Membrane Contact Sites.

Question 1

How are proteins transported around the cell?

Answer 1

By vesicles and tubules

Question 2

What is a key feature of tubules and vesicles?

Answer 2

They have a high surface area to volume ratio of tubules and vesicles

Question 3

What does the smooth endoplasmic reticulum do?

Answer 3

The is important for lipid synthesis and for calcium storage

Question 4

When looking at the RER and SER with an electron microscope, what are the dark parts?

Answer 4

Proteins, alway proteins. Remember this very simple fact!

Question 5

In neurones and muscle cells, which are calcium dependent, you see proteins.

What is important to note about electron dense areas shown in the SER? Considering the SER isnt responsible for protein sythesis

Answer 5

Presence of proteins in the SER may be linked to maintaining calcium homeostasis

Question 6

What is the SER referred to in muscle cells and what does it do?

Answer 6

Its referred to as the sarcoplasmic reticulum and it is an important calcium storer

Question 7

How do muscles contract? This goes for skeletal muscle

This relates to the movement of the sacroplasmic recticulum (SER) to the plasma membrane to form contact sites (which show up as dark on the electron microscope)

Answer 7

The ER is closely associated with the plasma membrane of muscle cells

When an action potential moves along a motor neurone this is interpreted by a muscle cell

As the sacroplasmic reticulum is in close contact with the plasma membrane you get a release of calcium, essential for muscle contraction.

You also get the replacement of calcium.

So note:
The contact sites (between the SR and the membrane) are electron dense meaning there must be proteins here important for calcium mobilisation and movement

Hence why the SER has these proteins which move to contact sites

Question 8

So what do SER contact sites do?

Answer 8

Helps with the release of calcium

Or the reuptake of calcium

Question 9

How does the sarcomplasmic reticulum detect a drop in calcium levels after muscle contraction?

Answer 9

A drop in calcium sensed by stim 1 proteins in the ER

This is a resident ER protein

This helps to move the ER to the type 1 transmembrane proteins

This is embedded in the membrane

These form contact sites for reuptake of calcium.

Question 10

What are normal calcium conditions in the ER?

Answer 10

High calcium levels

Stim 1 is inactivated

The stim 1 ensure that the ER is maintained away from the plasma membrane

Question 11

Going back to sewards lectures, what it the important phosphate on membranes? And how does this relate to the STIM 1 protein and ER movement?

Answer 11

PIP2 phospholipids

So after muscle contraction calcium stores depleted changing STIM 1 proteins

The stim 1 proteins become positively charged

It moves the ER to the plasma membrane of the cell because its attracted to the negative PIP2

Calcium can now enter via ORAI channels from outside of the plasma membrane to inside to the ER via contact sites.

The calcium contact site is key for calcium replenishment.

Question 12

How can we prove that STIM1 proteins are conserved between species?

Answer 12

They can be seen in both mammals and drosophila via RNAi screens.

Question 13

What is the difference between vesicles and contact sites?

Answer 13

Vesicles fuse with the plasma membrane

However stim 1 does NOT fuse with the plasma membrane. There is no fusion of it with the plasma membrane. Its just a contact protein

The membrane just comes very close to the stim1 protein

Question 14

What has electron microscopy helped us to see with reference to ER and contact sites?

Answer 14

Helps us to see size of contact sites (between ER and membrane) and how far apart these things are

Also helps us to understand the environment of membrane contact sites.

Question 15

So where do contact sites form and how far apart is the ER from the membrane?

Answer 15

The distance is small so calcium can go through quickly

Membrane contact sites also form in part of the ER where there are no ribosomes.

Question 16

How does the ER transfer material to the mito?

Answer 16

Via circulating it and transferring material

There is no fusion

Thus the ER is responsible for non fusion transfer of material

Question 17

What are the typical structures of ER contact sites?

Answer 17

Ribosomes are excluded from contact sites

Membranes are very close

Endoplasmic reticulum is long lived

Question 18

Functions of contact sites?

Answer 18

Calcium mobilisation

Lipid transfer

Signalling

Question 19

What happens to protein components in the plasma membrane / ER when contact sites bind?

Answer 19

These extends to allow bridges to form.

Question 20

Live cell imaging and electron microscopy is correlated by

correlative light and electron microscopy : CLEM

What does this stand for?

Answer 20

This allows for localisation of fluorescently labelled proteins to the membrane contact sites.

Question 21

LDL enters into cells via clathrin mediated endocytosis

What is LDL? And what in the cell breaks it down?

Answer 21

LDL is a cholesterol carrier

It is passed from the early endosome to the late endosome then to lysosomes

These lysosomes break down LDL particles and release cholesterol which is transported to other places in the cell. Important for plasma membrane permeability.

Question 22

Note endosomes can pull the membrane of the endoplasmic reticulum

What may be the benefit of this?

Answer 22

May help with the positioning of the ER in the cell

Question 23

Once LDL is broken down, cholesterol moves out of the ER. How does this happen?

Answer 23

This is facilitated by MPCI and II proteins

Question 24

Note 30% of cholesterol is taken up by cells and is delivered to the ER by the clathrin mediated endocytic pathway. Which is a contact site.

What is this transfer called? And in what direction is lipid transfer?

Answer 24

This transfer is called non vesicular transfer of lipids

This is unidirectional.

Question 25

What are the proteins called which are important in delivering cholesterol to the ER? Where are these proteins found ? What is the effect of mutations in niemann pick 2 and 3 proteins?

Answer 25

Niemann pick proteins.

These interact with proteins on the ER membrane for transfer of cholesterol

These proteins are found in the lysosomes

Mutations in these niemann pick 2 or 3 proteins cause niemann pick disease C. This affects the spleen, liver, bone marrow and brain. Leads to weak muscles.

Question 26

Membrane contact sites are involved in organelle fission.

What does this mean in terms of ER action on mitochondria

Answer 26

The ER helps the mito to split in two

The ER recruits the components needed for fission or the signals needed

Note the mito and ER dont connect

Question 27

What do the cytoskeleton do in terms of contact sites? What does organelle fission do?

Answer 27

Ensure contact sites are in the best positions.

Organelle fission helps cells divide and for new cells to be formed in the right places in the cell

Question 28

What do ER tubules do?

Answer 28

They help form contact sites for calcium mobilisation

Lipid transfer

Signalling

And organelle fission.