03 membrane fluidity
TRANSCRIPT
Lecture 3
Cell Biology
Membrane Fluidity
“Fluidity” is the capacity of
individual molecules
to move freely
Lipids behave as
fluids within the membrane
Lipid fluidity is
temperature- sensitive
Phase Transition Point (“melting
temperature”)
Different lipids have different “melting points”
Artificial membranes made with just one lipid type have a sharp MP
Real membranes with a mix of lipids have a
broad MP
OK – the lipids can moveBut what about the proteins??
Classic Indirect Evidence
that membrane proteins have and require freedom of motion
within the membrane
Create artificial membrane vesicles
made of pure dimyristoyl phosphatidyl choline
Add some familiar players
Hormone Receptor
Adenylate Cyclase (makes cAMP)“G” protein
These three work cooperatively so that a hormone signal turns on AC
Above 23°C, this works just fine
Below 23°C, it won’t work at all
This is why cold kills
What about fish that live in cold water?
More unsaturated fatty acids at low
temperature
In each pair below, the sat/unsat ratio is lower
at low temp
Classic Direct Evidences
that membrane proteins have freedom of motion within the
membrane
Antibodies can be used to locate lymphocyte receptor proteins
Antibodies can be used to locate lymphocyte receptor proteins
Initially spread all over the
cell surface
Becoming patchier over time
Eventually all gathered together
in one “cap”
Called “Patching”
& “Capping"
Patching can’t occur if the membrane lipids are gelled
4°C
37°C
Patching results because antibodies cross-link proteins
Each Ab can bind more than one protein, and each protein can be bound by more than
one Ab
Clearly these proteins must be free to move around
Does the antibody induce the mobility, or did it pre-exist????
Each Ab can bind more than one protein, and each protein can be bound by more than
one Ab
Experiment to demonstrate that at least some proteins are
free to diffuse naturally
Add some differently labeled antibody arm fragments (Fab)
But if you let them sit a while . . .
Rate of blending is temperature-
dependent
Wouldn’t it be nice if we could describe diffusion rates with
greater quantitative accuracy?
FLIP: Fluorescence Loss In Photobleaching
FRAP: Fluorescence Recovery After Photobleaching
FRAP
Read about
FLIP on your own
Finally – nowadays it’s sometimes even possible to
watch a single protein diffuse
The Immunogold Technique
So – multiple
evidences demonstrate that
membrane proteins are free
to move within the membrane plane
BUT let’s not go too far with that thought
If all proteins are freely diffusible
within the membrane, then
why do some proteins have
localized distributions?
(Guinea pig sperm labeled with fluorescent antibodies against different membrane proteins)
Integrin a highly
localized protein
The orange patches – localized with a fluorescent antibody
If some proteins have less than full freedom of motion
What are the restraints upon mobility?
Integrins bind to cytoplasmic actin network
This restricts their mobility
Answer #1Sometimes
membrane protein mobility is restricted by interactions with the
cytoskeleton
But other restraints upon mobility are
much more indirect
Let’s return to something we looked
at a little earlier
Band 3 Protein DiffusionCareful analysis of the film shows that Band 3
sometimes acts like it’s “corralled”
Careful trypsinization cleaves P-face of Band 3 protein – removes
“boxed in”-type motion
What are the main proteins that make up the network depicted in this diagram?
Answer #2Sometimes protein
mobility is restricted by interactions with other membrane proteins
Tight (Occluding) Junctions An example of membrane proteins restricting
each other's motion
Tight Junctions form connective seals between epithelial cells
Based on “claudin” transmembrane proteins
(Another important protein type is occludins)
Motion of claudins is restricted by intramembrane and
intermembrane interactions with other claudins
The multiple claudin strands fence off separate “pastures”
A similar example Septins in Yeast Cytokinesis
A similar example Septins in Yeast Cytokinesis
Some membrane proteins are found only
in the daughter cell
Septins Part of a peripheral protein
network at the mother-daughter interface
Are septins restricting mobility and creating separate membrane domains? How could you tell?
A temperature-sensitive septin mutant
Natural and engineered
mutations are another tool for your
box
FINALLY Sometimes protein mobility
is restricted by surrounding lipids
Lipid Rafts
Some “patches” of membrane stay
together after mild detergent treatment
Suggests their composition is different
from the rest of the membrane
Rafts are enriched in sphingolipids and cholesterol
Lipid rafts stained with Filipin (binds to cholesterol)
Lipid rafts are also enriched for certain membrane proteins
Especially (but not only)
proteins with fatty acid prosthetic
groups
Some protein enrichment may be due to physical thickness of raft
Rafts may act to “trap” wandering proteins of the right types
Proteins with longer-than-standard membrane-spanning domains
Thus, rafts influence functional associations between membrane proteins
The fungal pathogen Candida albicans (Filamentous fungal cells exhibit tip-growth)
Stained with filipin
For Next LectureCore-Level Things To Review in Advance:
What is the difference between "anabolism" and "catabolism"?
What is the structure and what is the metabolic function of glycogen?
What is the general effect in the body of epinephrine (adrenaline)?
What is glucagon, and how does its general effect compare to that of epinephrine?
What is the difference between ATP and cyclic AMP?
In which of the following is a hydrolysis reaction involved: 1) removal of, or 2) addition of the terminal phosphate group of ATP?
Does it require or release energy to remove the terminal phosphate of ATP?
Is the delta-G (change in free energy) of that reaction a positive or a negative value?
What is GTP? Have you ever run across a protein that binds to GTP?
Advanced-Level Things to Learn Independently Before Class:
What is the meaning of the term “signal transduction”? How would you define a “second messenger”? Be sure you do more than merely memorize a dictionary definition – would you recognize when something is or isn’t a second messenger?
Who was Rube Goldberg, and what was his cultural contribution?
Also
Read and understand the basic points made in pp. 169 – 177, paying particular attention to allosteric proteins and protein phosphorylation
NEXT TIMESignal Transduction
How cells sense and respond to their environments