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