What is intracellular trafficing?
its the transport event from a donor compartment (or membrane) to an acceptor compartment (or membrane) in a cell
What are the two major membrane trafficing pathways?
Biosynthetic/secretory pathway: starting from Endoplasmatic reticulum, sorting in golgi network and transport in vesicels to membrane (secretion)
Endo-lysosomal pathway: molecules are collected (from extra cellular matrix = endocytose) or transported from ER / Golgi and transported to early endosome and can then be recycled, or degraded via lysosome
What biological processes that requiere membrane trafficing?
-General maintenance of cell homeostasis in every cell/every tissue
-Insulin stimulated glucose uptake (if insulin receptor is triggered by insulin binding, glucose transporter are relocalized from specialized recycling endosomes to cell membrane to boost glucose uptake) —> rapid response
-Transcytosis (cellular process involving the transport of macromolecules or particles across a cell e.g. Antibodys from mother to milk lumen for the child to feed or transport of nutrients across epithilelial barriers from blood to brain)
-Receptor desensitization (reduction or loss of responsiveness of cell surface receptors to a particular ligand (signaling molecule) or degradation of a rezeptor through internalization
-Cell migration/organogenesis (recycling of integrins [cell-matrix adhesion] drives cell migration)
-maintenance of cell/epithelial polarity
Ways for the delivery of macromolecules across epithelial barriers.
E.g. from blood to brain
diffusion
paracellular transport (inbetween the cells)
carrier mediated transport
receptor-mediated transcytosis
adsorptive transcytosis
active efflux
How does intracellular transport affect neuronal viability?
Neurodegeneration:
-Perturbed autophagic flux (=impaired autophagic activity)
-Impaired lysosomal function
-decreased degradation
that leads to
Accumulation of: -damaged mitochondria -protein aggregates -plaques
that lead to decreased neuronal viability
How do coat complexes mediate cargo selectivity, enrichment and ultimately cargo transport?
The cargo is collected and sorted on the donor membrane the specifity of the coat compexes define what cargo is transported and where!
Examples for coat proteins are:
Clathrin: for endocytosis
COPII (Coat Protein Complex II): ER to Golgi transport (especially newly synthesized proteins)
COPI: Golgi to ER transport
Explain the basic mechanism behind Clathrin mediated endocytosis.
-Cargo binds to transmembrane receptors initiating formation of Clathrin-Coated Pits
-The Cargo recruits its own coat complex through Clathrin adaptors (adaptin)
-Formation of the coat further concentrates additional cargo (concentration)
-self re-inforcing process leads to effcient and cargo specific endocytosis
-membrane invagination and then Dynamin (GTPase to pinch of the vesicle)
-uncoating (recycling of coat proteins)
-transport of naked vesicle needs motility to reach target, find target selectively and fuse
—> Rabs GTPases mediate membrane identity and direct intracellullar sorting
What are Rabs and name examples.
Rab GTPases (Ras-related in brain proteins) are molecular switches and mediate membrane identity and direct intracellullar sorting, a human has more then 60 Rabs and they all have defined subcellular localizations
Rab1 = ER to Golgi complex
Rab5 = early endosome
Rab7 = late endosomes
Rab11 = recycling endosomes
How do Rab GTPases act as master organizers of membrane trafficking?
Rabs can bind to a multitude of effectors:
(Phospho)-Lipid Kinases/ Phosphatases —> Generate membrane (compartment) identity, establish microdomains
Motor proteins (Myosins, Kinesins), often via adaptor proteins —> Movement of transport vesicle along microtubule tracks
Tethering factors -long coiled coil proteins -large protein complexes (Garp/COG/Exocyst) —> Capture a moving vesicle and tether it to target membrane system
Snare Proteins (V-Snares T-Snares) —> Mediate fusion after vesicle has docked via tethering factors
-Rabs form microdomains
Rab proteins can organize most steps of vesicular transport, name them.
They enable all that by the ability to bind to various effectors:
1. Rab proteins can determine coat assembly (achieves cargo selcetion)
2. The Rab then organized Uncoating and motility (motor proteins)
3. The Rab recruits tethers to „tie“ the vesicle to a target membrane
4. Rabs recruit specific SNAREs to fuse the membranes
How is specific fusion achieved?
A multitude of SNARE proteins mediates Compartment specific membrane fusion
v-SNARE on donor/vesicle and t-SNARE on target organelle (acceptor membrane)
Mechanic of SNARE mediated fusion: Force generation through helix rearrangement intercoiling to pull vesicle close to membrane
N-ethylmaleimide sensitive fusion protein (NSF) and SNAP use ATP to recycle SNAREs the v-SNARE is transported back to donor via vesicle
In the Endo-lysosomal pathway there is recycling and degeneration what determines the fate of the molecule?
A multitude of recycling pathways shuttles cargo back to the cell surface via Rab GTPases (Rab4,11,14,22)
Ubiquitination determines degradation
1. A surface molecule is ubiquitinated by specialized ligases
2. On early endosomes, ubiquitinated cargo is sorted into intralumenal vesicles
3. Intralumenal vesicles accumulate in „older“ endosomes
4. Ultimately, mature late endosomes fuse with lysosomes to degrade cargo
Information about the Lysosomes
• Lysosomes = the major "digestive" organelles of the cell involved in receptorligand degradation, cholesterol metabolism via the LDL receptor, and organelle turnover.
• contain a large number of specific enzymes (hydrolases) involved in the degradation of proteins, lipids, carbohydrates, and nucleic acids.
• surrounded by a single bilayer that has unique characteristics
• One of the hallmark features of the lysosome is the low pH around 5 of its lumen
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