Collection of notes for 3-S2 "Membranes and Cell Communication"

Calcium Signalling

Key Concepts in Calcium Signalling Mechanisms

On/Off reaction: swinging of cytosolic calcium concentration between 100 nM (nano-level) and 1,000 nM (micro-level).

Calcium toolkit: different cell types use different sets of proteins (toolkits) to implement calcium signalling.

• Receptors. Notably GPCR - e.g., GqPCR including ETA/B (endothelin receptors).
• Transducers. Non-exclusive category - anything that transduces signals counts. For example, voltage-gated calcium channels.
• Channels. RyR and IP3R are most well-known.
  • TPC (two pore channel) on endo-lysosomal membranes, activated by NAADP.
  • MCU allows calcium influx into mitochondria. MCU inhibition is thought to be a potential mechanism to reduce cell death - high mitochondrial calcium concentration activates PTP.
• Pumps. Notably SERCA on the ER.
• Buffers. Bind free calcium. Calsequestin located in the lumen of SR helps pumps by reducing the concentration gradient.

SOCE: store-operated calcium entry. STIM on the ER oligomerize upon sensing low calcium concentration inside the ER lumen, leading to ORAI (a calcium channel) activation, directly loading extracellular calcium into the ER.

Diverse Characteristics of Calcium Signalling

Temporal: response times to calcium signalling are diverse.

• μs. Exocytosis.
• ms. Contraction.
• sec. Metabolism.
• min. Transcription.
• hr/day. Fertilization.

Spatial: calcium signalling can occur over differnt ranges of distance, determining differnt types of cellular responses.

Patterns: Same stimulus applied on different concentration levels can elicit calcium waves of different frequencies and amplitudes.

Clathrin Endocytosis

Clarification

• CCV: clathrin-coated vesicle.
• Accessory protein: could mean anything involved in CCV formation that's not clathrin.
• ΥXXΦ motif: an endocytic motif commonly present in the cytosolic side of transmembrane proteins.
• PIP2: PI(4,5)P2
• PIP3: PI(3,4,5)P3

Key entities

Adaptor Protein 2.

• Structure: trunk, 2 hinges, 2 appendages.
• Consisting of two large subunits (α, β), one medium subunit (μ), and one small subunit (σ).
• α trunk binds to PIP2.
• β hinge recruits clathrin through the clathrin box.
• μ recognizes the ΥXXΦ motif. Also binds to PIP2 - this is believed to help keep YXXΦ-binding site open.
• σ stabilizes the AP2 complex.
• α and β appendages interact with accessory proteins (e.g., Epsin, Eps15, AP180). β appendage also includes an additional clathrin binding site.
• There's also another endocytic motif that AP2 can recognize.

BAR-domain-containing proteins.

• The BAR domain recognizes PIP2 on a curved membrane.
• BAR-domain-containing proteins amphiphysin and syndapin recruit dynamin, thereby targeting it to membrane curvatures.
• FCHO1,2 contains the F-BAR domain.
  • the F-BAR domain recruits Eps15 and intersectin.
  • FCHO additionally introduces early bending of the membrane.

Epsin

• Epsin can bind to Eps15 and AP2.
• Epsin is a membrane bending protein that functions in a way analogous to forcing an incision into the membrane.
• Epsin is a cargo-specific adaptor for EGF receptors.

AP180

• AP180 can bind to clathrin.
• AP180 is a membrane bending protein.

Dynamin

• Dynamin is a twisting mechanoenzyme.
• PH binds PIP2 and PIP3.
• GED (GTPase effector domain) serves as a GAP (GTPase-activating protein). By triggering GTP hydrolysis, the GED functions as a switch-off mechanism.
• PRD (proline-rich domain) interacts with SH3, which amphiphysin and syndapin possess.

Synaptojanin

• Synaptojanin is an inositol-5-phosphatase. Therefore, it can dephosphorylate PIP3 and PIP2.
• Synaptojanin contains PRD.
• Synaptojanin is named after Janus, a Roman two-faced God, because it has two domains related to inositol phosphate metabolism. One of them is its inositol-5-phosphatase domain, while the other being the Sac1 domain.

Auxilin

• Auxilin readily binds to phosphatidylinositol monophosphates.
• Auxilin recruits Hsc70, an ATPase that uncoats the vesicle.

Stages

1. Nucleation. Presence of the nucleation module defines the site of clathrin recruitment and later budding action. This module contains FCHO, Eps15, and intersectin.
2. Cargo selection. The nucleation module recruits AP2. AP2 binds the cargo:
   • by interacting with the endocytic motifs present in the cytosolic domains of the cargo receptor or the cargo (which itself is a transmembrane protein).
   • by interacting with intermediate cargo-specific accessory adapter proteins.
3. Coat assembly.
4. Scission.
5. Uncoating.

Regulated Exocytosis

SNAREs

• SNARE abbreviates SNAP Receptor.
• A SNARE motif is an α-helix.
• v-SNARE. Found on the vesicle membrane.
  • Synaptobrevin, brevin, because it is really short. Also known as VAMP, Vesicle-associated membrane protein. Has one SNARE motif.
• t-SNARE. Found on the target membrane.
  • Syntaxin. Has one SNARE motif.
  • SNAP-25. Synaptosomal-associated protein (not soluble NSF attachment protein). Has two SNARE motifs.
• A trans-SNARE complex forms when four helical domains (SNARE motifs) from the v-SNARE and the t-SNAREs wrap around each other to form a stable four-helix bundle.

Accessory proteins

Synaptotagmin.

• A vesicular transmembrane protein that has two cytosolic calcium-binding and phospholipid-binding domains, namely C2 domains.
• Without it, regulated exocytosis cannot be triggered by calcium.

Munc18.

• Has a repressor motif that binds the SNARE motif on syntaxin.
• It is thought that syntaxin cannot reach the membrane fusion site without it.

NSF & SNAP.

• NSF (N-Ethylmaleimide Sensitive Fusion protein) is an ATPase that is involved in dead-end SNARE complex disassembly. NSF is needed to rescue membrane trafficking in NEM (N-EthylMaleimide) treated cells.
• SNAP abbreviates Soluble NSF Attachment Protein.
• NSF is a soluble protein whose function requires attachment to membranes. SNAPs are adaptor proteins that link NSF to membranes via SNAREs.

Complexin.

• Complexin delays the fusion event after priming.

Some Links

Sticky proteins

Laemmli buffer

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Protein domains

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