W9 L2 - Cell Membranes and Experimental Techniques

Flashcards about cell membrane's fluidity and experimental techniques.

What processes is membrane fluidity important for?

Signaling, cell division, membrane fusion, and redistribution of lipids and proteins.

What is the fluid mosaic model?

A model proposed in 1972 that underpins our understanding of cell membranes, emphasizing their fluidity and mosaic structure.

Who proposed the fluid mosaic model in 1972?

Singer and Nicholson.

What are the main components of the fluid mosaic model?

Bilayer structure with transmembrane and integral proteins, asymmetry between the outer and inner leaflets, and lateral diffusion of proteins and lipids.

What are transmembrane proteins?

Proteins that span both the outer and inner leaflets of the bilayer.

What are integral proteins?

Proteins that embed themselves into only one half of the bilayer.

What does the asymmetry of the bilayer refer to?

The top half of the bilayer looks different from the bottom half due to the arrangement of proteins and lipids.

What is lateral diffusion?

Sideways movement of proteins and lipids within the plane of the bilayer.

What is flip-flop movement?

Movement from one side of the bilayer to the other.

Why is flip-flop movement unlikely for proteins and lipids?

It is unlikely due to the amphipathic nature of the molecules, making it difficult to pass through the hydrophobic core.

Summarize the fluid mosaic model in one sentence.

A mosaic of protein molecules embedded within a fluid lipid bilayer.

What did Charles Overton's experiments show in 1895?

Molecules dissolved in lipids enter the cell more easily than insoluble ones.

What did Charles Overton propose in 1895?

He proposed that the cell was surrounded by lipids.

What did Ivan Langmuir find when making artificial cell membranes?

Phospholipids formed a monolayer when dissolved in benzene and evaporated.

What did Goulter and Grendel find when isolating phospholipids from red blood cells?

The lipid layer was twice the area expected for the surface area of red blood cells.

What did Goulter and Grendel propose?

Cell membranes are phospholipid bilayers.

What did Davson and Danielli propose about protein interaction with the bilayer?

Proposed that beta sheets interact with the hydrophilic heads of phospholipids, creating a sandwich model.

What did Robertson's electron microscopy show?

Saw "railway tracks" under electron microscopy, interpreted as protein layers sandwiching the phospholipid bilayer.

What do the "railway tracks" actually represent?

The dark lines are the hydrophilic heads, and the white space is the hydrophobic core of the bilayer.

What is lateral diffusion in the context of membrane fluidity?

Movement within the planes of the bilayer.

How do hydrophobic tails flex?

They can wiggle and move around.

What enzymes catalyze flip-flop reactions?

Enzymes like scramblase and flippase.

What was done in the hybridization experiment in the 1970s?

Fused mouse and human cells together using a virus to create a heterokaryon and observed the intermixing of fluorescently tagged membrane proteins.

What fluorescent tags were used in the hybridization experiment?

Fluorescein and rhodamine.

What did the intermixing of red and green signals suggest?

That membrane proteins diffused without restriction.

How can membrane protein movement be restricted?

Movement can be restricted by the cortical cytoskeleton, extracellular matrix, and tethering to other cells.

What is the cortical cytoskeleton?

Components of the cytoskeleton that sit just underneath the plasma membrane.

Give examples of components of the extracellular matrix (ECM).

Collagen and fibronectin.

What is the integrin family?

A protein family that links membrane proteins to the extracellular matrix.

Where can you find diffusion barriers in cells?

Epithelial cells of the gut linked together by tight junctions, creating diffusion barriers.

What is spectrin?

A protein found in red blood cells that supports the plasma membrane and gives it flexibility.

In what disease is the flexibility of red blood cells lost?

Sickle cell anemia.

What are ERM proteins?

Proteins that link transmembrane proteins to the cytoskeleton.

Give examples of ERM proteins.

Ezrin, Radixin, and Moesin.

What are the two conformations of ERM proteins?

Closed (inactive) and open (active).

What happens when an ERM protein opens?

Exposes a FERM domain that binds the protein of interest and an actin binding domain that binds the cytoskeleton.

What controls whether an ERM protein is closed or open?

Phosphorylation events.

What are microdomains?

Domains specialized for their function, resulting from saturated and unsaturated lipids.

What characterizes microdomains or lipid rafts?

Enrichment in cholesterol, leading to a more ordered domain surrounded by a more disordered domain.

What types of lipids are typically enriched in lipid rafts?

Sphingolipids and cholesterol.

What is the function of lipid rafts?

They help with signaling functions.

What does phase separation refer to?

The difference in whether lipids are in a fluid or less fluid state.

How has the fluid mosaic model been expanded?

Linking between transmembrane proteins and the cytoskeleton, linkages between proteins and the ECM, and specialized domains.

What does the lipid raft model propose?

Lipid rafts are enriched for certain types of lipids, and some proteins localize there.

What does the picket fence model suggest?

The cytoskeleton is the fence, and transmembrane-cytoskeleton interactions are the pickets, creating diffusion barriers.

What does the protein island model suggest?

Membrane proteins are always localized together in specialized domains, which may or may not be enriched in cholesterol.

What are lipid rafts?

Areas enriched in cholesterol.

What are protein islands that are not enriched in cholesterol called?

Non-raft islands.

What does FRAP stand for?

Fluorescence Recovery After Photobleaching.

How does FRAP work?

Using a strong laser to bleach an area of a cell with fluorescently labeled membrane proteins and observing how quickly the fluorescence recovers.

What is the immobile fraction in FRAP?

The fraction of proteins that do not recover fluorescence after bleaching.

What is the mobile fraction in FRAP?

The fraction of proteins that recover fluorescence after bleaching.

What is the half-time to recovery (T half)?

The time it takes for half of the fluorescence to recover after bleaching.

What are the normal diffusion units?

Micrometres squared per second.

What lipids were used in the supported lipid bilayer experiment?

DOPC and DPPC, which are lipids.

Which lipid is more fluid, DOPC or DPPC?

DOPC (unsaturated lipids).

What do supported lipid bilayers do?

Mimic cell membranes by forming a bilayer on a glass surface.

What does GUVs stand for?

Giant unilaminar vesicles.

What can GUVs be used for?

Look at things like cell growth and division, and also the different phases of the lipids, so like phase separation.

What is a limitation of FRAP?

Looking at a large number of proteins across a large area in the membrane.

What is single particle tracking?

Fluorescently tagging a protein and tracking its movement within a membrane.

What can proteins be labeled with in single particle tracking?

Fluorescent proteins, organic dyes, labeled antibodies, or gold particles.

What happens if there is too high of a labeling density?

If you label too many of those proteins, this will just, this whole cell will be red, right? And you won't be able to distinguish different things.

What is fluorophore brightness?

Have to think about fluorophore brightness. So, for the time that I image, is that fluorescence going to be there or as soon as I put it under the microscope, is that fluorescence going to disappear?

What issue can arise from the size of the tag used for labeling proteins?

Something really big on, that introduces what we call steric hindrance. So that means that you might interfere with diffusion of the protein or you might interfere with the biological function of that because it can't get to where it needs to be because it's got something big and bulky attached to it

What types of behaviors are observed for molecules tracked during single particle tracking?

Free to move, confined to a microdomain, or largely immobile.

In what type of membrane, artificial or cellular, do molecules move more freely?

Artificial bilayers because no barriers.

How do you quantify single particle tracking?

Calculating the mean square displacement.

What is displacement?

The displacement or the difference between two points.

What do different mean square displacement traces indicate?

Straight line indicates free diffusion; plateaus indicate constrained movement; directed movement is active transport.

How do you calculate diffusion from MSD?

Using the gradient to then get out your diffusion coefficient.

What is the purpose of formative assessments?

Designed to help you understand what you know and what you don't know.

What is the purpose of summative assessment?

Tests what you've learned over the course of the module, for example.