Biology 201 Notes 10/21/2012 3:40:00 PM
Cell biology is the study of cells:
Basic unit of biology.
Unicellular or multicellular.
Cells can grow, divide, specialize, respond to stimuli and adapt to environmental
In the 1600’s he discovered cells.
First to see cells.
1838 – improved magnification to see compartments within cells.
Types of Cells
Prokaryotic – cells which lack a cell nucleus or other membrane bound organelles.
Eukaryotic – cells that have membrane bound organelles. Includes years, plant cells,
animal cells, and fungi.
All organisms consist of one or more cells.
The cell is the basic unit of structure for all organisms.
All cells arise from preexisting cells.
Can be either prokaryotic or eukaryotic.
Includes bacteria, Archaea, protozoa, algae, fungi, and yeasts.
Many are pathogens.
Typically, they are too small to be seen with the naked eye.
Often consist of differentiated cells that perform specialized functions.
Includes plants and animals.
One millionth of a meter.
Eukaryotic cells are approximately 10-20 micrometers.
Prokaryotic cells are approximately 1-2 micrometers.
o Many exceptions to the size generality.
The cell consists of organelles, which are made of supramolecular structures. These
structures, in turn are made of macromolecules, which consist of small organic
One billionth of a meter.
Ribosomes, plasma membranes, microtubules, etc. can be seen using units of
Resolution of the Cell
We require magnification to see cells.
Resolution is the minimum distance that two objects have to be in order to be
distinguished as separate.
o Light microscope = 2oo nm.
o Electron microscope = 0.2 nm. The Light Microscope
Light is passed directly through the sample.
Light must interfere with the sample.
Shorter wavelength has increased resolution.
Shortening the wavelength allows you to se the object.
Both the objective and ocular lenses magnify the image. It is the sum of the
magnification, which is important.
o Bright field microscopy – to improve the contrast, you can stain the cells.
However, when the cell is stained, it has to die. But if you don’t stain it, then
there is reduced contrast.
In-phase waves result in brightness. Out-of-phase waves result in dimness.
Sample staining can change wavelength.
o Phase contrast microscopes – convert the changes in wavelength phase
differences into images.
o Refractive index – a measure of the path difference of how light travels
through the media.
Phase Contrast and Differentiated Interference Contrast (DIC)
Good for visualizing living cells.
Both techniques amplify slight changes in the phase of transmitted light as it passes
through a structure having a different refractive index that the surrounding medium.
o Phase Contrast – uses a phase plate to visualize.
o DIC – has a polarizer.
You are able to select what you fluoresce.
The background is black, so anything you colour can be seen. And, since you chose
what will be fluoresced, then you know that the colour is what you are looking for.
Fluorescence refers to the absorption of light by a molecule at a certain wavelength
followed by the emission of light with a longer (lower energy) wavelength.
You can select the microscope to only allow specific light wavelengths.
A labeled cell with fluorophore will emit the fluorescence.
Disadvantage: you must kill the cells in order to stain them. Also, sometimes the
fluorescence is not very specific.
Advantage: attaches directly to the antibody. Can use secondary antibodies to allow a
much brighter image.
Traditional and Confocal Microscopy
Traditional – unfocused light from above and below the plane of focus obscures the
Confocal – allows imaging of a single place of focus by means of a scanning
(dichromatic) mirror to dramatically enhance resolution. Images are recorded on the
computer. Then, when you are done, it fine-tunes the image for you.
o Disadvantage – immensely expensive and technically demanding.
Limitations of Using Light Wavelengths
Resolution is poor.
Electrons have a much higher resolution because the wavelength is shorter.
o Every sample must be dead.
o Expensive and technically demanding. o Lots of work to obtain the sample.
Replaces visible light and optical lenses with a beam of electrons that are deflected
and focused by an electromagnetic field.
Images are captured with a specialized detector.
The wavelength of electrons is much shorter than photons.
The limit of resolution of EM is much better.
Approximately 100 000 x magnification.
o Transmission electron microscope (TEM) – beam of electrons goes through
the sample. The electrons accumulate at different rates.
o Scanning electron microscope (SEM) – electrons bounce off when they hit the
sample and a detector picks them up.
o To tell them apart, SEMs typically look like they are in 3-D.
Centrifugation and Cell Biology
When an organelle or protein is subject to centrifugal force, its rate of movement
through a solution depends on its size and density.
Sedimentation Rate – rate of movement through a solution. Larger and denser
particles have a higher sedimentation rate.
Subcellular Fractionation – using centrifugation to isolate and purify organelles and
macromolecules based on their sedimentation rates and density.
Centrifuge – a piece of equipment that consists of a rotor the can be spun rapidly in a
circular motion by an electric motor.
Pellet – consists of larger and more dense components. Typically a solid.
Supernatant – smaller and less dense composition. Typically a liquid.
Separation and Isolation of Organelles
Break cells with high frequency sound.
Use a mild detergent to make holes in the plasma membrane.
Force cells through a small hole using high pressure.
Shear cells between a close fitting rotating plunger and the thick walls of a glass
Chill the cells when lysing.
Differential Centrifugation – a crude isolation technique where larger particles are
removed first. Allows you to obtain fractional samples. Ribosomes, viruses, and
macromolecules are typically the last fraction to obtain.
You can use the density to locate cellular components in a supernatant.
Technique for separation of proteins according to their electrophoretic mobility.
Linearizes proteins so they separate strictly by molecular weight.
Uses polyacrylamide gel.
Smaller proteins go farther than the larger ones.
Technique used for the detection of a specific protein in a complex mixture.
First uses SDS-PAGE, then the proteins are transferred onto a membrane, where
they are probed, using antibodies specific for the protein of interest. A secondary antibody recognizes the primary antibody, sits there, and the enzyme catalyzes a
Western Blotting has one discrete band and indicates protein presence.
Cellular Organelles and the Nucleus
Why do eukaryotic cells have organelles?
Larger eukaryotic cells need a way of maintaining adequate concentrations of
reactants and catalysts for the proper functioning of cellular activities.
Organelles are used as compartments specializing the in the enzymes and
compounds necessary for survival.
Many organelles are membrane bound, including the nucleus, mitochondrion,
ribosomes, Golgi complex, and chloroplasts.
Small, bean shape.
In cytosol – there may be a few or a lot.
o Muscle cell have lots.
Powers the conversion of glucose to ATP.
Efficient level of compartmentalization because double membrane and large surface
Sperm cells have lots of mitochondria to power the tail for movement.
A complex network of tubular membranes and flattened sacs.
Internal space is called the lumen.
Continuous with the outer membrane of the nuclear envelope.
Rough Endoplasmic Reticulum
o Covered in ribosomes.
o Proteins generated accumulate in the membrane of the ER lumen.
o Mainly secretory and membrane proteins produced.
Smooth Endoplasmic Reticulum
o No protein synthesis and no ribosomes.
o Synthesis of lipids and steroids.
o Cellular detoxification.
o Not associated with the membrane.
Site of secretory protein packaging and synthesis of polysaccharides.
Receives protein from the ER in vesicles.
One processed, the proteins are transported to other cellular compartments or the
o Initial stages of glycosylation occur in the lumen of the rough ER, but are
completed in the Golgi.
Modifies and sorts proteins.
Have specific compartments.
Littered throughout the cytoplasm.
Harsh enzymes with acidic pH. Compartmentalized to prevent cell damage.
Role in protein synthesis.
Smallest organelle found in cells ~ 30 nm in diameter.
The most numerous of all intracellular structures.
Can associate with other organelles, such as the mitochondria and the endoplasmic
Location of chromosomes (DNA).
Surrounded by nuclear envelope – double membrane.
Nuclear pores are important for the movement of water-soluble molecules such as
ribosomal subunits, mRNA, chromosomal proteins, and enzymes.
o Specialized pores dictate what enters and what leaves.
Nucleoli – the location of synthesis, process, and assembly of tRNAs with ribosomal
Darker chromatin, called heterochromatin, is much more densely packed.
Euchromatin is much more loosely packed. When a cell is dividing, the chromatin is
highly compacted. Chromosomes are the highest order of packaging.
DNA doesn’t exist without being complexed with protein – called chromatin.
Nuclear Lamina – provides structural support under the lower membrane. It also
associates with chromatin.
Nuclear Pores – specialized small cylindrical channels extending through both
membranes of the nuclear envelope, providing direct continuity between the cytosol
and the nucleoplasm.
o The nuclear pore complex is a channel lined with intricate protein structures.
o It is ~120 nm in diameter and composed of thirty proteins.
o It allows the diffusion of small macromolecules in and out of the cell. These
include rRNA, tRNA, and mRNA, as well as ions and proteins needed for
o Many nuclear porins make up the NPC. The nuclear lamina anchors the
nuclear pore complex. Transmembrane proteins anchor the nuclear porin.
o NPC is highly complex. The channel is not hollow.
Active Transport Through Nuclear Pore
o Importins/Exportins are important for moving the macromolecules.
o Macromolecules have an address stamp to bring into the nucleus (found on
alpha domain of importin/exportin complex). This is called the Nuclear
Localization Signal. To exit, a protein must have the Nuclear Export Signal.
o Active transport is needed when the molecules are too big.
o It is highly selective.
o Requires GTP as the source of energy.
o Ran-GTP is a small GTP binding protein belonging to the RAS superfamily
that is essential for the translocation of RNA and proteins through the NPC.
o The Import Cycle:
1. Importin binds NLS containing cytoplasmic protein.
2. Importin-NLS complex passes through the transporter region of the
NPC. 3. Importin binds to Ran-GTP and the NLS containing protein is
released into the nucleus.
4. Importin/Ran-GTP complex then travels back through the transporter
into the cytoplasm.
5. Importin is released from Ran-GTP, which undergoes a hydrolysis
reaction catalyzed by GAP.
6. Importin is now available to bind to other NLS-containing proteins.
7. Ran-GDP re-enters the nucleus and is converted to Ran-GTP by GEF.
o The Exportin Cycle
1. Ran-GTP binds to exportin in the nucleus.
2. Ran-GTP/Exportin complex promotes the binding of a NES-
containing nuclear protein.
3. Ran-GTP/Exportin/NES protein complex is transported through the
NPC and into the cytoplasm.
4. Exportin-NES protein complex is released from Ran-GTP, which is
mediated by the hydrolysis of GTP to GDP.
5. Exportin returns to the nucleus for re-use.
o The concentration gradient of Ran-GTP governs the direction of nuclear
transport. The net result is that the direction of transport for any given cargo
molecule is determined by the type of targeting sequence it contains (NES or
Chromosomes and Chromatin
DNA can be up to two meters long.
Packaging of DNA
o Nuclear DNA molecules are always associated with protein.
o DNA bound to protein is distributed throughout the nucleus and called
o Histones attract the DNA with their positive charge. The stabilization occurs
via ionic bonds.
o Non-histone proteins are also associated with DNA and are important for
enzymatic, structural, and regulatory processes.
o Nucleosome – a basic repeat structure containing ~200 bp of DNA associated
with a protein particle.
Not associated with histones.
Have the necessary machinery to replicate, transcribe, and translate DNA.
Still dependent on nuclear DNA for 95% of mitochondrial proteins.
Proteins are composed of amino acids:
They are polymers of amino acids.
Twenty different amino acids are used in protein synthesis.
Each protein contains these amino acids in varying proportions.
All amino acids have the same basic structure but are distinguished by their side
chains. Alpha carbon of amino acids is attached to carboxyl group, side-chain group, and an
amino group, as well as a hydrogen.
Amino acids are always in the L form, but they have an optical isomer in the D form.
Cysteine can form disulfide bonds.
Phosphorylation of tyrosine residues occurs.
Amino acids are linked together via peptide bonds. They are a stable, rigid bond,
where bending only occurs at the alpha carbon.
Every protein begins with methionine.
The backbone consists of both the alpha and carboxyl carbon, and the nitrogen. It
does not include the side chains.
Influences on Protein Folding and Stability
Disulfide Bonds – intra (one piece) and intermolecular (two pieces) bonds that occur
between cysteine amino acids.
Electrostatic Interactions/Ionic Bonds – noncovalent bonds help mold protein into
shape. Small changes in shape can cause huge conformational changes.
Van de Waals Forces
Hydrophobic Interactions – in an aqueous environment, hydrophobic side chains are
found on the interior of the folded protein.
Structure of Protein
Primary Structure – formal designation of the amino acid sequence.
o The order of amino acids that make up to polypeptide.
o Directly derived from the DNA sequence of the gene that encodes for the
o All other levels of protein structure are dependent on the amino acid
sequence of each polypeptide chain.
o Only the peptide bond is involved.
Secondary Structure – repetitive patterns of local structure.
o Predictable, repeating conformational pattern that derives from the repetitive
nature of the polypeptide.
o Patterns result from the hydrogen bonding between atoms in the peptide
bonds along the polypeptide backbone.
o Two main types:
1. Alpha helix – spiral shape
2. Beta sheet – extended sheet with peaks and troughs. They can be
parallel or anti-parallel.
o Does not have anything to do with the side chains.
o Some regions might not have secondary structure.
o Motif – the assembly of secondary structure.
Tertiary Structure – dependent on the type of amino acid side chains.
o Reflects the unique aspects of each folded polypeptide and how the side
chains of the amino acids will interact in order to form the stable 3D native
conformation of the protein.
o Not easily predicted as it involves the competing interactions between all side
o All types of bonds are involved in the formation of tertiary structure: Disulfide, Hydrogen, Ionic, Van de Waals, and Hydrophobic.
o Proteins do not have a random shape. They have evolved to have a specific
o Domain – a region known to do something.
o Slight changes in binding site can have a huge effect.
Quaternary Structure - subunit interactions and assembly.
o Only applies to multimeric proteins, such as hemoglobin.
o Dependent on the same bonds and forces as tertiary structure. 6
The cell synthesis of mRNA from the DNA template in the nucleus.
The cell converts the coding nucleotide sequence in mRNA to an amino acid using
ribosomes and tRNA.
Has a 5’ cap
Poly A Tail
Always read 5’ to 3’
Not polysystonic – one mRNA codes for exactly one protein. Compare this to
prokaryotic mRNA, which can code for more than one protein.
Translation Major Players
Ribosomes – performs polypeptide synthesis. It is the site of protein synthesis. It
consists of a large and a small subunit. The small subunit bind to the mRNA and
transfers 5’-3’. The large subunit catalyzes binding.
o E-site = exit site.
o A-site = where activated aa attaches.
o P-site = peptidal site (growth of pp chain).
Transfer RNA (tRNA) – aligns the amino acids in correct order along mRNA. There
exists a wobble site where there is flexibility in the ability to bind codons. The third
position is the flexible one. The anticodon of tRNA binds to the mRNA.
Aminoacyl-tRNA synthetases – attach amino acids to the correct tRNA. This is done
via the formation of the high-energy ester bond between the 3’ OH of tRNA and the
carboxyl end of an amino acid, which results in amino acid activation. The first step
is the adenation of the amino acid. Then, it is attached via an ester bond.
o Amino acid activation is done via ATP hydrolysis.
Messenger RNA (mRNA) – blueprint for the protein being synthesized.
Protein factors (such as chaperones) – prevent protein misfolding and promote
“proper” protein folding.
Three Stages of mRNA Translation
Initiation – mRNA is bound to the ribosome and positioned for translation.
o Initiation factor has GTP associated with it – elF2 is a dissociation factor.
When initiation factors dissociate, there is room for the large ribosomal
subunit to bind to the complex.
o Translation is an energy consuming process.
Elongation – amino acids are sequentially joined together via peptide bonds
according to the arrangement of codons in mRNA.
o Elongation factors use energy in the form of GTP. o If it is incorrect, it will dissociate.
o Systematic process.
o Large ribosomal subunit shifts ahead of the small subunit.
o This opens up the A site to allow binding.
o EF-Tu holds tRNA and prevents peptide bond formation unless
o EF-G promotes ribosome movement. Hydrolysis promotes realignment of
LRD for next addition.
Termination – the mRNA and the newly formed polypeptide chain are released from
o Release factor recognizes the stop codon.
o The whole thing dissociates and the end result is a polypeptide.
Co-translational Protein Folding
Proteins want to fold to produce secondary structure.
Chaperones assist some proteins or they can prevent them from folding, so they can
be delivered to the correct location to be folded properly.
Chaperones and Protein Folding
Molecular chaperones facilitate polypeptide folding.
Several different chaperones can be used to assist in protein folding.
Many serve to prevent polypeptides from premature (or incorrect) folding.
Incorrectly folded proteins can be very dangerous.
Hsp70 and Hsp60 are two common chaperones.
Hsp70 searches for hydrophobic regions. It requires energy.
The Hsp60-like protein complex places the cap, ATP, and the protein inside it and
the protein is slightly unfolded so that it can be refolded properly.
If the protein is not folded correctly, and attempts to correct it have been
unsuccessful, then the protein is targeted for destruction.
o Ubiquitation tags are added to the protein.
Proteosomes digest them.
Protein aggregates lead to disease.
Removal of localization sequence.
Chemical modifications of individual amino acid groups.
Formation of multimeric protein complexes.
Protein splicing – removal of intermediate piece to generate a completely new
Protein cleavage – the removal of a connecting polypeptide to activate a protein.
Example: proinsulin to insulin.
Protein Targeting and Sorting
o mRNA associates with free cytoplasmic ribosomes.
o If proteins are destined for the endomembrane system, then the ribosome will
attach to the ER.
o Growing polypeptide chains are then transferred across the ER as synthesis
proceeds. o It is called “cotranslational” import, because the movement of proteins into
the ER is coupled with translation.
o Transfer of proteins from the ER to their final destination involves vesicles,
the Golgi Apparatus, as well as other components of the endomembrane
o mRNA associates with free cytoplasmic ribosomes.
o Proteins that remain in the cytosol or proteins destined for mitochondria, and
the nuclear interior.
o Ribosomes remain free in the cytosol (unattached to any membrane).
o Once completed, the proteins will either remain in the cytosol or be
transported to specific organelles due to the presence of special targeting
o Proteins destined for the nucleus enter via the NPC, whereas proteins that
need to enter other organelles do so via a different mechanism.
Proteins destined for the ER have an N-terminus ER Signal Sequence. This sequence
directs the ribosome-mRNA-polypeptide complex to the surface of the rough ER.
o The ER signal sequence is positively charged, 15-30 aa, and consists of three
o The Signal Hypothesis – signal peptide is cleaved. Ribosome pushes the
Signal Recognition Peptide
o Interacts with large ribosomal subunit.
o Job is to bring to ER complex and assist in