Cell Biology Study Guide Exam 1
Foundations in Cell Biology
Whole Cell Techniques
Projection lens x Objective lens = total magnification
Magnification is a function of the microscope, which means that it is preset.
The objective lens is the first level of magnification and the projection lens is additional magnification, however it is usually no more than 10x
The specimen on the specimen stage must be transparent and extremely thin in order to allow the light to pass through and be seen by the
individual using the microscope.
Resolution (D) is the ability to distinguish two objects from one another. ∙ You want the minimum distance between two objects to be
The resolution in light microscopy is limited by the wavelength ( λ ) of visible light, which is 0.45 micrometers (violet) to 0.70 micrometers (red). This means that any beam of radiation cannot be used, it must be on the
visible light spectrum.
The distance between the two objects must be greater than or equal to the resolution of the microscope.
∙ If the resolution is X micrometers, the distance between the two
objects must be greater than or equal to X micrometers
The equation to calculate the resolution of a microscope is D=0.61 λ Nsinα
where Nsinα is the Numerical aperture. The numerical aperture
depends on the microscope.
∙ N is the refractive index of the fluid or the air between the object
and the objective lens.
∙ α is the angular aperture, which is actually half of the angle
from the specimen plane to the objective lens.
The best resolution that can be obtained from a light microscope is about 0.2 micrometers.
o Sample Preparation If you want to learn more check out econ 102 unlv
Fixation: freezing the sample, because cells are dynamic (which means they are always in motion)
∙ Crosslinking macromolecules
o Kills the cell
o Common agents: glutaraldehyde and formaldehyde (which
forms covalent bonds with free amino groups, such as the
N or C terminus and side chains)
∙ Partially permeabilizes cells, making the cells susceptible to staining.
Embed or Section (required for thick specimens only)
∙ Done using embedding wax or resins, then the specimen is cooled or polymerized. Don't forget about the age old question of chem 111 exam 3
∙ This gives the cell more structural integrity, which is important when the cell is sectioned
∙ Sectioning is done using a microtone, which is similar to a meat slicer seen at delis.
∙ Common stains include:
o Hematoxylin, which is a nuclear stain that appears blue/
violet. It is a base that binds to acids, such as DNA; thus, it
tends to make the nucleus of cells appear blue.
o Eosin, which is an acid that binds to base, such as proteins
present in the cytoplasm causing the cytoplasm of a cell to
o Benzidine binds to heme containing proteins, such as
hemoglobin in the blood.
o Hematoxylin and eosin, H&E, are commonly combined
during the staining process in order to create a contrast
between the cytoplasm and the nucleus of a cell.
∙ The darker the stain, the higher presence of the protein in that location, this is because the stain has more substance to bind to.
o Light Microscopy
o Simple and inexpensive
o No staining is required
o Allows for live cell imaging
o Very low contrast for most biological samples, which
makes the organelles difficult to separate
Cells are 70% water and 30% of everything else,
which allows them to be transparent without string
o Low apparent optical resolution because of the blur of out
of focused materials on the specimen lens. Don't forget about the age old question of Where does the end of the product life cycle fit in?
∙ However, there are optical, physical, and biochemical ways to improve the limitation of the bright field light microscope.
phase contrast uses dark and light bands to allow more intracellular details to be seen
DIC, differential interference contrast microscopy, illuminates the sample from the top and sides which results in shadows, creating a seemingly 3D effect
o Fluorescence Microscopy
The key of this technique is that the fluorescent compound absorbs light at one wavelength and emits light at a longer wavelength.
∙ Excitation λ is the wavelength at which the fluorescent compound absorbs the light.
∙ Emission λ is the wavelength at which the fluorescent compound emits the light; this value is always larger than the excitation If you want to learn more check out the central force that motivates us to mature is
A common compound used for this technique is fluorescein, which is excited around 492nm and emits around 525nm.
This technique requires a specific microscope and staining; however, it does have its limitations. A major issue with this technique is
∙ This problem is fixed by choosing a filter that blocks all light except for the wavelength being emitted by the target fluorescent compound. By angling the dichroic mirror a certain way, the Don't forget about the age old question of the cells that produce testosterone in the testis are called ________.
unwanted light can be blocked from the spectators view in the
o Immunofluorescence Microscopy exploits the antibodies (proteins that are produced by B cells that will recognize and bind to foreign antigens present in the body at any time). We also discuss several other topics like jeffrey morgan uh
The step by step process is as follows:
∙ This is usually done by inserting Protein X into a rabbit
which will elicit the B cells to create an antibody that
can recognize and bind to this target protein.
∙ The antibodies are then collected by isolating the serum from the rabbit about 8 to 10 weeks after injection.
∙ This antibody is then injected into the cell and binds to the target protein X. Then, a second antibody is created which has a
fluorescent tag on it and specific for the primary antibody.
∙ This second antibody will recognize the opposite end of the
primary antibody, which will allow the location of the target
protein to be seen through fluorescence microscopy.
A secondary antibody is used because it is expensive to add a fluorescent tag to a primary antibody because the process causes half of the antibodies to be denatured. Also, using a secondary antibody allows multiple secondary antibodies to attach to a single primary antibody, resulting in an amplification of fluorescence.
All primary antibodies must be derived from different animals, thus if you want to label the nucleus red and the cytoskeleton blue, the red label could attach to the rabbit derived antibody and the blue label could attach to the chicken derived antibody.
o Live Cell Imaging
o Confocal Microscopy or confocal laser scanning microscopy (CLSM), uses ordinary and fluorescent light to illuminate and focus on a thinly sliced specimen at a specific location, allowing for more detail to be shown.
Confocal pinholes allow only a small amount of light through, allowing the light from the point of focus to be seen and all of the light outside of the point of focus to be rejected (because it cannot come back through the pinhole).
The laser allows the thinly sliced specimen to be penetrated with light, thus thick specimens cannot be used for this technique which causes all 3 D information to be lost.
o Fluorescence Resonance Energy Transfers (FRET) are based on the distance between two fluorescent protein molecules.
The distance between these two proteins must be less than or equal to 10 to 100 angstroms in order for one fluorophore to absorb the light emitted from its neighboring protein. This results in the initial emission
wavelength to be the excitation wavelength of the other, when the two components are within the optimal distance range.
∙ The final emission wavelength will be a mixture of the two
fluorescent colors. Thus, if FRET occurred between a CFP (blue) and a YFP (yellow) the final emission color would be green.
This type of interaction can also occur intramolecularly. If a single protein contains two fluorescent compounds but is then phosphorylated (or another adjustment resulting in a conformational change) the two compounds can be brought into an optimal distance resulting in FRET to occur (see slide 44).
o Fluorescence Recovery after Photobleaching (FRAP), utilizes the dynamic of movement and flow in the cell.
The target protein is fluorescently labeled and then bleached with a laser; the photobleaching causes the label to lose the ability to fluoresce. After this you wait to see if the protein’s fluorescing ability recovers in the bleached section of protein, indicating the proteins mobility.
∙ The time that it takes the protein to recover will indicate the
mobility of the protein.
∙ This data is quantified in a graph (see slide 48), comparing the fluorescence before and after bleaching. Once the slope of the line reaches 0, which is a flat line, that indicates that all the protein is anchored. This value will allow you to quantify the percentage of protein that is mobile in the cell.
o There are ion sensitive fluorescent dyes, this means that the color of the fluorescence changes depending on the concentration of ions in the cell. For example, Fura2 is dependent on calcium concentrations in the cell: low [Ca] fluoresces blue, medium [Ca] fluoresces green, and high [Ca] fluoresces yellow/ orange/ red.
o Electron Microscopy
Transmission electron microscopy uses a beam of electrons, which have a wavelength much shorter than visible light, as radiation. This
microscope has a resolution about 2000x better than a light microscope, due to the small wavelength emitted by electrons.
∙ The resolution of an electron microscope is 0.1nm.
∙ This microscope is built in an almost identical manner as the light microscope, except the radiation beam is electrons, not visible
∙ Specimen preparation for the electron microscope (same steps as a light microscope):
o Fixate by using glutaraldehyde and osmium tetroxide,
which binds and stabilized the lipid bilayers.
o Embed by dehydrating the cell and placing it in a solid
o Section the cell into extremely thin widths of about 50nm to
100nm, because electrons have limited penetrating power.
This results in the loss of 3D image potential.
∙ The resulting contrast in the image is caused by the organelle’s ability to deflect electrons, this phenomenon is cause by the
o A larger atomic number indicates a higher probability of
electron scattering, thus causing the organelle or section of
the cell to be a darker color because little to no electrons
are able to pass through.
o A darker section also indicates that it had a greater ability
to pick up the osmium tetroxide stain than the lighter parts
of cell produced in the image.
Immunoelectron microscopy uses a secondary antibody, which is
labeled with gold, to bind to specific target proteins.
∙ Gold is extremely good at deflecting electrons, which means
anything bound with this labeled antibody will appear as a dark dot
on an image.
∙ This method is usually mixed with osmium tetroxide staining to
create varying contrasts in the image.
Scanning electron microscopy uses whole cells or unsectioned tissue specimen that are fixed, dried, and coated with a heavy metal that can
deflect electrons, resulting in a 3D image of the specimen.
∙ This technique requires a detector that will send the collected data
to a viewing screen and arrange it in a viewable manner.
∙ The 3D perspective of the structure is gained, however the ability
to have a high resolution is lost.
Cryoelectron tomography uses a rotating stage that can be tilted to
collect images of all sides and angles of the specimen, which are then
transmitted to a computer that will allow the visualization of a 3D image. ∙ This technique does not require fixation or staining, it uses
specimens frozen by liquid nitrogen in aqueous solutions.
Isolating Cells from Tissues
∙ Physical properties, such as density
∙ Physiological properties, such as affinity for a specific substance.
∙ FACS, Flow cytometry, which separates different cell types by taking advantage of cell surface antigens and sorting them through a machine that orders the cells in a single file line. The target antigen is labeled with both red and green fluorescent antibodies, these cells then gain a negative charge which passes through an electronic field and sorts the various cell types based on charge.
o The process ‘thinks’ of these questions: Is it a cell? Is it fluorescent? Is it red? Is it green? Is it both?
o This information is collected and recorded in a graph (see slide 72) where each individual dot represents a single cell
∙ Most cells can be cultured in the lab if the medium has the appropriate conditions and nutrient requirements.
o A rich medium is desired, this contains: 9 essential amino acids, vitamins, peptide and protein growth factors, and a negatively charged solid surface. The peptide and protein growth factors are usually supplied by the addition of fetal calf serum.
The negatively charged solid surface is required because it mimics
the extracellular interactions of an animal cell, which will allow for
ample cell growth.
∙ Primary cell cultures are cells that are prepared directly from the tissues of an organism. These cultures usually display the differentiated properties of the organs from which the cells were isolated. However, primary cells cannot divide an unlimited about of times, they can only double about 50 to 100 times before they die. (see slide 76)
∙ Transformed cells are cells that are derived from a tumor or another cell that has undergone spontaneous genetic change, this allows the cell to grow and divide indefinitely in culture, grow to high densities on a plate, and a solid surface is not required. These cells have gone through an oncogenic transformation. However, these cells will not necessarily mimic the organism from which they were isolated from. Techniques Addressing the Perturbation of Cellular Function
∙ Pharmacological methods
o Drugs and inhibitors are commonly used in cell biology in order to inhibit the function of specific organelles or proteins in cells.
o Some examples include: Brefeldin A inhibits the function of the Golgi apparatus, cytochalasin A inhibits actin in muscle cells, and Nocodazole inhibits the microtubules in muscle.
o Latruculin B inhibits the actin function in muscle; as Latrunculin B is increased the percentage of phagocytosis decreases, which indicates that actin is important in phagocytosis (see slide 81).
∙ Genetic methods
o At the level of protein
Dominant negative involves a mutation in the wildtype allele that will still allow the new mutant protein to bind to the enzyme, however it will
not elicit a response.
∙ The expression of a dominant negative version will compete with
the wild type protein.
∙ See slide 84.
o At the level of RNA
RNAi targets and degrades the message therefore reducing the number of proteins produced. This causes the mRNA to go through on of the
∙ In vitro production of dsRNA: the sense and antisense transcript
will find each other, resulting in dsRNA which is targeted by a
dicer and destroyed
∙ In vivo production of dsRNA: the mRNA will fold over on itself
and produce a hairpin structure that resembles dsRNA which is
targeted by a dicer and the message is destroyed.
∙ RISC chops up the dsRNA because it thinks that it is a virus
invading the cell.
o At the level of the genome
Gene replacement involves homologous recombination that will switch or “knock out” a specific sequence with a mutant sequence. This allows a new sequence to be inserted into the cell’s genome, however it does not
work efficiently in all cells
CRISPRCAS9 is an error prone repair that usually results in a frame shift, thus causing problems when building the protein from the amino
∙ CAS9 is a nuclease that allows a single gene to be targeted and
either knocked out or replaced or a new gene to be inserted.
∙ This method relies on guide RNA.
∙ Allows for whole genome editing to be achieved.
o Highly debated, especially in the current media due t the
researcher in China that claims he removed the CCR5
antigen on the surface of T cells, which will eliminate the
HIV’s ability to enter the cell. However, all organisms
contain the CCR5 antigen on the T cell surface, thus the
consequences of its removal are unknown.
Techniques Addressing the Purification and Characterization of Organelles ∙ Purification of organelles
o Centrifugation techniques
The cells are centrifuged. This method is more specifically called
Differential Velocity Centrifugation, which utilizes the density of each organelle in order to sediment each one around its corresponding density.
Then the remaining organelle fraction goes through Equilibrium Density Gradient Centrifugation. Using a linear or step gradient of sucrose the
fraction is centrifuged again; however, this time the organelle will remain in the section that is equal to its density.
Finally, the fractions of each density are collected in sections of unique densities.
o Purification of endosomes by magnetic fractionation
This is a onestep purification process!
Endosomes are phagocytic cells
∙ Endosome will engulf an inert iron particle into their phagocytes.
∙ The cell is then lysed, allowing the phagocytes and other
organelles to homogenate.
∙ The mixture is then run through a steal wire column attached to an
∙ When turned on, the electromagnet will catch the endosome’s
phagocytes that contain the iron particle, allowing all other cell
debris to flow through the column.
∙ When turned off, the iron particles will no longer be attracted to
the magnet which will cause the phagocytes to flow through the
column. This gives a pure sample.
Internal Organization of the Cell
∙ Important, but not all, membrane functions are as following
o Selective permeability
o Localize biochemical reactions
o Membrane Proteins
Respond to signals, energy transduction, ATP synthesis, and structural anchors.
o Membranes limit the cell in both eukaryotes and prokaryotes
∙ Major components of Cell membranes
Lipids are amphiphilic (polar) molecules
∙ Hydrophilic head
∙ Hydrophobic fatty acid tails
A fatty acid tail is saturated when it is comprised of all single bonds. However, if the fatty acid chain has at least one double bond it is
∙ 50% of all fatty acid tails are unsaturated.
∙ When the tail is bent, as indicated above, that indicates that there is a double bond, which causes the whole direction of the tail to bend.
They are great building blocks for biological membranes
∙ Why are lipids such a great building block?
o Because they are amphiphilic; they can spontaneously form
higher order structures where the hydrophobic tail is on the
inside to avoid the aqueous solution, and the hydrophilic
head is in the solution.
o They can form micelles or bilayer sheets, or monolayers.
o The type of higher order structure that will form is
determined by the individual phospholipid structure.
∙ Major types of Lipids
Hydrophilic head: Glycine (this position varies), phosphate, Glycerol Hydrophobic tails: long fatty acid chains
Common Phospholipids include: Phosphatidylethanolamine,
phosphatidylserine, phosphatidylcholine, and phosphatidylinositol.
∙ Phosphatidylethanolamine and phosphatidylcholine have a net
∙ Phosphatidylinositol is not important to an organism’s structural
membrane; however, it is an important signaling molecule.
The charge of the phospholipids is extremely important to the structure of the membrane.
Hydrophilic head: a variable polar group
Hydrophobic tail: sphingosine
Hydrophilic head: a variable polar group
Hydrophobic tail: nonpolar hydrocarbon tail
In between the head and the tail, there is an inserted steroid ring structure. This structure is comprised of four flat and rigid rings.
∙ Lipid SelfAssembly
The donut is an extremely versatile structure; however, it does not form naturally. This structure is created in labs and used to study cell delivery and trapping substances in the middle of the structure.
o Biological monolayers add more structural integrity to the bacteria, and benefit the bacteria’s living.
More specifically the bacteria are thermophiles, which are a type of bacteria that live in extremely hot environments.
The unique tails of the lipids are joined together to form the monolayer, this process is extremely messy because the tails of the phospholipids are constantly in motion.
These can also form spheres, such as LDL particles which deliver
cholesterol from the liver to all of the cells. The sphere encases the
∙ Lipids are Fluids
o Movement of lipids
Lipids can actually move in a bilayer sheet.
∙ Lateral diffusion is when the phospholipids move horizontally in the membrane. This process is viscous, more so toward the heads
because the tails are packed into the membrane tightly.
∙ Flexation is when the fatty acid chains of the hydrophobic tails
change the distance between them.
∙ Rotation is when the individual phospholipid rotates in its
∙ Transverse diffusion is when a phospholipid moves from one side of the bilayer to the other, which is rare. This requires a flipase
enzyme because the hydrophilic head must be pushed through the
hydrophobic tail region of the bilayer.
o Transition temperature
The fluidity of the bilayer is temperature dependent.
∙ Below the transition temperature, the membrane has a gel like
consistency but over the transition temperature gives the
membrane a fluid like consistency.
∙ Transition temperature is directly proportional to fatty acid chain length and the degree of saturation.
o A longer chain and higher saturation cause the transition
temperature to increase.
∙ Long, saturated fatty acid chains decrease the fluidity of the bilayer because the tails are more highly ordered and more difficult to
melt. Unsaturated tails are more disorder and less stable, which
makes them easier to break.
∙ Which property, saturation or length contributes more to the
fluidity/ transition temperature of the membrane?
o Saturation has a much bigger influence than the length of
the fatty acid chain.
∙ The fluidity of the membrane is also influenced by the sterol
content, because the sterol inserts itself between the phospholipids
and sphingolipids. And due to the rigidity of the sterol group, the
membrane will also become rigid.
o Does cholesterol make the membrane more or less fluid?
At physiological temperatures cholesterol decreases the
fluidity of the membrane, however at lower temperatures
cholesterol actually makes the membrane more fluid. Thus,
it is temperature dependent.
∙ Composition of lipids leads to
o an asymmetry in charge
Biological membranes are asymmetric; thus, the outer and inner
membrane composition of lipids are not identical. There is usually a
charge differential across the membrane.
o regulates curvature
The composition of the membrane matters! It contributes to the width of the inner and outer leaflets and the curvature of the membrane.
∙ A group with a smaller head is used in the inner leaflet because it
causes a curve and bends the membrane inward.
∙ Membrane Associated Proteins
o Integral membrane proteins are tightly associated with the membrane
Transmembrane proteins go all the way through the membrane. Inside of the membrane is usually the hydrophobic region with alpha helices. ∙ These proteins can either be single pass: go through the membrane one time or multiple pass: goes through the membrane two or
∙ Bacteriorhodopsin is 7 pass and the photosynthetic reaction center is 11 pass.
∙ They could also have beta barrels that pass through the membrane, creating a cylinder. These are known as porins.
∙ These proteins are extremely difficult to isolate from the rest of the membrane.
Lipid anchored proteins are covalently attached to the membrane by a lipid on the protein, which is then buried into the membrane.
∙ Acylation (fatty acid anchors) has an amide bond between the fatty acid tail buried in the membrane and the protein.
∙ Prenyl anchors have a thioester bond between the fatty acid tail
buried in the membrane and the protein.
∙ These are named by the number of carbons present in the tail.
∙ The lipid anchors can be attached during or post synthesis
o Peripheral Proteins noncovalently interact with the transmembrane proteins or lipid binding domains.
∙ Isolating proteins
Integral membrane proteins can be solubilized with detergents, such as SDS and TritonX100.
It’s difficult to study these proteins because of the hydrophobic section of the molecule; it is hard to create a hydrophobic environment in a test tube. By adding a detergent, the detergent molecules will insert themselves into the membrane to create a soluble proteindetergent complex. This complex has both the monomers from the detergent and some phospholipids/
sphingolipids from the membrane.
These are east to study in lab settings because peripheral proteins can be separated from the rest of the membrane due to their noncovalent
interactions. Once these interactions are broken, the peripheral proteins can ‘fall off’ and be isolated.
∙ Detecting the movement of proteins in membranes
o Cell fusion
∙ Cells can constrain the movement of proteins
o Epithelial cells
o Lipid rafts are rich in sphingolipids and cholesterol, which form wide and tall stable structures. The proteins are free to move in the lipid raft; however, it cannot leave the lipid raft.
Certain proteins have an affinity for lipid rafts.
These structures are often used for cell signaling.
o Selfassembly into large aggregates would completely immobilize the protein in the cell membrane Interaction with intracellular macromolecules
o Interaction with extracellular macromolecules
o Interaction with proteins in neighboring cells
Tight junctions often line ducts and tubules, it rescripts the protein’s movement because it cannot cross the tight junction. The protein is free to move within its domain but not into any other domains.
∙ These junctions are formed when there are interactions between
proteins on neighboring cells.
∙ Sodium is able to cross tight junctions
Movement of Small Molecules across membranes ∙ Passive diffusion is protein independent, which means that no energy is consumed and no protein is used.
o No energy is required because substances will move down the gradient, so particles will move from high concentrations to low concentrations.
o Gases and small uncharged polar molecules are transported using this method ∙ Transportermediated movement of small molecules across membranes requires the help of a protein transporter in the membrane
o Facilitated diffusion
No energy is required because substances will move down the gradient, so particles will move from high concentrations to low concentrations.
Uses the help of a uniporter, which will move one molecule in one
direction. The uniporter actually helps catalyze the transport of the
particles and limits the site of the diffusion.
Amino acids, nucleosides, and sugars are transported using this method. GLUT1 is a uniporter that transports glucose into most mammalian cells. It goes through a conformational change that will transport the glucose
molecule into the cell where it is immediately made into glucose6
phosphate which allows the gradient to remain. It has a higher affinity
than GLUT2 and passive diffusion (see slide 53) because its km is higher. o Active transport consumes ATP through ATP hydrolysis.
∙ The sodium potassium pump requires both sodium and potassium
to bind before it undergoes a conformational change. It pumps
3Na+ out of the cell and 2K+ into the cell, against its concentration
Fclass and Vclass pump
ABC superfamily pumps are found in both prokaryotes and eukaryotes. A wellknown pump is MDR1.
∙ This pump was discovered during cancer research because tumor
cells had a large amount of MDR1. These pumps were pumping
out the medication, which means that there was no accumulation of
drugs in the cellular space of the tumor cells. It flips a small, polar
molecule and exports it from the cell.
Coupled transport consumes energy that was stored in a concentration gradient.
∙ Pumps two molecules, one against its gradient and one with its
gradient. The pump exploits the energy from the molecule moving
down its gradient to pump the second molecule against its gradient.
∙ The sodium/ glucose symporter pumps 2Na+ down its gradient
while pumping one glucose up its gradient. It uses energy by
making both the sodium and glucose bind before the symporter has
a conformational change that allows transport.
∙ Transepithelial transport
o Coordination of multiple transport systems
o utilizes all pumps, this allows all parts of the cells to work together and cause a chain reaction.
∙ Cystic Fibrosis
o Cystic Fibrosis is a recessive trait that mainly affects Caucasians. The chloride ion channel is mutated, and doesn’t allow the chloride ion to pass from the lung lining into the airway lumen.
Symptoms of CF is basically that small ducts become clogged with mucus, more specifically they include:
∙ clogging and infection of lungs
∙ plugging of small bile ducts in the liver which impedes digestion
∙ plugging of pancreas ducts which impedes digestion
∙ obstruction of the small intestine
∙ males are infertile due to the block in the vas deferens
∙ malfunctioning sweat glands
Collins and Tsiu discovered the CFTR gene located on chromosome 7. CFTR, cystic fibrosis transmembrane conductance regulator, is an active transport protein that requires ATP to bind in order to induce a
conformational change that would allow a chloride to pass through.
∙ 2/3 of CF patients have a mutation at position 508, either the
phenylalanine is missing or the protein is misfolded and results in
CFTR being absent from the surface of the cell. This means that
the transporter is not in the membrane and cannot allow the
chloride ion channel to open, and no chloride ions can pass
∙ If the chloride ion does not pass through then water cannot pass
either, which means the mucus on the lining of the airway lumen
will not be hydrated, resulting in thick mucus to build up and house pathogens to reside and grow.