BIOL3402 Cell Biology & Cell Technology

Dr. Heath E. Johnson - Not Health!

School of Biological Sciences

5S-15 Kadoorie Biological Sciences Building

Tel: 2299-0877
Email: [email protected]
 Learning Outcomes

To provide a coherent understanding of the

structure and function of cells, and the
principles and applications of cell culture in
biology and technology.
Contents
1. Cell Biology (10 lectures by Dr. H. E. Johnson)
Cellular membrane. Cellular transport; Protein and RNA transport.
Ions transport and ion channels. Membrane potentials. Cell-cell
interactions. Extracellular matrix. Cell signaling.

2. Techniques in plant cell culture (6 lectures by Dr. P. Wang)

Light microscopy. Principles of confocal microscopy and image
analysis. Root and shoot culture. Explant regeneration. Protoplasts
and cell fusion. Secondary metabolites.

3. Techniques in animal cell culture (6 lectures by Dr. W. Y. Lui)

Metabolism and growth of somatic cells. Media formulation and
design of serum-free medium. Primary cell cultures, immortal cell
lines and cell growth parameters. Cell fusion and expression of cloned
gene in cultured cells. Sterilization and laboratory facilities.
Cryopreservation and freezing and thawing of cells.

Assessment: 20% Quiz, 30% lab report, 50% exam

Alberts, B. et al.:
Molecular Biology of the Cell
(Garland, 2014, 6th/7th ed.)
Notes, zoom link, and Date Lecture Topic

lecture recordings: 4-Sep 1 The Cell/Membrane (Chapter 10)

Moodle 2 Nuclear transport (Chapter 12)
Transport across the plasma
Slides are available on 11-Sep 3 membrane(Chapter 11)
Moodle now, but I may 4 Ion channels and optogenetics (Chapter 11)
make small updates to
25-Sep 5 Junctions (Chapter 16)
them closer to the class
6 Cellular Adhesion Molecules (Chapter 19)
so check them closer to Cell migration and thecytoskeleton (Chapter
the day of class. 2-Oct 7 16)
8 Cell Signaling (Chapter 15)
9-Oct 9 Cell Signaling (continued)
10 Quiz

Laboratory Session: Sept 30, Oct 7, Nov 11, and Nov 18 (Mon): 2:30
pm – 5:20 pm (Lab A/B/C)

Assessment:
One 2 hour written examination (50% weighting)
Laboratory practical works (30% weighting) – 3 reports 10% each (Lab
1 & 2 report is together)
Quiz (20% weighting) –1st Oct 11th (10%)/ and Nov 29th (10%)
The Cell
Membrane Structure

The lipid bilayer

Membrane proteins
Cell membranes act as selective barriers


Membranes are barriers, allowing the inside of
cell to be different from the outside, or for
differences in different compartments.
 The compartments of a
cell are surrounded by
membranes:
endoplasmic reticulum,
nucleus, peroxisome,
lysosome, Golgi,
mitochondrion, and
transport vesicles.

Two of these have

double membranes.

the nucleus and mitochondria (also chloroplasts)

Why?
 The lipid bilayer

At EM level, it can be seen as a dark line because of staining lipids.

2D (or 3D) representations show bilayer with two layers and

integrated proteins
Membrane lipids are amphipathic molecules


that there are two opposing properties, i.e.
a hydrophilic head and hydrophobic tail.


hydrophilic heads (polar) form hydrogen
bonds with water.


hydrophobic tails (non-polar) are excluded
by water molecules.


the most common lipid in mammalian
membranes are phospholipids.
 Three major classes of membrane lipids

Polar head groups

 “Like dissolves like”

Polar molecules dissolve in other polar molecules

 The shape in which lipids pack
controls the structures they form
Different membranes have different compositions
All phospholipids of the cells are only synthesized
on the cytosolic half of the ER membrane.
New lipids do not flip
spontaneously from one
monolayer to another, but
membrane-bound phospholipid
translocator proteins
(‘flippases’) are required.
 Cellular membranes are asymmetric

- - - - - - - - - -


phosphatidylcholine (red), sphingomyelin (brown), glycolipids (with
blue hexagons) are in outer leaflet, phosphatidylserine (light green),
phosphatidylinositol (dark green), and phosphatidylethanolamine
(yellow) are in inner leaflet.

Phospholipids and glycolipids are distributed asymmetrically in the
plasma membrane lipid bilayer
 Fluidity of the membrane


Lateral diffusion occurs rapidly within the plane of the
membrane (in an order or 2 mm/sec (length of a bacterial cell)

Individual phospholipids may rotate axially
How does this compare to the cytosol?

Flip-flopping from one side to the other is very rare as it is
energetically unfavorable
 The composition of a membrane regulates
the degree of its fluidity


the more closely packed the hydrocarbon tails, the less fluid is the
membrane.

shorter HC tails and unsaturation increases fluidity of membrane.

the presence of cholesterol in the membrane makes it less
deformable, decreasing permeability, but not necessary making it less
fluid
 Cholesterol

Prefers to associate with saturated lipids (I.E. Sphingolipids)

This can form rigid structures and decrease fluidity

These interactions are critical to the formation of lipid rafts

However, the “bent” tail prevents these saturated lipids from

packing too tightly
Cholesterol acts as a buffer for temperature changes
in membrane fluidity
Lipids and fluidity
Demonstration of membrane fluidity
FRAP to measure lateral diffusion rate on the
membrane
 Membrane proteins
• Proteins compose ~50% of the membrane
• ~1 protein:50 lipid molecules
• Glycoproteins
• Membrane proteins perform many functions
Membrane proteins, like lipid molecules, do not
tumble (flip-flop) across the bilayer, but they are
able to move laterally within the membrane.

Which moves faster?

Membrane proteins associate with the
bilayer in different ways
Transmembrane proteins usually span the
bilayer using a-helices
 Some membrane proteins use b-sheets to
cross the bilayer


b-sheets arranged in
cylindrical conformation are
known as a “b-barrel”


hydrophilic amino acid
residues face towards the
pore, hydrophobics face the
bilayer
Spectrin links the membrane to the
cytoskeleton
 The cytoplasmic side of the membrane is
called the cell cortex

• Meshwork of transmembrane proteins and filaments (spectrin)

• Mechanical support for the membrane and cell shape
Intracellular transport of proteins
- Transport through nuclear pores
- Transport across membranes
- Transport by vesicles
How does the cell sort the newly synthesized proteins?
Intrinsic information carried by the proteins: signal peptides

Receptors/transporters are required for recognition of these signal peptides.

Two ways that a sorting signal can be built into a protein

Signal peptide
- cytosol ER, mitochondria, chloroplasts, peroxisomes, and nucleus

Signal patch
- Golgi lysosomes, nucleus
Nuclear transport
the transport of materials in and out of the nucleus
Nuclear Envelope

The nuclear membrane is a barrier that

prevents the free passage of molecules
between the nucleus and the
cytoplasm.

The nuclear membrane is made up by

two lipid bilayers, underlining with
nuclear lamina and with nuclear pore
complexes incorporated.

The outer membrane is continuous

with the ER so that the space between
the outer and inner membranes is
directly connected with the lumen of
ER. Ribosomes are present on the outer
nuclear membrane.
The sole channels that allow transport of molecules in
and out of nucleus is nuclear pore complexes

Ions, proteins and RNAs need to travel between the nucleus

and cytoplasm through the nuclear pore complex.

A large complex of approximately 125 million daltons and a

diameter of 120 nm.

It is composed of a hundred or more distinct proteins.

 50 nucleoporins
Octagonal

Variable numbers of
pores (3000-4000)
depending on transcription

Can move about 100 histone molecules

per minute per pore

6 large and small ribosomal

subunits per minute per pore
EM micrographs of nuclear pore complexes
Dependent of the size of the molecules being transported,
molecules can travel through the nuclear pore complex by one
of the two mechanisms:

Passive transport: If the size of the molecule is smaller than

20 kDa (ions, amino acids, nucleotides, small proteins), they
can pass the nuclear pore complex rapidly in both directions by
passive diffusion. It is estimated that there are open channels of
10 nm diameter of this mode of transport.

Active transport: Most proteins and RNAs are too large for
passive transport. Special recognition sites on these
macromolecules and energy is required for active transport. In
response to appropriate signals, the nuclear pore complex can
open to a diameter of at least 25 nm. Active transport is
unidirectional (RNA out, protein in).
 Small molecules are able to rapidly pass through open
channels in the nuclear pore complex by passive diffusion

Results from injection:

< 5 kDa: fast diffusion Ribosome 30 nm

17 kDa: 2 minutes
> 60 kDa: cannot enter DNA, RNA polymerases
100-200 kDa subunits
Channel is 10 nm in diameter
15 nm long
Nuclear localization signals

Experiment using
recombinant DNA technique:

Nuclear localization signals usually

contain a short stretch of basic amino
acid residues (Arg, Lys). Mutation of
these amino acid residues stops the
import of the protein into the
nucleus.

Immunofluorescence micrographs
showing
T-antigen localization
NLS/NES can be used to control the balance of a protein
between cytosol and nucleus

Singh et al., Cell Rep., 2022

 Shuttling proteins

Shuttling proteins (nuclear import receptor and nuclear export receptor)

are required for the import/export of the target proteins (cargos). These
receptors carry two binding sites for the cargos and to the FG repeats on
the nucleoporins, respectively. They move back and forth between the
nucleus and the cytoplasm, with the help of a GTPase (Ran).
 Ran is a molecular switch with two
conformations – GTP or GDP binding

GAP (cytosol): GTPase activating protein: promotes hydrolysis of GTP to

GDP.
GEF (nucleus): Guanine exchange factor: promotes the exchange of GDP
with free GTP.
Cytosol: Ran-GDP and Nucleus: Ran-GTP.
Ran is required for both nuclear import and export:
interaction with Ran and their cargos
Import:
1. (Cytosol) binding of cargo to the import receptor initiates the
import of the complex.
2. (Nucleus) binding of Ran-GTP promotes the release of cargo
and outflow of import receptor Ran-GTP.
3. (Cytosol) GAP activates the conversion of Ran-GTP to Ran-
GDP. The released import receptor is ready for another round
of traffic.

Export:
1. (Nucleus) binding of cargo and Ran-GTP to the export
receptor induces the outflow of the complex.
2. (Cytosol) GAP activates the conversion of Ran-GTP to Ran-
GDP. This release the cargo and the Ran-GDP from the
export receptor.
3. Free export receptor returns to the nucleus and is ready for
another round of traffic.
How do importins pass through the nuclear pores?

So how is the directionality ensured?

 The import of nuclear protein is under regulation

Proteins with the nuclear localization signals are not always immediately
imported once they are synthesized. Their imports are regulated mainly
by phosphorylation and dephosphorylation or by dissociation of
inhibitory subunit.
Toettcher JE, et al. Cell (2013))
Transport of RNA out of the nucleus
- The export of RNAs (tRNAs, mRNAs and rRNAs) is also an energy-
dependent process that requires the GTPase (Ran).

- RNA is exported as a large RNA-protein complex, but not naked

RNAs.

- Pre-mRNAs and mRNAs are associated with more than 20 proteins to

form hnRNPs (heterogeneous nuclear ribonucleoproteins). At least
one of these proteins contain the nuclear export signal.
Assembly of ribosome involves protein import and ribosome export.
Nucleoi are the “factories” which produce ribosome subunits
Nucleoi are muti-phase membraneless organelles that
partition through liquid-liquid phase separation

Feric et al. Cell, 2016

 Liquid-liquid phase separation plays a role in membrane
partitioning as well

phosphatidylcholine / sphingomyelin +cholesterol

 Nuclear Lamina

Nuclear lamina is composed of one or more related proteins called lamins.

- There are 4 different lamins, A, B1, B2 and C.
- Molecular weight of 60-80 kDa.
- Homology to the intermediate filament proteins of cytoskeleton.
- B-type lamins is covalently linked with a lipid molecule (prenylation) at its C-
terminus cysteine residues.
- The lamina attaches to the inner nuclear membrane through prenylation and
interaction with integral membrane proteins.
- The nuclear lamina serve as a site of nuclear attachment.
Breakdown and reformation of the nuclear envelope during mitosis

phosphorylation of lamins disassembly of the nuclear lamina

dephosphorylation of lamins fusion of nuclear envelope vesicles