Cell membranes and cell junctions

Question 1

Schleiden and Schwann

Answer 1

Famous for cell theory, suggested “barrier” between cells

Question 2

Overton

Answer 2

Stated that cell barrier has to be lipid soluble

Question 3

Langmuir

Answer 3

Famous for Langmuir Trough- took some phospholipids and put them on a water surface so they would form a layer. Pressure vs distance graph- first part is a monolayer, then the dip in the graph represents the molecules flip flopping. The last part is a bilayer. These results suggested that cell membranes are either monolayers or bilayers

Question 4

Gorter and Grendel

Answer 4

Examined the surface area of lipids using red blood cells and found that the surface area was 2 times the size of the cell. They concluded that the cell membrane was made of a phospholipid bilayer.

Question 5

Davson-Danielli cell membrane model

Answer 5

Danielli and Davson carried out the Mudd and Mudd experiments- used WBCs and RBCs and put them in an oil/water mixture. WBCs preferred the water and RBCs preferred the oil. This suggested that WBCs have more proteins than RBCs. Contributed that proteins are involved and form distinct layers, like a sandwich

Question 6

JD Robertson cell membrane model

Answer 6

According to Robertson, the unit membrane consisted of a bimolecular lipid leaflet sandwiched between outer and inner layers of protein organized in the pleated sheet con­figuration. He demonstrated that membranes had a characteristic tri-laminar appearance con­sisting of two parallel outer dark (osmiophilic) layers and a central light (osmiophobic) layer

Question 7

Dan Branton freeze fracture

Answer 7

Branton used freeze fracture and electron microscopy to study cell membranes and found that the “bumps” in the membrane of RBCs were actually proteins, which is the “mosaic” part of the fluid mosaic model. The P and E faces are the sides of the membrane viewed from inside the cell.

Question 8

In the 1970s, which 4 key experiments were critical and allowed us to move forward without new ideas about cell membranes?

Answer 8

1. Freeze fracture
2. Cell fusion
3. Cell patching (capping)
4. FRAP- fluorescence recovery after photobleaching

Question 9

Cell fusion experiment

Answer 9

Frye and Edidin fused mouse and human cells and examined the distribution of membrane proteins after staining with anti-mouse and anti-human antibodies labeled with different fluorescent dyes. Immediately after fusion, the proteins were located in different halves of the fused cell surface, but after a brief incubation at 37 C the proteins were intermixed. This demonstrated that the proteins could diffuse laterally in a fluid membrane.

Question 10

Cell capping/patching experiment

Answer 10

Fluorochrome tags are used to observe cell capping. Monoclonal antibodies can be tagged with fluorochromes and the antibodies will bind to the antigen they have an affinity for, which is a protein on the cell membrane. This technique can provide information about which proteins are present on the cell membrane and their concentration.

Question 11

FRAP experiment

Answer 11

This technique is used to examine the lateral movements of specific plasma membrane proteins and lipids. To monitor lipid movement, phospholipids containing a fluorescent substituent are used. For proteins, a monoclonal antibody with an affinity for the POI is tagged with a fluorescent dye (ConA). This reveals the rate at which membrane molecules move as well as the proportion of molecules that are laterally mobile

Question 12

Which factors limit lateral plasma membrane protein mobility? (2)

Answer 12

1. Cytoskeleton- the cytoskeleton is composed of fibrous proteins giving the cell strength and mobility. It also provides tracks for organelles to move on, making sure they’re in the correct location
2. Extracellular matrix- coordinates cellular functions and activates intracellular signaling pathways

Question 13

Fluid mosaic model (Singer and Nicholson)

Answer 13

The membrane is composed of a bilayer of phospholipids as well as membrane proteins that give the cell membrane its unique functions. Phospholipids can move and spin laterally within the membrane- the membrane has a fluid olive oil-like consistency. The hydrophobic core of the bilayer prevents water soluble molecules from moving across the membrane without assistance.

Question 14

How are RBCs important to our knowledge of cell membranes?

Answer 14

RBCs are critical to the development of cell membrane theory- they are plentiful and an easy population to purify. There are few cell proteins and few contaminating organelles- just a bag of hemoglobin. You can make inside out and right side out vesicles- something you can’t do with other cells. Gave us more information regarding the topology of integral membrane proteins (or how they’re arranged in the membrane).

Question 15

RBC ghosts

Answer 15

Cells are lysed by swelling and bursting in a hypotonic solution, leaving behind membrane ghosts (membrane remnants). Ghosts can be leaky (open) or sealed, and if the membrane is disrupted and resealed, you can create right side out and inside out vesicles

Question 16

Spectrin

Answer 16

Cytoskeleton protein located in the peripheral membrane, anchors proteins in place like glycophorin.

Question 17

Glycophorin

Answer 17

An integral membrane protein- principal RBC glycoprotein

Question 18

RBC membrane protein network

Answer 18

The cytoskeleton is a network of proteins on the inner surface of the plasma membrane that is responsible for maintaining the shape, stability, and deformability or flexibility of the RBC

Question 19

Hereditary spherocytosis

Answer 19

Causes a defect in a protein that forms the outer membrane of the RBC- this results in the RBCs being spherical instead of their usual disk shape. This is a hereditary hemolytic disorder where the blood cells are destroyed more quickly. Clinical features include anemia, jaundice, fatigue, and splenomegaly. In elliptocytosis, the cell shape is elliptical rather than spherical

Question 20

Vernon Ingram

Answer 20

The “father of molecular medicine”. He found that a single change in DNA sequence can result in sickle cells instead of normal red blood cells

Question 21

Formation and study of pure phospholipid bilayers

Answer 21

Biological membranes are prepared with an organic solvent, like chloroform and methanol to stabilize the phospholipids and cholesterol. If the extracted materials are put in water, they will form a liposome with an internal aqueous compartment. If phospholipids are dissolved in solvent and applied to a small hole in a partition separating two aqueous compartments- this system can be used to study the physical properties of bilayers like their permeability to solutes.

Question 22

Permeability of pure phospholipid bilayer

Answer 22

The combination of lipids and proteins in the phospholipid bilayer gives it its permeability capabilities. A pure phospholipid bilayer is impermeable to almost all ions, amino acids, sugars, and other water soluble molecules. Only a few gases and small, uncharged water soluble molecules can readily move across the membrane

Question 23

Classes of lipids making up a phospholipid bilayer (3)

Answer 23

1. Phosphoglycerides
2. Sphingolipids
3. Sterols

Question 24

Phosphoglycerides

Answer 24

Most abundant type of phospholipid, derived from glycerol 3-phosphate. Consists of a hydrophobic tail made of two fatty acyl chains and a polar head attached to the phosphate group

Question 25

Phosphatidylserine

Answer 25

Type of phosphoglyceride that is most common in the plasma membrane. The head group consists of a positively charged choline (an alcohol), which is bound to a negatively charged phosphate

Question 26

Sphingolipids

Answer 26

Derived from sphingosine, an amino acid. Can increase transmembrane electrical resistance, found in cell membranes and the myelin sheath

Question 27

Multiple sclerosis

Answer 27

Sphingomyelin is the most common type of sphingolipid. It is degraded during multiple sclerosis

Question 28

Cholesterol

Answer 28

A type of sterol. It has a 4 ring structure and lacks a phosphate head group, so it isn’t a phospholipid. It provides a structural role in the cell membrane, making it fluid while maintaining its rigidity. It is also the precursor for some important bioactive molecules, like steroid hormones

Question 29

Atherosclerosis

Answer 29

Excess cholesterol in LDL causes atherosclerosis- statins decrease LDL by inhibiting HMG-CoA reductase (an enzyme). HMG-CoA reductase is the rate-limiting enzyme for cholesterol synthesis. Competitive inhibitors of the reductase induce the expression of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL and lowers the plasma concentration of cholesterol

Question 30

Synthesis of phospholipids

Answer 30

Phospholipids are synthesized on the endoplasmic reticulum- the last stages take place at the interface between a membrane and the cytosol. Flippase proteins catalyze the movement of phospholipids from the cytosol to the exoplasm- they get their energy from ATP hydrolysis.

Question 31

How was flippase discovered?

Answer 31

Using fluorescent probes, fluorochromes, liposomes, and quenchers.

Question 32

Quenchers

Answer 32

Molecular beacons that stay outside the cell and quench fluorescence, end up with micelles

Question 33

Micelles

Answer 33

Closed lipid monolayers w/a hydrophobic fatty acid core + hydrophilic polar surface that are spherical.

Question 34

How are cholesterol and phospholipids transported between organelles? (3)

Answer 34

It’s not clear, but there are 3 possible mechanisms.
1. Vesicles transfer lipids between membranes
2. With direct contact between membranes, membrane embedded proteins mediate lipid transfer
3. Transfer is mediated by small, soluble lipid transfer proteins

Question 35

Why do phospholipids and cholesterol need to be transported out of the ER?

Answer 35

The final steps in the synthesis of phospholipids and cholesterol take place in the ER. The plasma membrane and other membrane bound organelles require these molecules, so they must leave the ER by some mechanism

Question 36

Which factors determine the thickness of the cell membrane?

Answer 36

The lipid composition of a bilayer influences its thickness, which also influences the distribution of other membrane components like proteins. Sphingomyelin associated into a more gel-like and thicker bilayer than phospholipids do. In addition, cholesterol and other molecules that decrease membrane fluidity increase membrane thickness. SM tails are optimally stabilized, so cholesterol has no effect on the thickness of those membranes.

Question 37

Which factors determine the curvature of the cell membrane?

Answer 37

Curvature depends on the relative sizes of the polar head groups and nonpolar tails of its constituent phospholipids. Lipids with long tails and large head groups are cylindrical in shape, so bilayers with these lipids are relatively flat. Lipids with small head groups are cone shaped, so these bilayers are more curved. The effect of lipid composition in bilayer curvature may play a role in forming vesicles from the plasma membrane and viral budding

Question 38

Nanopore technology- minION

Answer 38

Nanopore sequencing is a technology that enables real-time, rapid analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence. Alpha hemolysin is involved

Question 39

Glycophorin

Answer 39

Integral membrane protein that forms a dimer- has a coiled-coil shape stabilized by der waals interactions between adjacent side chains. There is an extracellular and cytosolic domain. It is a single pass transmembrane protein that contains only one membrane spanning alpha helix. Bind to carbohydrate groups like CON A

Question 40

Bacteriorhodopsin

Answer 40

Consists of 7 transmembrane alpha helices. Passes through the plasma membrane and has extracellular and cytosolic domains

Question 41

Porins

Answer 41

A class of transmembrane proteins whose structure is different from that of other integral membrane proteins. They are found in the membranes of bacteria such as E. coli, mitochondria, and chloroplasts. Each subunit has 16 beta strands that twist into a barrel shaped structure with a pore in the center

Question 42

Hydropathy profiles/plots

Answer 42

Hydropathy profiles can identify the amino acid sequences of newly synthesized (nascent) integral membrane proteins. They are generated by plotting the total hydrophobicity of each segment of 20 consecutive proteins. Positive values indicate hydrophobic portions of the protein, negative values indicate hydrophilic portions. Used to explain the multipass nature of integral membrane proteins

Question 43

Channels

Answer 43

One type of membrane protein- gates that allow movement of specific ions or water down their electrochemical gradient. They have the fastest amount of time between opening and ions passing through and can be ligand or voltage gated

Question 44

Categories of membrane transport proteins (3)

Answer 44

1. Channels
2. Transporters
3. ATP powered pumps

Question 45

Transporters

Answer 45

Facilitate movement of specific small molecules or ions. There are 2 types- uniporters, symporters, and antiporters

Question 46

Uniporter

Answer 46

Transport a single type of molecule down its concentration gradient.

Question 47

Cotransporters

Answer 47

Symporters and antiporters- catalyze the movement of one molecule against its concentration gradient. This is driven by the movement of one or more ions down an electrochemical gradient

Question 48

Pumps

Answer 48

Use the energy released by ATP hydrolysis to power movement of specific ions or small molecules against their electrochemical gradient

Question 49

Na+/K+ ATPase

Answer 49

Pump that consumes 50% of ATP. Pumps Na+ out and pumps K+ in

Question 50

Transmembrane potential

Answer 50

Transmembrane domains of channel proteins determine which ions can pass through the membrane, which creates a tightly controlled electrical potential. The inside of the cell is more negatively charged than the outside. In animal cells, the combined force of the sodium concentration gradient and membrane electric potential drives molecules against their concentration gradients by symporters and antiporters

Question 51

Sodium concentration gradient

Answer 51

The Na/K ATPase establishes the sodium/potassium concentration gradient, with sodium being at a higher concentration outside the cell. The pump pumps Na out of the cell. Na/lysine is sodium/amino acid cotransporter that moves 2 Na into the cell (with concentration gradient) along with one lysine (against concentration gradient)

Question 52

Cholera toxin

Answer 52

Found in unsanitary conditions- causes massive and quick dehydration, causing death. Patient secretes massive amounts of Cl- into the intestine. Big amount of water follows it, causing dehydration.
Treatment- feed glucose and sodium in high concentrations- water rushes back into cells to establish equilibrium. Uniport, symport, and Na/K pump work together

Question 53

Adherens junctions

Answer 53

Connect the lateral membranes of adjacent epithelial cells and are usually located near the apical surface. Principal CAMs are cadherins. Functions- shape, tension, signaling, and force transmission. Actin and myosin filaments form a complex with adherens to act as a tension cable that braces the cell and therefore controls its shape

Question 54

F-actin

Answer 54

Actin filaments of the cytoskeleton, used to strengthen the adherens junction

Question 55

Desmosomes

Answer 55

Snap-like points of contact connecting 2 cells. CAMS- desmosomal cadherins. Responsible for maintaining the integrity of the skin epithelia

Question 56

Hemidesmosomes

Answer 56

Found on the basal surface of epithelial cells. Anchor the epidermis to the dermis (cell to matrix adhesion). Uses integrin.

Question 57

Pemphigus

Answer 57

An autoimmune disease. Antibodies target desmosomes and disrupt adhesions between epithelial cells, which causing blisters of the skin and mucous membranes

Question 58

Tight junctions

Answer 58

Hold the tissue together, has a barrier function to separate lipids, and control the flow of solutes through extracellular spaces (prevent the diffusion of some substances). Tight junctions are mostly found in kidney cells, intestinal epithelial cells, bladder cells. CAMs are occludin, claudins, and JAMs

Question 59

Sodium fluorescein leakage assay

Answer 59

Measures the efficacy of tight junctions. MDCK cells used to reach confluence and sodium fluorescein dye was added. Damage to tight junctions between cells is determined by dye leakage.

Question 60

Fluorescein angiography

Answer 60

Can be used to diagnose disorders of the retina such as macular degeneration, macular hypertension, and retinal occlusion. The tight junctions in the blood vessels of the retina are not permeable to the fluorescent dye, so it can be used to view the blood vessels in the retina.

Question 61

Lanthanum hydroxide

Answer 61

An experiment demonstrating the role of tight junctions as permeability barriers. The lanthanum hydroxide could not pass through the tight junctions of pancreatic cells

Question 62

Gap junctions

Answer 62

Allow the movement of small water soluble molecules and ions between the cytosols of adjacent cells. The passage of ions allows cells to communicate, as some neurons are connected by gap junctions and are able to pass ions between them. CAMs are connexins, innexins, and pannexins.

Question 63

Electrical synapses

Answer 63

Where neurons are connected through gap junctions, allowing ions to pass between them. Information travels across these synapses much more quickly than it does in chemical synapses. Can’t really modify behavior

Question 64

2 types of gap junctions

Answer 64

1. Rectifying- passing ions in one direction only
2. Non rectifying- pass ions in both directions

Question 65

Neurochemical synapses

Answer 65

Releases neurotransmitters. Axon terminals where neurotransmitters are released contain voltage gated calcium channels. Calcium triggers the fusion of vesicles so neurotransmitters can be released. Modifies behavior, contributes to learning