Cell Biology 250

9.1.15
Lysozome
We discussed the lysosome where proteins come and eventually get
degraded.
There are a few different ways to put stuff into the lysosome:
autophagy, endocytosis and phagocytosis
Tay-sachs disease we have a deficiency in one of the enzymes (beta
heaminodase A) where the protein has a different structure than its
supposed to so it cant get into
Enzyme b hemanidosa a: degrades gangliosides
What we can do: screen for drugs that can alter enzyme/protein shape
so that it can get transported inside the lysosome.
Cell
Has polarity (in a direction), going in one way. None of this is by
accident.
How do these movements occur: cytoskeleton. Gives polarity.
In EM, we would see vesicles containing proteins going outside, etc.
Trans golgi is what step? Last! Step
Nucleus: what does it do?: regulate movement of molecules into
cytoplasm/ outside.
Mitochondria:
disease angle (mutation in mitochondrial DNA)
green mitochondria, red lysosome
where cell gains ATP
defects in mitochondria: human diseases associated with defects
Doug Wallace: discovery of human disease with mitochondrial
Loss of function in oxidative phosphorylation
Tissues that require energy, like retina, leads to blindness.
Not making enough ATP: Major issue

What is being done: Ways to do an end-run in mitochondrial defect.

Mitochondrial donor!!!
Pro-nuclear transfer
Remove nucleus from donor egg. Take donor oocyte enucleated .
but you can have carryover of bad mitochondria, when she goes on to
have children, some have defective

Fluorescence Microscopy
Tagging Proteins: GFP
Pick up an order of magnited
9-12: track a fluorescent protein. Actin hooked up to GFP. Use
fluorescence
Tag a protein with GFP. Nuclear envelope protein, lamin: Regulatory
DNA. Create an animal with an extra gene with same lamin sequence
with fluorescence. Its where lamin is expressed.
In other words: you can add a gene (GFP, fluorescent protein). Could
affect the type of protein that you are making. Make a very different
structure, sending it to the wrong place of the cell.
Tag a protein: Can have consequences in the cell.
GFP: gene that can glow green, protein coded by a single gene.
Inserted correctly, rna can initiate transcription
Fluorescence: where is the protein found\function of protein is not
changed by fusion. See localization of the GFP tagged protein, but view
distribution in a living cell over time.
Tagging Protein: TetraCysteine tag of protein
Connexin (Gap junction connect cell), tag on connexin cell. After
binding of a dye, can we see where it localizes. Tetracysteine tag. Done
localization and process to EM,
Tetracysteine allow you to create electron dense precipitate too. Can
see EM of the exact same structure. Higher EM magnitude.
Localized and then in finer detail
Immunofluorescence microscopy:
Purify protein
1. Produce antibody
2. Localize to tissue (incubate with tissue, want to

3. Fix or immobilize (formaldehyde, cross-linking agents)- stop

enzymatic activity.
4. Make tissue permeable.
5. Primary antibody could be fluoresced (Easy). Fluorescent tag
6. generate second antibody (Goat Ab) amplified response, get
localization
What if you cant reproduce Ab? Some proteins suck! Some ways
around it, we
would have to tag the protein probs. Create GLUT with tag
p. 409
Detect specific proteins in fixed cells.
Antibodies are proteins secreted by WBC bid with high affinity to their
antigen
Drosophila w. fluorescence microscopy
Wg localized with red fluorescence
Co-localization. Express proteins at the same time, at the point of their
intersection, can see whereve they overlap and they eliminate.
You can see the point of co-localization.
Red independent proteins are localized
Localized, where are the proteins interacting all with fluorescence
microscopy
book: Limitation of fluorescence
fluorescent light emitted plane of focus and molecules above and
below, blurry pic. Difficult for determine actual molecular
arragenemnts.
Confocal Fluorescence Microscopy
Look at small plane.
Obtain images from a specific focal plane and exclude light from others
3d can be seen. NO BLURRING from other background fluorescence.
Blocks all light except where you are focused.
Deconvolution Fluorescence Microscopy
Get rid of light, without pinhole? Remove fluorescence remove how
much fluorescen it contributes. Detail without

Gets reassigns out of focus photons to their site of origin.

Tirf Microscopy
Internal reflection. Only looking at an excitable at an angle, so light
barely pierces the crystal. Happens at the contact point between cell
and right at contact.
We can look at proteins at adjesion sites ad look at kinetics with
microtubules.
Electron Microscopy
Magnets condense beams. Electrons pass through. Metal binds tightly
to tissue. Not blocking electrons. Tissues are dead. Localize enzyme in
very fine detail. Localization
Immuno + EM
Localize catalysae, generate an antibody. And with EM, find electron
dense staining. Use gold molecules. To protein A. and get very fine
localization.
Bridge chem + bio with these techniques

Summary: Major functions of cell that welll discuss.

1. Tay-Sachs Disease: gangliosides, B-Hex A; make drugs that allow
enzyme to change structure to go into lysosome.
2. Cell has polarity: cytoskeleton, EM shows us this,
3. Mitochondria, defects in human disease; oxid phosphor; pro-nuclear
transfer for mitochondrial

9.4.15
Electron Microscopy
Transmission, block through
Scanning- sample coated with metal and reflects electrons, so we get a
surface view, structure of head of drosophila etc. different levels of
organization.
TEM with collagen
SEM: whole cell, fairly fine level at cellular level (not sub-cellular)
FIX SAMPLE, DEHYDRATE, IMPREGNATE WITH PLASTIC, CUT SECTIONS
STAINED WITH HEAVY METAS. THIN SLICE OF CELL.

Electron dense marker

FACS
Live cell, incubate with AB, and fluorescence. Sort cell by level of
fluorescence. Look at multiple markers (Red and green aB), select for
cells with diff proteins
TRANS-GENIC FISH, neural protein that can make it glow. Make brain
cells.
Cell mixture incubated to a dye, linked to an antibody
All about intensity and different Cool graph with FACS
Proteins
Secondary structure: do something different, structure and function
Alpha helix: face on one face, periodicity where R groups are.
Beta sheet: pleated, kinked series of steps, R group in DIFFERET
DIRECTIONS
Hydrogen bond across sheets (looks like dry wall). Cells
due use this
to their advantage to make channels or pores
Alpha helix: create a face with hydrophobic amino acids. In solution,
they come together, alpha helix, tightly bonded. Highly charged
surface too.
Beta turns: structure and reverse direction (Transcription factors) have
helices that bind together tightly. Attached to other domain. Helix turn
and another domain. DNA bound tightly to dimer. ]
Motifs (Little)
Charged groups face outside
Coil-coil motif and we create a dimer.
Charge on one side and other amipathic helix, dimerizes with
hydrophobic residues. Leucine zipper (Transcription factor) high
frequency on one side. One motif.
Calcium sensing
EF hand/helix-loop-helix motif
Interacts with calcium. Super important signaling molecules. Want low
Ca in cytosol. Come from outside or within cell

Calmodulin- interacts with EF hand motif, interacts with AA. Coordinate

with calcium, changing shape of protein!
Calcium + calmodulin -> protein diff conformation, different areas.
Zinc-finger motif
Dna binding proteins regulate transcription
Domains (Big protein)
Flu-virus protein that mediates infection. What types of domain that
interacts with receptor?
Globular domain
Trans-membrane domain, specificity
Two domains separated: fibrous domain, not highly structured (length)
Often site of recombination, re-assortment of domains.
Quaternary Structure
all bout the combination of proteins together!
3.2 Protein Folding
Chaperones
Most stable configuration, why do we end up with one structure. CELL
helps nascent proteins. Chaperone proteins help to make folds
correctly.
HSP 70
HSP 70 HEAT Shock protein?
Traumatize a protein at a high temp denature and it might not fold
right back up the same way
Every organism has a heat shock response to refold proteins, fold
nascent proteins.
Two domains
1 atp, AND GROWING INTERACTS at atp form, when it hydrolses.
Hydrophobic pocket occupies resides and helps it find most stable
hydrophobic associations.
Proteins can crash to each other. Chamber where AA slowly tested
without negative effects. Chamber where you can form proteins.

Protein folding fails, sometimes. Even with normal proteins equence

fails, or a mutation in a polypeptide sequence, which predisposes
protein to misfold during the process or with chaperon.
Alzheimers
Alzheimers plaque: proteins misfold. Neurodegenerative disease,
Wild type: alpha- helix,
Mutation or spontaneous conform. Change. Second conformation it can
multimerize, main issues with disease, or intermediates are toxic
Protein: change in conformation, crash out of solation.
Three ways to prevent or treat neurodegenerative
disease:
1. Drug one: slow or prevent alpha helix flipping into beta sheet
2. Between beta sheet structures and prevent them from forming. The
multimers
3. TOO Late: Multimers in a disease state, consider: split apart or
DISAGGREGATE. One possible mechanism.
Read abstract: HSP 70, exploit chaperons up regulate HSP 70. We
would reverse
Multimeric assemblies with and without HSP. Up regulate in tissue,
fibrils begin to degrade
FIRST STEP TOWARDS TREATMENT OF NEURODEGENERATIVE DISEASE
Degradation
Regulate protein: how much synthesized, degraded, activity level of
enzyme
Aberration in protein. Protein mutant shape, aberrated: recognized and
destroyed. Regulate at a certain or critical level.
Main players: Ubiquitin and Proteasome
Attach Ubiquitin at a polypeptide Lysine residue. Ubiquitin going to add
on to that protein. Also has lysine residues. Red flag on protein. Poly
ubiquination.

Goes to proteasome that has a cap on two ends that recognize

polyubiquitin, once it sees it and binds to protein through ubiquitin. Cut
after basic amino acids
Normal protein not folded by chaperon- ubiquiting ligase target
proteins and efficient at recognizing motifs and domains. Needs
ubiquitin on
Lysine residues.
Nascent-> cytoplasmic protein goes to proteasome.
9.8.15
How to isolate proteins; purify.
Centrifugation
Separate Organelles and proteins: centrifugation. Nuclei go to down.
Supernatant on top. Or separate them in a gradient.
SDS Gel
Use SDS: turns monomers, opens up a polypeptide to stretch out
through length
Detergent SDS binds hydrophobic areas on protein and add negative
charges
SDS -> NEGATIVE charges. Got rid of multimers. FROM NEGATIVE TO
POSITIVE
Separate by SIZE.
Many times we grind up a cell.
Identify a single polypeptide on a WESTERN blot.
Two-dimensional gel electrophoresis Isoelectric focusing, protein
moves under charge.
Two polypeptides with exact same length. One positively charged, one
neg. charged.
Separate by charge not weight.
Create a gel without SDS, but use ampholytes with a ph Gradient.
Isoelectric focusing, protein moves under charge.
Separate proteins and by size

Grind up cell lysate w. a complex mix of proteins

1. run isoelectric focusing separating acidic vs. basic class
2. attach to sds, layering the first physically on top of first
3. separate by size.
Get a lot of detail. Mutant vs. wildtype: do spots appear or disappear?
Sequency polypeptide in spot and sequence that dna sequence to
isolate gene, and you know what gene is used.
Liquid Chromatography/ gel filtration/ column
Large and small. Gell filtration
With beads and diff sizes.
Large proteins fly through column and ignore the beads
Small want to be with beads
Separate by size.
Big guys quickly, sds they are slow
Ion Charges
opposite charges attract. With electrostatic.
Adding a higher concentration of sodium that outcompetes a neg
charged and collect different fractions. Like isoelectric focusing. Fine
separation.
Antibody-Affinity Chromatography
A little easier to purify or isolate a complex the multimeric association.
Grind up cell with a column. Bead with antibody. Separate protein
recognized by antibody.

Membrane Structure
1. Proteins through membrane: integral (outside and in) attached to
membrane
2. Lipid-anchor protein: doesnt go through completely.
3. Peripheral membrane: outside or inside, and come off easily from
membrane. Add salt they are removed easily (Electrostatic
interactions)

HIV, buds off from lymphocytes from cell membrane. The more we
know, huge considerations.
Not memorize structures.
Lipids
Membrane lipids, amphipathic:
phosphoglycerides, are in bilayer with carged head groups (face
extracellular side) acyl hydrocarbon chain. Dont have any OH,
Sphingolipids will bond better to other lipids. Make the protein be less
mobile
In general: these are stiffer (less fluid) in the membrane. The chemistry
depends how fluyd. produced in golgi and face extracellular domain in
plasma membrane.
Tay0sachs cant clear out sphingolipids (glycolipid)
Cholesterol- highly or less likely fluid
Ampipathic, OH on side of aqueous. Key constituent of membranes.
Three classes of lipids
Saturations: are important
Liposome
1. use to deliver drugs (Fuse with cells) Can see what you can alter
with liposome.
Purify proteins out of membranes with an organic solvent.
Vesicle
Bi-directional. Whatever is in the lumen is extracellular
Something is endocytosis, cytoplasmic whatever is from outside the
cell is in the lumen.
Modifications (Enzymatic level) is done in the lumen.

Fluidity
Temperature dependent, can be measured.
FRAP (fluorescence recovery after photobleaching)
Determine rate of recovery and extent of proteins or lipids

Labeling membrane proteins with a fluorescent tag on it.

How fluid is membrane
Bleach area and see how efficiently bleach molecules turn back to
fluorescence.
Results: never really achieve 50% of the fluorescence.
Liposome with only fluorescent protein we get 100%
Proteins and lipids in cell membranes dont flow around, there are
restrictions to flows.
What modifies fluidity?
(Temp, sphingolipids, cholesterol, how long the tails are,
saturated or
unsaturated, and interactios of proteins)
Lipids Rafts
Idea that cell membranes arent a mix.
Within cell membrane, area distinguishable by less mobile (lipids rafts)
Enrichment of sphingomyelin(lipids) and cholesterol.
Those two make you a lot slower and denser in plasma membrane.
These areas are where proteins in cell signaling are enriched.
FRET: MEASURING DISTANCE
If molecules proteins are close to each other, you can emit a
wavelength that is higher.
When we add calcium binds to calmodulin and CTP and YFP go to FRET
Going to measure calcium concentrations. Look to see if you get FRET
or not.
Extent of calcium conc. A lot of 535.
Cell signaling + lipid rafts + fret
Experiment: calmodulin + RGS4 protein responds to calcium. Targeted
to specific part of membrane or not?
ECFP (Attaches to calmodulin)
Raise calcium, calmodulin changes shape and interacts with RSG4
CFP and Venus are close enough to make a FRET reactions
CFP and YFP brought within 10 nm. Calmoduin interacts with protein it
regulates.
You can deplete lipid rafts!!!!!

If you deplete lipid rafts you should see less activity. Calmodulin + rgs4
want to find each other.
Another experiment
CAV1 protein binds to cholesterol
Notch sits in rafts. Its not everywhere.
Signaling molecules function within lipid raft regions of membranes.
Occurs in a subset of cell membrane.
9.10.15
Complexity of membrane structure. Disproportion. Domains of activity
in key places in cell.
Phospholipids, susceptible to enzymatic activity
Phospholipases C cuts.
Enzyme cuts used to degrade. Particular classes are signaling
molecules. Enzymatic. Occupy. Not just structural.
Membrane Protein Components and Function
33% gene encode a membrane protein,
1. Proteins through membrane: integral (outside and in) attached to
membrane
2. Lipid-anchor protein: doesnt go through completely.
3. Peripheral membrane: outside or inside, and come off easily from
membrane. Add salt they are removed easily (Electrostatic
interactions)
Transmembrane protein
Distinct run of uninterrupted hydrophobic amino acids
Talk about ampipathic (coil coil)
Hydrophobic residues, leucine zippers. Transmembrane to exist in lipid
bilayer, transmembrane hydrophobic surfaces on both sides.
Hydrophobic on both side
Glycophorin: integral membrane protein
Protein can pass by membrane many times