Cell Bio Midterm #1 Flashcards
Lec 1 - 10
what do cell cultures allow us to do
propagate and maintain cells in the lab
what does cell cultures refer to
the maintenance of cells outside of an organism (in vitro)
what are cells bathed in during a cell culture and what does it do
bathed in a culture medium that mimics the extracellular fluid
what does the culture medium contain
nutrients, ions, growth factors, and often antibiotics
what are culture dishes coated with
proteins that are found in the extracellular matrix
what degree Celsius and CO2 levels are most mammalian cells incubated at
37 celsius and 5% CO2
what does our genome carry
the necessary instructions for the differentiation of a single fertilized egg into all of diverse cell types in the human body
what makes up the cell culture environment
temp, pH, ionic strength, nutrients, special surfaces, antibiotics
what are the two main forms of cultured cells
primary cells and cell lines
what are primary cells
cells that are directly generated from multicellular organisms
do primary cells divide
finite division and some do not divide at all
what are cell lines
cells that were originally primary cells but have acquired mutations that allow them to grow and survive indefinitely
what do these “transformed” cells have the characteristics of?
cancer cells
what is passaging
extracting transformed cell lines and transferring them to another culture dish to expand that culture
what does microscopy do
enables us to magnify objects so that we can observe them in greater amount of detail
what is cryopreservation
freezing a subset of these transformed cells for later use
what is the average cell length
tens of microns (millionth of a meter)
which lens provides the greatest amount of magnification
objective lens
what is the quality of the magnified image affected by
contrast and resolution
what is contrast
the ability to distinguish between the sample and the background
what is resolution
the ability to distinguish between two closely apposed objects
how do brightfield microscopes illuminate specimens
use white light (all wavelengths of light) to illuminate
how do fluorescence microscopes work
they direct light of a single wavelength of narrow range at the specimen
what are the two orientations of microscopes
upright (objective lens above and light below) and inverted (objective lens below and light above)
what does a fluorophore possess
an electron that can be readily excited by light of a narrow range of wavelengths (excitation wavelengths)
what is fluorescence microscopy used for
detection of fluorescent molecules (fluorophores)
what is a dichroic mirror and what microscope uses it
a fluorescence microscope uses it and a dichroic mirror reflects the excitation wavelength toward the specimen and passes emission wavelength produced by the specimen to the eyepiece
what type of fluorophores can be used in live cells
membrane-permeable fluorophores
how doe sa fluorophore stay inside the cell once it enters
it is enzymatically cleaved so it can no longer cross the membrane
the gene encoding a specific protein can be modified to produce what
the normal protein fused to a fluorescent protein
what is immunocytochemistry used for
used to fluorescently label specific proteins in preserved cells
(i.e., what cells expressed the protein, when the protein was expressed, and where it is located)
what is specificity provided by
antibodies
through what are antibodies produced
naturally produced by the plasma cells of the mammalian immune system
what is an antigen
the specific binding region of a antibody
what are fluorophores attached to
antibodies
what does direct immunocytochemistry do
uses a single antibody to label the protein of interest
what is antibody specific to
target protein and contains a fluorophore
what does indirect immunocytochemistry do
uses two antibodies to label the protein
what does the primary antibody do
binds to antigen on target protein
what does secondary antibody do
binds to antigen on primary antibody and contains fluorphore
how is signal amplified
when multiple secondary antibodies bind to a primary antibody
what do conventional fluorescence microscopes do
pick up emitted light from all depths in the cell
- causes blurring of resultant image
what do confocal fluorescent microscopes pick up
only detected emitted light from a single focal plane
- multiple images from different planes can be combined to make 3D image
what does electron microscopy do
directs a high-velocity electron beam at the sample instead of visible light
electrons are directed at sample by what
electromagnetic lenses instead of optical lenses
what happens in flow cytometry
- isolated cells are labelled with antibodies that possess fluorophores
- cells are sent single file through a tube into flow cytometer
- light is directed at the cells to excite fluorophores
- flow cytometer detects emitted light
- flow cytometer also determines the degree to which light is scattered by the cell
what is fluorescence-activated cell sorting
an application of flow cytometry that can be used to separate cells based on their expression of surface molecules
what does flow cytometry allow you to do
culture a pure population of cells
what is the process of a western blot
- a heterogeneous mixture of proteins isolated from wells or tissues
- protein mixture is incubated in sodium dodecyl sulphate (SDS)
- SDS coated proteins are then loaded into a well of a polyacrylamide gel
- a potential difference is applied across the gel and proteins will move toward the positive end
- proteins on polyacrylamide gel are transferred to a nitrocellulose membrane
- membrane is incubated in antibiotics
- gel is placed on an imager that excited the fluorophores and detects emitted light
- only the protein of interest is visible on gel
how are proteins separated in a western blot
based on weight
biomembranes consist of:
a lipid bilayer, embedded proteins, glycoproteins, and glycolipids
what does the lipid bilayer provide
selective permeability
embedded proteins participate in
cell signalling, cell-cell and cell-matric adhesions, energy transduction
what is the lipid bilayer primarily made up of
phospholipids
what are phospholipids
amphipathic molecules that contain a hydrophilic phosphate head group and a hydrophobic tail group
what is the most abundant phospholipid in eukaryotic biomembranes
phosphoglycerides
what does each phosphoglyceride consist of
phosphate group attached to variable head group (R), glycerol, fatty acid
what are two other amphipathic molecules commonly found in eukaryotic biomembrances
sphingolipids and cholesterol
each layer of the lipid bilayer is known as a what
leaflet
how does the lipid bilayer exhibit fluidity
they readily move laterally within each leaflet
- can also rotate or wave
what is degree of fluidity dependent on
temperature and composition of the bilayer
what is the membrane like at each temp
gel-like at low temps and more fluid at high temps
what are fatty acids
long hydrocarbon chains
what do saturated fatty acids not contain
carbon-carbon double bonds
what do unsaturated fatty acids contain
one or more carbon-carbon double bond that place a kink in the hydrocarbon chain
how do unsaturated fatty acids affect fluidity
the greater the amount of unsaturated fatty acids in the bilayer, the greater the fluidity
what do long fatty acids provide
more intermolecular interactions between phospholipids
how do fatty acids affect fluidity of the biomembrane
the greater the proportion of long fatty acids, the lower the fluidity
what do longer fatty acids also do
increase the thickness of the lipid bilayer
what does cholesterol do
stabilized the membrane and prevent sit from becoming too fluid or too gel-like
what does fluidity of the lipid bilayer allow
allows embedded proteins to move laterally within the membrane
- enables proteins to interact with their target
what are lipid rafts
small segments of the lipid bilayer that contain a high degree of cholesterol (and are therefore more stable)
do all regions of lipid bilayer exhibit the same degree of fluidity
no
what do integral proteins do
spam plasma membrane
what are the three domains in an integral protein
extracellular domain, transmembrane domain, cytosolic domain
where are lipid-anchored proteins anchored
the hydrophobic core
what do peripheral proteins attach to
hydrophilic phospholipid head groups, integral membrane proteins or lipid-anchored proteins
how are phospholipids transferred between leaflets of the biomembrane
moved by membranes known as flippases
what are the three types of flippases and which ones require ATP
flippase - require ATP
floppase - require ATP
scrambles - no ATP requirement
what are glycoproteins
integral membrane proteins that are covalently bound to carbohydrates
what are glycolipids
membrane lipids that are covalently bound to carbohydrates
what are biomembranes
dynamic structures that grow and retract according to changing cell needs and ongoing cellular processe
what needs to happen for a biomembrane to grow
new phospholipids, sphingolipids and cholesterol must be synthesized and incorporated into an existing biomembrane
process of fatty acid synthesis
- synthesized in the cytosol from two-carbon acetyl groups present in the acetyl-CoA
- Acetyl-CoA carboxylase converts other acetyl-CoA molecules to malonyl-CoA (3-carbon molecule
- Growth continues via addition of malonyl-CoA until full fatty acid is produced
what is the process of phosphyglyeride synthesis
- Fatty acid is converted to fatty acyl-CoA in the cytosol
- Enzymes embedded in the membrane of the smooth endoplasmic reticulum catalyze the reaction between 2 fatty acyl-CoA molecules and glycerol 3-phosphate
- This generates phosphatidic acid which is inserted into the cytosolic leaflet of ER membrane
- Head groups are then added to phosphatidic acid to generate the phosphoglyceride
- Fatty acids can acquire double bonds via desaturase enzymes that act on phosphoglycerides
sphingolipid synthesis
- Occurs in the smooth endoplasmic reticulum
- Palmatoyl-CoA (a 16 carbon fatty acyl CoA) enters ER and binds to serine
- Second fatty acyl CoA then binds to palmatoyl-serine to form ceramide
- Ceramide is sent to the Golgi where a head group is added to form a sphingolipid
- Sphingolipids are sent from the Golgi to final destinations in cell
cholesterol synthesis
- Cholesterol precursors are synthesized in the cytosol
- These precursors become embedded in the cytosolic leaflet of the smooth ER membrane
- Enzymes in the ER membrane convert these precursors into cholesterol
what are the 5 things rate of simple diffusion is affected by
- temp
- magnitude of the concentration gradient
- surface area of the biomembrane
- hydrophobicity of the molecule
- size of the molecule
what is facilitated transport
movement of molecules down their concentration gradients using integral membrane proteins, including uniporters and ion channels
what is active transport
the movement of molecules against their concentration gradient by ATP-powered pumps
what is co-transport
the movement of one molecule against its concentration gradient while the other molecule moves down its concentration gradient
when do transporters exhibit saturation
when there is a high concentration of the molecule to be transported
what is the main source of energy for eukaryotic organisms
glucose
where does energy production occur
inside the cell
where is glucose transported from and where does it go to
transported from extracellular fluid into the cytosol
what are GLUT proteins
uniporters that move glucose down its concentration gradient
how is a low concentration of glucose achieved inside the cell
rapid conversion of glucose to glucose-6-phosphate
how many different isoforms of GLUT proteins are there
14
how do cells modify the amount of glucose uptake
regulate the number of GLUT proteins in the plasms membrane
what do sodium-coupled glucose transporters do
move glucose against its concentration gradient and sodium down its concentration gradient
what are glucose transporters important for
epithelial cells lining the gastrointestinal tract that want to absorb glucose from food and send it to the bloodstream
how to determine the flow of water during water transport
look at the osmolarities of the solutions on both sides of the membrane
what is osmolarity
the number of solute particles per unit volume
what is concentration
the number of molecules per unit volume
what is water
a polar molecule that is only slightly permeable to the lipid bilayer
how can water transport be accelerated
aquaporins
what influences cell size
movement of water across the membrane
what do ATP-powered pumps generate
concentration gradients in the cell
how do ATP-powered pumps work
use energy from ATP to move molecules against their concentration gradients
what are the 4 main classes of ATP-powered pumps
P-class pumps
V-class proton pumps
F-class proton pumps
ABC superfamily
what is ATP used to phosphorylate
the cytosolic face of the pump protein
what do P-class pumps do
move ions against their concentration gradients using energy from ATP
what do V-class proton pumps do
pump protons (H= ions) against their concentration gradient using energy from ATP
what uses V-class proton pumps
lysosomes
what can ABC transports do
transport certain drugs out of the cell
what are ion channels
transmembrane proteins that create pores in the plasma membrane that are permeable to select ions
what are ions selectively permeable for
K+, Na+, Ca2+
what are the 4 categories of ion channels
- non-gated ion channels
- ligand-gated ion channels
- mechanosensitive ion channels
- voltage-gated ion channels
what do all eukaryotic cells contain
ion channels
what are leak channels
non-gated ion channels that allow K+ to freely pass across the membrane
what is potential difference
the inside of the cell becomes more negative than the outside when K+ leaves
what is membrane potential
the potential difference across a cell membrane
how does the electrical gradient become greater
more K+ leaving the cell
what is the equilibrium membrane potential for K+
-90 mV
what are leak channels largely responsible for
setting the resting membrane potential
what is resting membrane potential
refers to the membrane potential of a cell in the absence of any stimulus
where is Na+ and Ca2+ more concentrated
outside the cell
what are Ca2+ ions important in
signalling molecules
what is Ca2+ involved in
molecule signalling, neuronal communication, exocytosis, gene expression, muscle contraction
what are excitable cells
specialized cells that possess gated ion channels that open in response to specific stimuli
what are the three specific stimuli that excitable cells respond to
- changes in voltage
- stretching of the membrane
- presence of extracellular or intracellular signalling molecules (ligands)
what is depolarization
membrane potential becoming more positive
what is hyperpolarization
membrane potential becoming more negative
what are voltage-gated ion channels sensitive to
changes in membrane potential
what is the main energy currency for eukaryotic cells
adenosine triphosphate (ATP)
where does ATP store energy
in the bonds connecting its phosphate groups
what releases the greatest amount of energy
hydrolysis of the terminal phosphate group
what are the two main mechanisms through which ATP production occurs
- substate-level phosphorylation
- oxidative phosphorylation
what does oxidative phosphorylation involve
the transfer of electrons from nutrients to high-energy intermediate molecules
how do F-class proton pumps function
opposite of V-class proton pumps where protons move down their concentration gradient
what is the outer mitochondrial membrane permeable to
ions and most small molecules
what does the inner mitochondrial membrane require
protein transporters to move most molecules
what is a Cristal
the inward folding inner membrane
what is the mitochondrial matrix
the innermost region of the mitochondrion which is surrounded by the inner mitochondrial membrane
where does pyruvate decarboxylation, the citric acid cyle, and B-oxidation occur
mitochondrial matrix
what do electron shuttles do
shuttles high energy electrons from NADH produced in the cytosol into the mitochondrial matrix
when is oxidative phosphorylation not possible
in the absence of O2
what do we need a constant supply of to keep glycolysis going
NAD+
when oxygen is not available what does glycolysis produce in animal cells
lactic acid
during pyruvate decarboxylation what happens in the presence of oxygen
pyruvate is transported into the mitochondrial matrix
what does the citric acid cycle represent
a series of enzymatic reactions that catabolized acetyl-CoA to CO2 and strip electrons from acetyl-CoA to form NADH and FADH2
what is succinate dehydrogenase attached to
inner mitochondrial membrane
what does the electron transport system consist of
4 multiprotein complexes (I-IV) and 2 electron carries known as Coenyme Q and cytochrome C
what do the electron transport proteins assemble into
supercomplexes
what does Respirasome consist of
complexes I-IV, coenzyme Q, and cytochrome C
what does the electron transport system use to pump proteins into the membrane space
the energy from electrons from NADH and FADH2
what is a redox potential
affinity of a molecule for electrons
(more positive the redox potential, the higher the affinity for electrons)
what is NADH oxidized by
complex I
what is the proton motive force
an electrochemical gradient established by the pumping of protons into the intermembrane space
how is ATP synthesized
using the energy stored in the proton motive force
how do protons move
through the enzyme ATP synthase down their electrochemical gradient
what type of pump is ATP synthase
F-class proton pump
what does F0 do
forms the transmembrane subunit that spans the inner mitochondrial membrane
what does F1 do
binds to ADP and Pi and catalyzes the synthesis of ATP
how many protons must pass through ATP synthase for each molecule of ATP synthesized
at least 2
what does B-oxidation refer to
the enzymatic catabolism and oxidation of fatty acyl-CoA
what are the 4 parts of a neuron
dendrites, cell body, axon, axon terminals
what part of a neuron can self-propagate along an axon
action potential
what does action potential do
depolarize the axon terminals, activating the Ca2+ channels, allowing Ca2+ to enter
what is exocytosis
the process through which elevations in intracellular Ca2+ levels promote neurotransmitter release
