Introduction to Cell Biology Samuel Tobias
What is cell biology?
Cell Biology is an academic discipline that studies cells, the basic structural, functional and biological unit of
all known organisms.
Cell biologists look at all aspects of the cell:
cell organelles and membrane trafficking
cell cycle, division and death
What is a cell?
Ultimate goal is to understand how macromolecular systems and organelles work and cooperate to enable
cells to function autonomously or as tissues.
How do we study cells?
Know the tools and methods to isolate and maintain cells in vitro
to know what we’re looking for
how to separate organelles
identify and study how proteins drive a cell’s biological processes.
Once we have the tools, we can start to ask and answer questions about our ~75 trillion cells in our body.
Cell culture is the technique used to grow cells or tissues outside the organism under strictly controlled
Much easier to grow single cell organisms than animal cells, because they come from multicellular
Cells isolated from any tissue by breaking down the cellcell cellmatrix interactions. (mechanical
fragmentation, trypsin, EDTA)
Trypsin chews apart extracellular matrix
turns a clump of cells into individual cells
EDTA reacts with Calcium ions, which make the outside of the cell sticky.
Cells are supplied with proper nutrients (amino acids, minerals, vitamins, salts, glucose, etc) and serum
(insulin, growth factors) and grown usually at 37°C in a CO in2 bator. (Body conditions)
Serum is the liquid component of blood
lacking in cells
contains insulin (for uptake of glucose)
growth factors stimulate cell growth
Medium in which cells are grown is red because phenol red indicator is used. Indicates pH, and therefore
Cells can grow as adherent cell cultures or suspension cell cultures.
Adherence cells are stuck to the bottom of the vessel
Suspension cell flasks have a spinner to prevent adherence to the sides of the container.
Primary cell culture refers to cells taken directly from an organism. These cells usually divide a limited
number of times (~50 generations, Hayflick limit). Also undergo contact inhibition.
they’re densely packed, cannot move around.
When exposed to trypsin, the monolayer falls apart, and the process can be started again.
cells get close, they stop dividing
Cell line refers to cells which are transformed and are able to grow indefinitely. Also known as immortal
cells. Less likely to exhibit contact inhibition.
HeLa cells come from cervical carcinoma of Henrietta Lacks
instrumental in forming vaccines, etc 3
Birth, Lineage, and Death of Cells
Stem cells can make more exact copies of themselves
Cell differentiation is when cell takes on different
attributes/properties than a stem cell
typically have a finite life cycle
As seen in the diagram ▯
Stem cell can either differentiate
or self renew
Embryonic Stem Cells (ES cells) can be maintained in culture and can form differentiated cell types. 4
Feeder cells secrete factors that help
inner cell mass cells grow
These ES cells differentiate very easily
very hard to keep in
In suspension, ES cells form embryoid
Embryoid bodies give rise to all
3 germ layers of embryo:
endoderm, mesoderm, ectoderm
ES cells can be induced to differentiate into precursors for various cell types
can be grown indefinitely under right conditions, and are pluripotent
Adult Stem Cells
Most tissues contain adult stem cells
Adult stem cells are required to maintain and repair tissue
Capable of generating a limited number of different cell types 5
Adult stem cells are located in the stem cell niche (adjacent cells provide signals to either self renew of
Is the path a one way trip?
We CAN take differentiated cells and
return them back to stem cells
Insert genes that code for
turn back time to ES cell state
Called “induced pluripotent cells”
future of transplantation medicine
Origins of Cancer
Genetic mutations can arise that result in
uncontrolled cell division or prevent programmed
cell death (apoptosis)
These mutations can occur in Stem Cells or Normal Cells
Reprogramming Normal Cells
to Stem cells could
potentially promote the
Cancer cells may arise from proliferating
normal cells and also from stem cells
Remember: Stem cells have no specific function,
other than to differentiate into other cells. Imaging in Cell Biology Samuel Tobias
Anton van Leeuwenhoek Imaging in Cell Biology Samuel Tobias
Built many simple, single lens microscopes
First to observe living protozoa and bacteria, which he called “animalcules”
Went on to visualize human red blood cells and sperm
With great skill at grinding lenses, naturally acute eyesight and lots of patience, he was able to achieve
magnification of 200X
Features of a Modern Compound Microscope (“BrightField”)
condenser to focus light on specimen
objective lens to collect light after it has passed through specimen
ocular or eyepiece lens to focus imagine onto eye
typical light microscope magnification is 401000X
only structures with high refractive index (ability to bed light) are observable
Darker structures bend or slow down light more (higher refractive index)
Resolution of Microscopes
Resolution: the ability to distinguish between two very closely positioned objects as separate entities
A conventional microscope can never resolve objects/cellular features that are less than ~0.2µM apart
Smaller resolution is better
Making numerator small or
resolution gets better Imaging in Cell Biology Samuel Tobias
Lens getting closer to specimen
Obtaining contrast in light microscopy: Exploit changes in the phase of light
Certain parts of the cell (eg. nucleus) refract
light more than other parts
Cellular constituents with high refractive
properties can slow the passage of light
by a quarter wavelength (~1/4λ)
Phase Contrast Microcopy
used to examine live ‘unstained’ cells
small differences in refractive index & thickness
within the cell are further exploited and converted into contrast visible to the eye.
Differential Interference Contrast Microscopy (Normarski Microscopy)
used to examine live ‘unstained’ cells
small differences in refractive index & thickness within the cell are converted into contrast visible to the eye.
uses polarized light
DIC microscope is equipped with polarizers Imaging in Cell Biology Samuel Tobias
Defines the outline of large organelles such as nucleus and vacuole, and provides better detail of cell edge.
Good for imagine live cells
shows 3D topographical features
Brightfield Phase Contrast DIC
note the Phase Contrast ‘halo’ around the outside of the cell
Uses a property of certain molecules to fluoresce, i.e. to emit visible light, when they absorb light at a
specific wavelength. (eg. invible UV light)
Location of fluorescent dyes or fluorescent protein molecules can be imaged.
Can visualize more than one protein or cell structure
we can do multiple types of labelling to discern different structures, proteins, organelles, etc.
Dyes, or “fluorophores” absorb energy kicking electrons (e ) into a higher orbital.
Instability causes e to drop into its normal orbital, releasing energy as visible light. “Fluorescence.” Imaging in Cell Biology Samuel Tobias
Stoke Shift is the difference between the optima of excitation and emission.
How are fluorescence images obtained?
Light doesn’t pass
through the specimen, Imaging in Cell Biology Samuel Tobias
it is reflected off
Dichroic mirrors reflect
most light, but let specific
wavelengths pass through
The many colours of flurophores
signals are bright on a black background
A variety of fluorophores exist with different excitation and emission wavelengths, that allow labelling of
more than one protein or organelle at the same time.
DAPI binds to DNA, emits blue
Mitotracker Red shows mitochondria, emits red.
A dye can be conjugated with antibodies to localize molecules of your interest in cells (immunofluoroescent
Bcells in mouse, recognize synthetic protein (antigen)
as foreign, and produce antibodies
Find these antigens in spleen cells, and fuse these cells Imaging in Cell Biology Samuel Tobias
with mutant mouse myeloma cells
Fusing the immortal myeloma cells with mouse spleen
cells, will hopefully cause cell to be immortal, and
continue to produce the antigen
HAT medium only lets the fused cells survive.
therefore, after culturing the cells in HAT medium, we get a pure population of immortal ‘Hybrid’ cells that
produce the antigen.
Antibodies are made to specific proteins
(eg. microtubules, actin)
Antibodies are added to cells fixed
on a slide which bind the specific
protein they were designed to recognize
Secondary antibodies with attached
fluorophores are added and bind to the
Each fluorophore has a unique excitation
and emission wavelength that can be
detected with appropriate filters in the
Use appropriate microscope
filter set for each fluorochrome Imaging in Cell Biology Samuel Tobias
then digitally overlay images
microscopy is performed on
fixed cells (ie. dead)
Fluorescent imaging in live cells: Green Fluorescent Protein (GFP)
Derived from a naturally occuring protein found in a jellyfish capable of bioluminescence.
The protein containsa short sequence of amino acids (chromophore) that are capable of fluorescing when
excited with blue light.
Gene was isolated and heavily modified so it would encode a protein with properties ideally suited for live
cell fluorescent imaging.
GFPFusion Proteins allow fluorescent imaging in live cells.
plasmid DNA needs to be transfected into the DNA of a cell.
Fluorescent proteins come in many different flavours! Imaging in Cell Biology Samuel Tobias
By mutating various amino acids in GFP, new types of fluorescent proteins were created with different
excitation and emission profiles
Two or more different fluorescent fusion proteins can now be visualized in live cells
Laser Scanning Confocal Microscope
Gives very good resolution, and can comine layers to produce 3D reconstructions of cells.
Laser uses a very precises
wavelength of light
focuses at a very small point
at a certain point in the cell
Fluorescence microscopy is extremely powerful due to its ability to show specifically labeled structures
within a complex environment and also because of its inherent ability to provide threedimensional
information of biological structures.
However, this information is blurred by the fact that, upon illumination, all fluorescently labeled structures
emit light no matter whether they are in focus or not. This means that an image of a certain structure is
always blurred by the contribution of light from structures that are out of focus. Imaging in Cell Biology Samuel Tobias
Confocal microscopy generates the image in a completely different way to normal "widefield" microscopes.
Using a scanning point of light instead of full sample illumination confocal microscopy gives slightly higher
resolution, and significant improvements in optical sectioning by blocking the influence of outoffocus light
that would otherwise degrade the image by means of specific pinhole, which is located in front of the
detector. Confocal microscopes use laser as the source of illumination. Laser scans the sample within the
focal plane. Confocal microscopy can be used where 3D structure is important.
A computationally intensive math procedure to remove fluorescence contributed from outoffocus parts of
the stained sample
It considers socalled point spread function which determines the degree of blurriness by comparison to a
reference set of tiny fluorescent beads
Images are taken at different focal planes (called a Zstack)
Images restored by deconvolution display impressive details without any blurring.
Basically, images are taken at different layers throughout the cell, and computationally, light from other
layers which would be seen as blurry is removed from the layer you are focussing on. (Making a dirty image
clean, by digitally subracting out the blurry stuff.
Fluorescence resonance energy transfer (FRET) to measure protein interactions in live cells
If no protein interaction occurs then excitation of cyan fluorescent protein (CFP) will only result in cyan
fluorescence (480 nm)
If protein interaction occurs then excitation of CFP will result in yellow fluorescence (535 nm)
In picture, protein interaction detected at front of migrating cell
If the proteins do not interact,
we will only see the blue
If the proteins do happen to interact,
then we will see yellow fluorescence Imaging in Cell Biology Samuel Tobias
From Fluorescence to Electron Microscopy
EM provides better resolution than FM.
EM, however, needs fixed and sectioned samples or metalcoated samples, i.e. living cells cannot be
Electron Microscopy Essentials
1. wire filament is an electron source & when it’s heated, electrons accelerate towards anode
2. a magnetic (not glass) condenser focuses electrons on specimen
3. specimen is stained with electrondense heavy metals (lead, uranium, osmium tetroxide)
Has to be done in a vacuum.
Doesn’t use glass as a lens, uses magnets to focus beam on specimen
The fundamental principles of EM are similar
to those of light microscopy; the major difference
is that electromagnetic lenses focus a
highvelocity electron beam instead of visible light
used by optical lenses.
In a transmission electron microscope (TEM),
images are formed from electrons that pass
through a specimen. Electrons are emitted from
a filament and accelerated in an electric field. Imaging in Cell Biology Samuel Tobias
A condenser lens focuses the electron beam on
the sample; objective and projector lenses focus
the electrons that pass through the specimen and
project them onto a viewing screen or other detector.
In a scanning electron microscope (SEM), images
are formed from electrons that are scattered
from a metalcoated specimen. To coat samples such heavy metals as platinum and gold are used. SEM
produces images that appear to be threedimensional.
Images are formed from electrons that pass through a specimen (TEM) or are scattered (SEM) from a
metal coated specimen
TEM intracellular structure
SEM outside of cell structure
Important that entire tube in both TEM and SEM is maintained under an ultrahigh vacuum because atoms
in air would absorb electrons.
Resolution of a TEM
Very fine D
No N, as light is replaced by electrons in a vacuum
sin a is now a since electron scatter is ~0
Theoretical resolution is 0.005nm;
the effective resolution is 0.1nm
2000X greater than light microscopy
Sample Preparation for TEM Imaging in Cell Biology Samuel Tobias
Fix cells, then embed in plastic, then make sections with an apparatus
Take the sections, stain with heavy metal
How is imaged formed?
electrons hit specimen but deflect due to metals deposited on organelles
unobstructed electrons are focused by lenses onto a phosphorescent screen
crystals in the screen, excited by the electrons, give off energy as visible light
B&W image is made up of shadows where electrons failed to penetrate
4. Areas that take up less stain appear lighter.
Detection of specific proteins using immunoelectron miscroscopy
Antibodies recognized a specific protein, called an antigen
Antibody can be linked with a heavy metal (gold) with another protein
Wherever the antigen is inside the cell, the gold particle is bound to it, therefore showing dark dots where
the protein are. Isolation and Analysis of Organelles and Molecules Samuel Tobias
Labelling live cells with fluorescent antibodies or stains
Antibodies made against specific cell surface proteins can be linked to fluorophores
Membrane permeable fluorescent dyes can be used to label intracellular structures (i.e. Hoechst stain binds
DNA in nucleus)
Cells with bound antibodies or that have taken up the dyes can now be sorted and counted Isolation and Analysis of Organelles and Molecules Samuel Tobias
Cell Cycle Analysis by FACS
Cells that have replicated their DNA but not fully divided (G2) will have twice the Hoechst stain fluorescence
intensity of nondividing cells (G1)
Quantifying number of cells in each
stage of the cell cycle
▯ All eukaryotic cells contain membranelimited
compartments termed organelles.
The isolation of organelles based on their
different density and different sizes.
Among cell organelles, the nucleus,
mitochondrian, and chloroplast are bounded by two bilayer membranes. All other
organelles are surrounded by single membrane.
How do we isolate Cell Organelles? Isolation and Analysis of Organelles and Molecules Samuel Tobias
STEP ONE: DISRUPTION of CELL PLASMA MEMBRANE
i) mechanical homogenization
ii) sonication (ultrasound)
iii) pressure (cells are forced through a very narrow valve)
iv) nonionic detergents i.e., Triton X100
v) placing cells in hypotonic solution
STEP TWO: CENTRIFUGATION of CELL HOMOGENATE
ii) equilibrium densitygradient
Safe centrifugation requires balanced loading of the centrifuge rotor. Imbalanced rotors can lead to damage
to centrifuge and rotors.
Differential centrifugation is a common first step in fractionating of a cell homogenate.
Differential centrifugations means
centrifuging a homogenate at different
speeds and times.
Cell organelles which differ in size and
mass travel to the bottom of the
centrifuge tube (undergo sedimentation) Isolation and Analysis of Organelles and Molecules Samuel Tobias
at different rates.
spinning homogenate yields
pellet & supernatant
increasing centrifugal force (gravity)
to isolate organelles based on mass
Equilibrium DensityGradient Centrifugation
Further separation of organelles based on density
separation based on density
homogenate is applied to a gradient of sucrose
at high speed/several hours, organelles migrate to sucrose layer equal their own density and remain there
How are proteins separated from the organelle?
have a hydrophobic end and a hydrophilic end
hydrophobic part is good at disrupting lipid bilayers
Triton X is a little weaker, leaves Transmembrane protein together
SDS breaks apart protein interactions Isolation and Analysis of Organelles and Molecules Samuel Tobias
SDSPAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels and called
polyacrylamide gel electrophoresis
PAGE is usually carried out in the presence of the negatively charged detergent SDS and called SDS
SDS is denaturing and binds to and destabilizes the hydrophobic side chains within the core of proteins
All polypeptide chains are forced into extended negatively charged conformations with a similar chargeto
The mobility of the SDSprotein complexes are influenced primarily by molecular size, i.e MW=daltons
How do we quantify the actual proteins?
Two types of stains:
Coomassie Brilliant Blue
Relative mobilty is proportional to size
small proteins move fast
How to detect a specific protein?
Apply an electric current to gel, and the proteins
are transferred to the membrane, which makes them
more accessible to antibodies
Put membrane in an antibody solution and shake,
leave time for antibodies to bind to protein of choice Isolation and Analysis of Organelles and Molecules Samuel Tobias
Then, use secondary antibody, linked to an enzyme, that binds to the primary antibody. After an enhanced
chemiluminscent substrate reacts with the enzyme, the secondary antibody will glow.
Then, after placing an xray film on it, we have a permanent version of the blot.
We can detect the bands using the luminescent procedure, and the degree of darkness is proportionate to
the amount of protein that the antibody detected
Bigger band = more protein expression Protein Synthesis and Transport Samuel Tobias
Protein Sorting and Targeting
Typical mammalian cell:
Must be localized correctly
Newly made peptides must be directed to the correct destination
Directing proteins to the right destinations (organelles)
During or after synthesis
Direct proteins to the secretory pathway (ER, Golgi, lysosomes).
General Principles of Protein Synthesis, Targeting and Storing
Many proteins are synthesized just by cytosolic ribosomes:
Those which remain in the cytosol
Those which are targeted to intracellular organelles such as (ER), mitochondria, chloroplasts,
peroxisomes, and nucleus (they have a specific signal sequence)
typically found at amino end of peptide
can be found elsewhere in peptide, however
Other proteins are synthesized by ribosomes attached to ER (the rough ER):
Those which reside
in ER and proteins
which are sorted to
and lysosomes. Protein Synthesis and Transport Samuel Tobias
Accordingly, two major
are known: nonsecretory
Some proteins are fully made in the cytosol, then directed into an organelle
Some proteins are made and imported at the same time as it being translate at the ER
Proteins use a translocation channel (a pore) that allows a protein to get through a membrane
Endoplasmic Reticulum Structure
First part of it is a direct continuation of nuclear membrane
uninterrupted membranous tubules & vesicles separated from cytoplasm (cisternae) stacked on eachother
RER has ribosomes on cisternae
Stringing Concepts Together!
identify cellular features by microscopy, isolate & homogenize them to free organelles
sucrose densitygradient centrifugation of homogenate allows for isolation of microsomes & ribosomes
microsome is small part of ERM that has broken off and refromed on itself
functional because still associated with ribosome
SDSPAGE is used to identify newly translated proteins Protein Synthesis and Transport Samuel Tobias
After separating/purifying with sucrose, split that amount
half treat with detergent, exposing proteins found
within the microsome to an added protease.
protease will chew up all protein
Other half, no detergent, add protease
will eat up proteins outside the microsomes,
however leaving the internal proteins unharmed
This reveals that the ER is where proteins are secreted are
have to go through ER
Endoplasmic Reticulum Functions
Secreted & membrane proteins are sorted through the RER
Sugars/carbohydrates are added to the polypeptide
disulphide bonds are formed
proteins are folded by chaperones
these are all posttranslational modifications
Translocation and Translation Occur Simultaneously
If you make a protein outside of the ER, it won’t go inside
But if you mix all the stuff at the same time, then the protein will be found inside the micrsome Protein Synthesis and Transport Samuel Tobias
missing the signal sequence, therefore a mature protein
RER: What are the major players?
i) amino terminal signal sequence of newly initiated polypeptide (nascent proteins)
ii) SignalRecognition Particle (SRP)
iii) SRP receptor embedded in ER membrane
iv) translocon: protein channel
v) cleavage site where signal sequence is cut by a signal peptidase
Done in a cellfree (in vitro protein translation) system
The mRNA codes for a protein that is secreted
It is recognized by ribosome in the cytosol, and starts to get translated
However, at the amino terminus, there is a unique sequence of amino acids that shows that it is destined for
A protein can recognize the sequence, and binds to it, and the ribosome Protein Synthesis and Transport Samuel Tobias
The whole complex will move towards the SRP receptor
GTP binds to SRP receptor, gives it a higher affinity for the complex
Once it makes it to the receptor, the receptor is in close proximity to the translocon. A pore.
When the complex gets to the receptor, it triggers the translocon to relax a bit, and the pore opens up
The protein can then be imported as it is translated,
A membrane associated peptidase will recognize the signal sequence, and cuts it off, the protein is still
translated into the ER
When it is completed, it dissociates from the receptor, and the protein is now inside the ER
Protein Modifications in the ER
Specific proteolytic cleavage
Formation of disulphide bonds
Formation of polypeptide chains Protein Synthesis and Transport Samuel Tobias
most secreted proteins are glycosylated
NAcetylglucosamine, mannose, glucose
branched sugars, not monosaccharides
Enzymatic transfer of 14residue oligosaccharide precursor from dolichol carrier (a glycolipid) to an
ASPARAGINE (Asn) residue of a nascent polypeptide by oligosaccharyl
Several glycosidases work to subsequently modify Nglycan.
Glycosylation functions: protein folding, confer protein stability, cell adhesion, etc.
Proteins with attached carbohydrates are known as glycoproteins.
Nlinked oligosacharides: carbohydrate chain is attached to the amide nitrogen of ASPARAGINE.
Oligosaccharyltransferase will recognize sequence of certain apragine residues of nascent polypeptide
stick big complex branched sugar onto it
certain glycosidases selectively chew off a few of the sugars from the branch.
some of the glycosylation confers charges to the protein, and insures the protein folds properly
also stability, and if it gets to cell membrane,