Unit 1 Flashcards
What do you have to understand in order to have a molecular understanding of cells?
Biochemical and genetic analysis of the cell
Why are disrupting agents needed to create a cell culture
Need to disrupt the intercellular attachments
What types of interactions are broken by things like EDTA, trypsin, and collagenases?
protein -protein interactions and the ECM
How does EDTA disrupt cellular attachments?
pulls off Ca ions
After treating a tissue with EDTA what are you left with?
A heterogeneous population of cells
What conditions do cells need to grow in culture
Correct pH, essential amino acids, vitamins, growth factors, negatively charged surface, antibiotics and antimycotics
What does a negatively charged solid surface mimic?
extracellular interctions
How are CAMS (cell adhesion molecules) like collagen and fibronectin inactivated?
by removing calcium ions
Define primary cell culture
cells prepared directly from tissues of an organism
Fibroblasts secreting collagen
Muscle cells contracting
Nerve cells forming synapses
Are all examples of what?
Primary cell cultures displaying the characteristics of the tissue from which they are created
Define a cell strain
lineage of cells from one initial primary culture. Continued through “passage of cells”
What is the approx life of primary cells?
50 - 100 doublings
Another name for contact inhibition
Confluency. THIS IS NOT SEEN IN CANCER CELLS>
Characteristics of immortal cells
Grow to a high density
Genetically altered
Solid surface not always required
Result from cells undergoing mutations that do not die.
AKA transformed cells (not like bacteria)
Which phase do you want to study cells in?
Which phase do mutations start to accumulate?
Phase 2
Phase 3
What is the definition of a cell line?
What is one problem with studying a cell line?
Cells that have undergone transformation and are immortal.
**May not accurately represent origianl cell type
True of false: Mouse cells transform much more than human cells
True
What part of an embryronic blastocyst has the stem cells?
ICM (inner cell mass)
Removed (step that destroys the embryo)
What are the additional requirements of embryonic stem cells?
fibroblast feeder cells (hormones and growth factors)
Cytokines (transcription factors)
**give feeder cells or supply cytokines
What are the methods of separating a specific cell type from a heterogeneous mixture of cells?
Physical proerties (ex. size / density)
Physiological properties (affinity / charge)
Flow cytometry
What is the goal of flow cytometry
separate cell types using cell surface antigens
How does flow cytometry separate cells using antigens?
make “tags” that only attach to the antigens on one specific cell type.
For “polar” cells (epithelial) what does the apical surface do?
Passes materials
For “polar” cells (epithelial) what does the laterial surface do?
touches the next cell
For “polar” cells (epithelial) what does the basal surface do?
attaches to cell surface
What are hybridomas used to produce
Produce monoclonal antibodies
How does this picture relate to B cells and antibodies
Polyclonal antibodies are created in B ells agains multiple epitopes of a protein (antigen)
A monoclonal antibody is created by ______________against a ______________________.
Single B cell
Single epitope
What is this picture illustrating?
Formation of hybridomas.
Fuse mutant (immortal) mouse myloma cells that can’t survive on selective medium with mouse B cells that have been exposed to antigen X (and therefore are making antibodies against antigen X).
What does Monastrol do?
inhibits microtubule - based moto kinesis - 5
This is needed to separate poles of mitotic spindle
**Tested as anti-tumor drug
What parts of the microscope are responsible for magnification?
collector
condenser
mirror
Cell doctorine by S and S
All plant and animal tissues are aggregates of individual cells
What is the size range of an animal cell
10 - 30 micrometers
5X smaller than the smallest particle visible to the naked eye
What is the limit of traditional light microscopes
The resolution of light
Why is there limitation to light waves being used for microscopy?
a beam of light can’t probe structures small than its wavelength. so lambda sets resolution
lambda of violet light = 0.45 nm
Define resolution
minimium distance between 2 distinguishable objects
Condenser vs. Objective
Condenser focuses light on specimen
Objective collects light (cone) to create an image
Best resolution of a light scope
0.2 micrometers
What are the three types of methods that can be used to improve light microscopy resolution?
What are the optical methods to improve light microscopy
Brightfield Scopes
Phase Contrast Scopes
What are Physical and Biochemical Methods for improving light microscopy
Fixation
Embed specimen
Section
Staining techniques
What are the advantages to bright field microscopy
simple
inexpensive
no staining
image live cells
What do you have to add in order to create a phase contrast microscope
annular diaphragm
phase plates
These combine refracted and unrefracted light
How does phase contrast microscopy work
Different parts of cell have different refractive indexes.
the anular diaphragm increases light on specimen
the phase plate alters light by 1/4 lambda
Uses polarized light to create ALMOST 3D pictures
Can use living cells!
What materials can be used to “fix” cells?
cross - linking agents
(glutaraldehyde and formaldehyde)
What does fixation do to cells?
cross links macromolecules (free amino groups)
kills cells
partially perabilizes cells
How do cellular stains stain different parts of the cell
use charge to bind
positively charged stains will adhere to negatively charge groups
In order to “see” the sample under a fluorescent microscope what is needed?
A filter that blocks all wavelengths other than the emission wavelength
What is the light source for a fluor. scope
laser
what do you have to add to create a fluor scope
dichroic mirror
How can you see more than one color with direct fluor staining
overlay images from using multiple dyes.
Ion senstive fluorescent dyes can
help observe cell movements
different colors in different concentrations of ion
Ca ions start / involved in many cellular processes
What is the primary antibody binding?
What is the secondary antibody binding?
Why do you need secondary antibody?
Primary: binds to X
Secondary: binds to Fc **not X
the fluoroflor destroys about half of the primary antibody. Secondary amplifies the signal.
Double label fluor. microscopy allows you to
see location of 2 proteins (WRT eachother)
Ex.
Epitope tags are added to proteins through
Transormation (like GFP)
Antibody is made against the EPITOPE not the protein
GFP allows what
locatlization of proteins in living cells
T or F: deconvolustion fluor. micro can create a 3D image
True. By consolidating data taken from multiple slices / images
Confocal microscopy includes what
laser scanning micro (for static object)
Spinning disc (dynamic event)
Laser scanning confocal micro addresses what problems
can use thick specimen (focus light from only one plane and reject light from others)
Gives info on location of organelles wrt eachother (lost in regular b/c of flat image)
T or F:
LSCM can create 3D images of thick specimens without sectioning
True
How does spinning disc lscm work?
spread laser light on 1st disc
light focused on pinholes in 2nd disc (spins)
multiple points on specimen are illuminated at once
lights up entire specimen
Two photon excitation micro uses what
2 lower energy photons out of visible spectrum
No light scattering!
What can you see with 2 photon excitation microsocpy
cells up to 1 mm deep in live specimen
live brain images.
What is total internal reflection fluor micro used for
single focal plane (eg. cells growing on cover slip)
most light is reflected back through the specimen
What does FRAP allow you to see
movement of molecules in real time
How does FRAP work
Hit samples with a laser at the excitation wavelength and the flurofor will eventally stop fluoresceing
Turn off laser
watch where bleached proteins move
How does FRET work
(Fluorescence Resonance Energy Tranfer)
Determines if two molecules are close together or interacting in the cell
Pairs of fluorofor molecules. Donor emits at acceptor excitation wavelength.
See different colors depending on how close molecules are. Have to be closer than 10nm
WHat are the different types of super resolution microscopy
Structured illumination microscopy
Stimulated depletion microscopy
photoactivated localization microscopy
Light sheet microscopy
Structured illumination microscopy (SIM) gives you what resoltuion?
How does SIM work?
up to 100 nm
Passes up theoretical optical limit of resolution using math / computer programs
Illuminate specimen in stripes / rotate. Create a picture without interfenece patterns.
What is the resolution range of STED
(Stimulated emission depletion microscopy)
How does STED work?
resolution = 30 nm
Scan the sample with laser / donut beam. Combine to increase resolution
What is the resolution of photoactivated localization miroscopy (PALM)?
How does PALM work?
resolution up to 5 nm
Merge 1000s of images, but a few GFPs are excited in any given picture.
Use only the “highest level” fluor in each pic
How does light sheet miroscopy work?
illuminate one plane (light sheet) at a time and repeat to get 3D image.
Can be used in living tissue / live specimen
Why do electron microscopes give such a higher level of resolution?
decrease wavelength (electron vs. light) means increased resolution.
wavelength for electrons is 0.005 nm
D = 0.61* lambda / (N*sin alpha)
Steps to prep sample for TEM
Fixation with glutaraldehyde and osmium tetroxide
Embedding sample (epoxy)
Sectioning (50 - 100 nm thick)
Negative staining
How do heavy metal stains work for TEM?
heavy metal stains are electron dense so electrons can’t pass through. Structures appear dark.
Different organelles pick up the stain differently.
Lots of stain = lots of deflected electrons
How does immuno electron microscopy work?
primary antibody binds to protein of interest
primary antibody bound to gold atom
gold is electron dense and appears very dark
T or F
Cryo-electron microscopy is a type of transmission electron mmicroscopy
True
How does cryo EM work?
Like a CT scan
No stain, No fixation
Rotate sample stage, collect multiple pics, merge
Gives 3D picture at atomic level
What is the overall process to separate cellular organelles?
Homogenize tissues
Differential centrifugation
density gradient centrifugation
Mass spectrometry
What methods can be used to break open cells without destroying organelles?
ultrasonic vibrations (sonication)
tissue homigenizer
high speed blender
force through pores of a filter (8 micrometer)
What needs to be added to whole cell extract before separating organelles?
Protease inhibitors
Differential Velocity Centrifugation
Density Gradient Centrifugation
What can mass spectrometry be used for?
Identifying different molecules from mixtures.
Define lysophospholipid
has only one fatty acid chain
Define leaflet
single layer of phospholipid
Identify the functions of the cell membrane
selective permeability
compartmentalize cell
localize biochemical reactions
hold membrane proteins
Identify the function of membrane proteins
respond to signals
energy transduction
ATP synthesis
structural anchors
What are the three classes of membrane lipids
Phospholipids
Sphingolipids
Sterols
Basic structure of a phospholipid
Head = choline + phosphate + glycerol
Tail shape dependent on bond type
Kinks in tail from double bonds
Phospholipids can have multiple phosphorylation states
Why?
1 , 2, or 3 phosphate groups
Abundant in membrane for signal transduction not structure
Sphingolipids
When attached to carbohydrate are NOT phospholipids
Found in mylein sheeth
Sterols
found in animals
Cholesterol
Steroid ring makes them flat and rigid (changes properties of membrane)
Single layers of phospholipids form
Micells
Double layers of phospholipids form
bilayer sheets
How can lipids move in the membrane
flip flop: very slow needs flipase
lateral diffusion (side to side)
rotation (change with lipid next to it)
Flexion (cross tails)
What happens at the transition temperature of a membrane
Above: lipids move and membrane is fluid
Below: lipids become more “solid” and lipid tails straighten and restrict movement
Short / unsaturated fatty acids
Long / saturated fatty acids
more stable
Less fluid
more solid
increased energy to melt
Which property has a larger impact on fluidity
degree of saturation
How does cholesterol impact membrane fulidity?
At 37C dereases fluidity
Below 37 C increases fluidity
Where does lipid composition differ?
Cytoplasmic face and exoplasmic face
Which direction do charged lipids face?
cytosol
Lipid composition of membrane can determine what things
Curvature (also proteins binding)
thickness
charge differences
Match up inner / outer leaflet with cytoplasmic / exoplasmic face
Inner = cytoplasmic (cytosolic)
Outer = exoplasmic
For single layer organelles math faces
Cytosolic = outer layer
Exoplasmic = inner layer
*Inner sections of organelles are like the outside of the cell
Which organelles have double bilyer membranes?
How many leaflets do these have?
Nucleus
Mitochondria
Chloroplast
Have 4 leaflets
*Innermost space is cytoplasmic space
Review enzyme faces!
What are the 3 classes of membrane proteins?
Integral / Transmembrane
Lipid anchored (tight with lipid in leaflet)
Peripheral (less tightly associated/sit on surface)
Typical structure of transmembrane protein?
alpha helices span across hydophobic section of the membrane
Single Pass Transmembrane Protein structure
dimeric
each half (monomer) passes membrane 1X
Hydrophobic residues interact with lipids
Multipass transmembrane proteins structure
all sections span membrane are alpha helices
molecules can bond to regions and cause conformational change
Ex. 7 pass
11 pass
Beta barrel structure
AKA porins
roll up to form pores
phobic on outside
philic on inside
Lipid anchor protein structure
Enire aa chain associated with one leaflet
Associated with lipid anchors
What are the 4 types of lipid anchors
Myristoyl anchor
Palmitoyl anchor
Farnesyl anchor
GPI anchors
Pheripheral membrane structure and removal
Associate by non-covalent interactions
Associate with heads of phospholipids
To remove: change salt conc. or pH
How can you remove transmembrane proteins
Add detergent:
Ionic: will denature proteins
Non-ionic: Will not denature proteins
What forms when a detergent is added to membrane
Micells of detergent monomers and lipids.
Tails of detergent micells interact with tails of membrane and pull them out
Non-ionic will dissolve protein without forming micells
What process was able to determine that proteins are free moving in the membrane?
FRAP
Also showed that cells can keep proteins from moving.
How do cells keep proteins from moving
tight junction
different sides of cells have differnet proteins
confine proteins to lipid rafts
How do lipid rafts keep proteins from moving
have cholesterol that makes them very rigid
GPI anchored proteins move within the raft but stay in raft
rafts have long saturated fatty acid tails
What are the ways cells immobilize proteins
self assemble into large aggregates
interact with extracellular macros
interact with intracellular macros
interaction with proteins on neighbor cell
Forms of transport that require energy
active transport
coupled transport
bulk movement (endo / exocytosis)
3 classes of protein pathways through membrane
channels (gated or open)
transporters (uniporter, symporter, antiporter)
atp powered pumps
K= partition coefficient
Relationship between K and ability to cross membrane
Increase K = more permeable becuase more soluble in lipids
Why do proteins move molecules across membranes faster?
remove rate limiting step of interaction with membrane
Facilitated diffusion is specific, fast, happens in a limited region of the cell
How does the cell keep GLUT 1 active
Turns glucose into glucose 6 phosphate as soon as it enters the cell
How do different cell types all use GLUT trasporters?
They express different types of GLUT transporters that have different affinites for glucose / other molecules
Two key types of uniporters
Glucose (GLUT)
Aquaporins (not beta barrel)
Identify the classes of active transporters
P class (ions)
V class (H ions)
F class (H ions)
ABC superfamily
V class vs. F class pumps
V class generate low pH (high H conc) in vaculoes and lysosomes
F class work in reverse from V and drive ATP synthesis (mitochondria)
Na / K Pump
Pump 2 K into cell
Pump 3 Na out of cell
Works by changing confirmatio and affinity for ions
How do ABC Superfamily proteins work?
Fliip substances across leaflets and out of cell
Have flipase activity
Can pump chemo drugs out of cells.
How do you treat congestive heart failure
inhibit Na/K pump to increase Ca conc in cells
how does coupled transport work?
use energy from gradient to move a substace against gradient
E not from atp
move 2 substances at once. one up and one down
