Biological membranes

Question 1

How does size difference between eukaryotes and prokaryotes/viruses effect diffusion?

Answer 1

Ratio of surface area to volume becomes smaller as the cell gets larger. Since eukaryotes are larger they have a a smaller surface area to volume ratio than viruses and prokaryotes. Therefore size can effect the diffusion of metabolites.

Question 2

Why is compartmentalization advantageous? Name 3 examples

Answer 2

It allows different reaction to be separate from one another.
1. Production/ use of energy
2. Synthesis / degradation
3. Oxidation / reduction

Question 3

What are 4 Functions of a membrane?

Answer 3

1. Semi-permeable barrier
2. Assembly-line for metabolic rxn’s and enzymatic machineries
3. Give identity to cell types and intracellular organelles
4. Signalling organelles and cells

Question 4

What does a semi permeable barrier allow to pass?

Answer 4

gases and small uncharged molecules but machines are needed for ions or larger molecules.
Ex. Signalling molecules such as reactive oxygen species(ROS)

Question 5

Give examples of an organized assembly line and enzymatic machineries.

Answer 5

-Glycosylation (sugar-adding or modifying) machinery of ER
-ATP-producing respiratory chain within mitochondrial cristae

Question 6

What is a example of membrane recognition

Answer 6

Recognition of the ER membrane by the signal peptide

Question 7

Give an example of signalling interactions between organelles

Answer 7

When the ER tubules wrap around mitochondria

Question 8

What do biological membranes consist of?

Answer 8

Membrane lipids such as
-phospholipids
-shingomyelin and cholesterol
-and small amount of glycolipids

Question 10

What is the difference between PE and PC phospholipids?

Answer 10

PE: are more conical, limits fluidity and adds curvature
PC: cylindrical, less curvature

(This is because the head group determines the charge/ extent of saturation determines membrane fluidity)

Question 11

Function of cholesterol?

Answer 11

Formation of rafts and membrane stiffening

Question 12

What two things determine asymmetry of membrane?

Answer 12

1. Asymmetric lipid composition, aka when amino acids (PS and PE) are enriched in the inner leaflet where as glycolipids are only on the outer leaflet
2. Proteins have unique orientation in membrane which is established during their initial insertion

Asymmetry can allow proteins to execute distinct functions on either side

Question 13

What is the histology protocol?

Answer 13

Histology protocol:
1.Formaldehyde fixation to cross-link proteins on lysine residues

2. Paraffin or plastic embedding
3. Washing and dehydration with alcohols
4. Clearing with xylol or toluol to remove alcohols
5. Block creation and cutting in thin slices
6. Mounting on glass coverslip
7. Rehydration
8. Staining with hematoxylin (nuclei) and eosin
9. Dehydration

10.Mounting and imaging

Question 14

Immunofluorescence protocol

Answer 14

1. Formaldehyde fixation
2. Incubation with primary antibodies
3. Washing
4. Incubation with secondary fluorescent antibodies
5. Washing
6. Mounting on glass coverslip and imaging

Question 15

What is the difference between primary and secondary antibodies?

Answer 15

Primary antibodies bind to antigen well secondary antibodies bind to primary, secondary antibodies usually are marker coupled

Question 16

Wide field vs Confocal

Answer 16

Wide field: is achieved by focusing light in a single spot in the centre of the pupil plane

Confocal: the pupil plane is filled, which generates a focused spot in the image plane to be scanned across the field view, Thus emitted light is either detected simultaneously using camera or point by point using photo detectors (200nm)

Question 17

Super resolution microscopy: STED

Answer 17

STED: excitation beam is overlaid by a depletion laser beam, with atleast one local intensity minimum (usually in focal centre) to inhibit or deplete fluorescence emission, apart from the local intensity minimum. This restricts spontaneous fluorescence emission to that region and shapes the effective scanning spot size to sub-diffraction scales. (30-100nm)

Question 18

EM protocol

Answer 18

1. Formaldehyde/glutaraldehyde fixation
2. Secondary fixation with odium tetroxide
3. Staining in uranyl acetate
4. Dehydration
5. Plastic embedding, block creation and cutting in thin slices
6. Optional incubation with primary and secondary (gold labelled antibodies)
7. Mounting and imaging on electron microscope

Question 19

Live cell imaging protocol

Answer 19

1. Introduce cDNA expressing GFP-fused protein of interest on glass coverslip attached cells. Wait 1-2 days
2. Mount on microscope with water-specific lens, immersed in growth medium
3. Imaging

Reveals both shape and time dependent movement using organelle markers