L13 - PIns signalling cascades

• What can be used as a model to view co-ordinated actin polymerisation?

  Phagocytosis - coat latex bead with IgG to attach to receptors on macrophages and drive phagocytic responses

  Actin polymerisation extends from the cell towards the bead - start of phagocytosis

  Polymerisation further extends around the bead - once the bead is fully surrounded by the membrane, there is a dramatic loss of actin polymerisation at the bottom of the bead

  Once the bead is fully engulfed - complete loss of actin polymerisation
• What specific domains bind to PIP2, PIP3 and DAG respectively?

  PIP2 - PLCd1-ph

  PIP3 - GRP1-ph

  DAG - C2-PKC
• What does phagocytosis via actin polymerisation enrich?

  PIP2 levels - as the bead is engulfed into the cell
• What happens to the PIP2 levels when phagocytosis ends?

  Completely reduce due to lack of actin polymerisation - PIP3 and DAG levels increase
• What inhibitors can be used to stop the loss of PIP2 after phagocytosis ends?

  PI3K inhibitor and PLC inhibitor - can block phagocytosis and maintain levels of PIP2

  Maintains actin rich pseudopods (temporary extension of the cell towards the cell its engulfing)
• How does a PI3K and PLC inhibitor block phagocytosis?

  Inhibits the ability of PIP2 to become PIP3
• How do the levels of PIP2 reduce during phagocytosis?

  PIP3 activates PLC-gamma1 - has a PH domain with high affinity for binding to PIP3

  PLC-gamma1 hydrolyses PIP2 - actin polymerisation stops and levels of PIP2 disappear
• How is PIP5K recruited to the phagocytic cup?

  Likely through recruitment to activated Fc receptor

  Converts PI4P to PI4,5P2 to start actin polymerisation at the beginning of phagocytosis
• What is the role of endocytosis?

  Acts to selectively monitor the external environment and to internalise receptors and membrane proteins for degradation and recycling
• How are endocytic vesicles formed?

  Via clathrin lattice structures - regulated by PIP2

  Lattice structure forms around the vesicle to allow it to grow and form - clathrin adaptors attach it to the membrane and bring in cargo to be endocytosed

  Dynamin protein (regulated by GTP) pinches the membrane once the vesicle is formed, allowing it to bud off.

  Clathrin lattice structure breaks down via PIP2 degradation, leaving the fully formed endocytic vesicle
• Where are endocytic vesicles formed?

  At the plasma membrane
• What does PIP2 do to the clathrin adaptor proteins (CAPs)?

  Causes clustering of AP2, AP180 and Epsin

  Clustering provides the energy to deform and destabilize the curved membrane
• What further induces and senses curvature of the membrane?

  Endophilin - through its BAR domain
• What does Endophilin recruit?

  Synaptojanin 2 - a PI4,5P2 5-phosphatase

  It degrades PIP2 only in the vesicle and induces uncoating
• What is the different in the process of vesicle budding in the TGN compared to on the plasma membrane?

  Requires the clathrin adaptor protein AP1 instead of AP2
• How does AP2 affect the levels of PIP2?

  AP2 is able to interact with a specific isoform of PIP5k - increases the levels of PIP2
• How does Dynamin cause pinching/scission of the endocytic vesicle?

  Binds to PIP2 - causes dynamin to oligomerise (join together to form an oligomer)

  Able to cause scission
• What can vesicle trafficking from the plasma membrane or Golgi fuse to form?

  Early endosomes
• What is the endosome?

  A sorting organelle

  Deranged in cancers, neurological diseases and parasitic infections
• What are endocytic vesicles covered in?

  PI3P
• How does PI4,5P2 coated vesicles become coated in PIP3?

  Synaptojanin converts PI4,5P2 to PI4P (phosphatase)

  PIK3C2A converts PI4P to PI3,4P2 (kinase)

  PI4-phosphatase converst PI3,4P2 into PI3P
• What type of family are the Myotubularin family?

  Phosphatases - there are about 12

  Often deregulated in human diseases
• What drives endosomal trafficking?

  Synthesis and degradation of PI3P
• What does coincidence signalling/detection enable?

  Low affinity interactions driving specific functions - will most likely need another component to help drive the cellular outcomes
• What 2 domains are important sensors for PI3P?

  FYVE and PX domains
• What are the features of the FYVE domain?

  Acts as a dimer

  Has a hydrophobic loop to insert into the membrane to stabilise interactions

  Mainly interact with PI3P but some can interact with PI3,4P2

  Low affinity
• What are the features of the PX domain?

  Has a hydrophobic loop to sit in the membrane to stabilise interactions

  Interacts with PI3P through the pocket formed between its beta-sheets and alpha helix

  Mainly interact with PI3P but some can interact with PI3,4P2 and PI4,5P2

  Variable affinity
• What is the coincidence detection involving EEA1?

  Endocytic vesicle to Early endosome

  EEA1 is a large protein with a low affinity for PI3P - needs 2 components to bind with stability to Rab5 vesicles and PI3P

  It has a FYVE domain to bind to PI3P and a Rab5 binding motif to bind to Rab5 - acts as a dimer

  Tethers the vesicle and the early endosome - along with snares, it enables fusion
• What is FYCO1?

  A protein - FYVE and coiled coil domain autophagy adaptor 1

  Can bind to PI3P, Rab7 and LC3 (protein involved with autophagy) - targets the vesicle to a microtubule to then take it to the lysosome for degradation
• What is Rab5?

  A small molecular weight G protein

  Acts as a marker for endocytic vesicles that come in from the plasma membrane and need to go into the early endosomal system
• What is the coincidence detection with HRS1?

  Early endosome to Lysosome for degradation.

  Has a FYVE domain to bind with PI3P and a ubiquitin interaction motif (UIM) to bind to ubiquitin motifs on an EGF receptor.

  Only stably associates after the recognition of the ubiquitin motif - after recognition, it engages the ESCRT complex.

  Uses 2 low affinity interactions to drive the stable association.
• What is the ESCRT complex?

  ESCRT 0, I, II and III

  Drives proteins for lysosomal degradation and is involved in membrane scission
• What is the BAR domain?

  Part of Endophilin

  Highly conserved protein dimerisation domains

  Banana shaped - can mediate interactions with membranes (thought to act as sensors)

  N-BAR type domains have an alpha-helix before the BAR domain and can mediate membrane tubulation - often found together with PX domains in the SNX family
• What is the coincidence detection with SNX1?

  Early endosome to Trans Golgi Network retromer recycling.

  Has a PX domain that recognises PI3P and a BAR domain that senses membrane curvature.

  The BAR domain tubulates the membrane as well - allows budding to occur and for the vesicle to go to the TGN.
• What is the role of the Retromer complex?

  Recognise cargo to be recycled back to the Trans Golgi Network

  Eg SNX1
• How could you strengthen a monomers interaction with the membrane?

  Oligomerise it - now can interact with the membrane much more strongly