L15 - membrane contact sites

what is the function of membrane contact sites?

• intracellular signalling

• lipid metabolism

• organelle division

the discovery of membrane contact sites

• porter and palade discovered membrane contact sites using electron microscopy

• through observing that organelles like the ER and mitochondria often sat very close together without fusing

• suggesting a structural and functional link

• electron microscopy showed contact sites that are electron dense

• membrane contact sites were discovered in sarcoplasmic reticulum in muscle

what does contact sites mean?

• organelles are in contact with each other but no fusion is occurring

what are the two ER domains?

• rough ER

• smooth ER

what is the function of the rough ER?

• ribosomes attached to the ER membrane

• site of protein synthesis

what is the function of the smooth ER?

• no ribosomes

• site of lipid synthesis

• roles in calcium storage

• membrane contact sites occur at smooth ER

what is the structure of ER contact sites?

• no ribosomes on contact sites

• membranes very close

• ER contact sites can be short or long lived

  —> either will form and break quickly or will be stable and last for longer

what is the functional importance of contact sites (on smooth ER)?

• calcium regulation - intracellular calcium signalling

• smooth ER acts as the main intracellular storage of Ca2+ ions

• the ER stores calcium at high concentrations inside its lumen

what is the sarcoplasmic reticulum (SR) of muscle cells?

a specialised form of smooth ER adapted for storing and releasing calcium to control muscle contractions

what is Stim1?

• calcium sensor anchored in the ER membrane

• senses the levels of calcium in the ER lumen

• transmembrane protein that has a calcium sensing domain in the ER lumen

what are phosphoinositides?

• low abundance lipids

• role in cell signalling and organelle identity

• consists of a glycerol backbone, 2 fatty acid chains, myoinositol head group

what gives rise to different versions of phosphoinositides?

• phosphorylation of the myoinositol head group at different sites

steps of how Stim1 works in the ER

1. when calcium is high - Stim1 stays inactive and is evenly distributed across the ER membrane

2. once calcium is released - Stim1 senses the drop in calcium and clusters together forming a complex

3. this complex migrates to a particular part of the ER membrane - the site of the plasma membrane where it forms is enriched in PIP2 - which allows interaction between Stim1 and plasma membrane bringing them together but no fusion

4. recruitment of Orai1 (calcium channel) which opens and allows calcium to flood back in replenishing the ER calcium store

why is the Stim1 mechanism important?

• it prevents a transcient increase in cytoplasmic calcium - calcium kept low within cytoplasm

• important to prevent:

  1. toxicity

  2. unwanted activation of signalling pathways

  3. maintain a steep gradient allowing precise calcium signalling

role of membrane contact sites in intracellular signalling - ER tubule interacting with late endosome

• endosomes form membrane contact sites with the ER - these contact sites can reposition sections of the ER

• membrane contact sites help regulate movement of EGFRs into multivesicular bodies

• through ER-endosome contact - PTP1B protein can modulate receptor signalling from a distance

• endosomes can drag sections of the ER

• the PTP1B protein dephosphorylates EGFRs on nearby endosomes through contact sites

role of membrane contact sites in lipid transport

• lipid transport can occur at membrane contact sites through non-vesicular transfer of lipids

• lipid transfer is unidirectional

• lipid transfer proteins (LTPs) are needed to ensure that the movement occurs

• the LTPs use concentration gradients of lipid to promote lipid transfer

how does a gradient of PI4P (phosphoinositide) allow transfer of cholesterol from ER to Golgi?

• there’s a high concentration of cholesterol in the Golgi than the ER (cholesterol is made in ER)

• cholesterol is moved against its concentration gradient to the Golgi - this movement occurs through the OSBP protein which uses PI4P gradient

• the OSBP binds cholesterol and delivers it to the Golgi where it picks up PI4P and takes it back to the ER where it’s hydrolysed into PI

what are the two main structural types of proteins that bring membranes together at membrane contact sites?

1. tethering proteins (long extended proteins)

2. protein complexes (bridging)

what are hydrophobic conduits?

• lipid transfer proteins with tunnel like domains

• they shield lipid tails from aqueous cytosol

what are protein tethers?

• proteins that bring two membranes close together

• visualised by electron microscopy (EM)

what can mutations in proteins important for membrane contact sites lead to?

Niemann Pick disease C

• due to defects in proteins in the lysosomal membrane that are important for cholesterol release from lysosomes

membrane contact sites in organelle fission

• the membrane contact sites are involved in organelle fission as they help coordinate and position the machinery needed to divide organelles