a resting potential
across the plasma membrane of between -20 and -200 mV.
 A typical vertebrate neuron

Figure 11-28 Molecular Biology of the Cell (© Garland Science 2008)

 Three types of ion channels

Figure 11-21 Molecular Biology of the Cell (© Garland Science 2008)

 Action Potentials
* Voltage-gated cation channels are responsible for generating action
potentials (AP).
* An AP is triggered by a depolarization of the plasma membrane; i.e. by a
shift in the membrane potential to a less negative value.
* This initial depolarization is generally achieved by the opening of
neurotransmitter-gated Na+ channels.
* This initial depolarization causes voltage-gated Na+ channels to open,
resulting in a further depolarization of the plasma membrane.
* The AP is propagated down the neuron because the local depolarization
of the plasma membrane is sufficient to also depolarize neighboring
regions of membrane.
* The voltage-gated Na+ channels possess an automatic inactivating
mechanism that causes the channels to rapidly reclose even though the
plasma membrane is still depolarized.
* These channels remain inactive until several milliseconds after the
membrane potential returns to its initial negative value.
Figure 11-29 Molecular Biology of the Cell (© Garland Science 2008)
 Three states of the voltage-gated Na+ channel

Closed
+++ +++
Membrane
polarized
--- ----

Membrane
depolarized

Inactivated Open
Figure 11-30a Molecular Biology of the Cell (© Garland Science 2008)
Model for the rapid inactivation of a voltage-gated K+ channel

Figure 11-31 Molecular Biology of the Cell (© Garland Science 2008)

Triggering the initial depolarization event of the AP

Time (milliseconds)

Figure 11-29
 Transmitter-gated ion channels
* Transmitter-gated ion channels convert chemical signals into
electrical ones at chemical synapses.
* Neuronal signals are transmitted from cell to cell at specialized
sites of contact known as synapses.
* Neurotransmitter release is triggered by a change in of electrical
potential in the presynaptic cell.
* Transmitter crosses synaptic cleft and is bound by transmitter-
gated ion channels.
* This produces a local change in membrane permeability and
hence in membrane potential. If the change is strong enough,
nearby voltage-gated cation channels will be opened and an AP
initiated.
 Chemical
Synapse

Figure 11-35b Molecular Biology of the Cell (© Garland Science 2008)

 Transmitter-gated
Channels

Figure 11-35a Molecular Biology of the Cell (© Garland Science 2008)

 The technique of patch-clamp recording

Figure 11-33 Molecular Biology of the Cell (© Garland Science 2008)

 Membrane Transport - Summary

* Transport proteins allow the cell to take up essential

nutrients, expel metabolic waste products and regulate
intracellular ion concentrations.

* Membrane transport proteins allow the cell to maintain

different concentrations of a variety of solutes across any
given membrane bilayer.

* The ionic concentration differences allow cell membranes

to store potential energy in the form of electrochemical
gradients.

* This energy is used to drive various transport processes,

control cell volume, convey electrical signals in the nervous
system and to produce the majority of the cell's ATP.