chapter 5 - endocytosis Flashcards
5.1. [endocytosis] what are the different endocytic pathways that can be used for synaptic recycling?
reproduce image and check it.
- endocytosis of sv via clathrin-coated pits (CCPs) from the plasma membrane and its deep unfolding
in other words: after sv is formed ccps are being released. This leads to acidification of sv and nt will be pumped in sv - kiss and run (still unsure if it happens)
in other words: sv only partially fuse with the pre synaptic membrane and the fusion pore is briefly opened and closed
———more on it:
> “kiss and run”: this mechanism is debated/controversial; only indirect evidence exists - bulk endocytosis by sv formation from endocytic intermediates (EI)
in other words: endocytosis of larger parts of the pre-synaptic membrane leads to the formation of EI (potentially sv could be regenerated through this mechanism) - it can be either clathrin-dependent or independent
—— more on it: Bulk endocytosis
> operates most prominently under strong stimulatory conditions, when a large number of synaptic vesicles fuse with the plasma membrane within a short time interval
> excess plasma membrane is rapidly captured via the formation of plasma membrane infoldings, which then undergo fission to generate intracellular vacuoles and cisternae (endosome-like intermediates, El)
> this is a non-selective mechanism of membrane uptake, but the resulting Els may be enriched with intrinsic SV membrane proteins.
These Els subsequently give rise to new SVS
> The molecular mechanisms of bulk endocytosis are largely unknown:
* the fission of deep membrane infoldings is dynamin-independent
* the actin cytoskeleton and proteins that couple membrane deformation to actin dynamics may be involved e.g. the F-BAR protein syndapin
——————– - housekeeping membrane recycling involving clathrin-mediated endocytosis and canonical early endosomes (EE), as well as traffic to the cell body via late endosomes (LE) and multivesicular bodies (MVB)
in other words: housekeeping membrane recycling involves clathrin endocytosis, which leads to the formation of EE which can then be converted into LE that can travel retrogradely to the cell’s cell body
———— more on it: > nerve terminal endosomes: the role of “canonical” early endosomes (organelles downstream of clathrin-coated vesicles and other vesicles that form directly from the plasma membrane) remains a poorly explored topic
5.2. [endocytosis] what are the methods to study sv endocytosis? Describe them and infer which method is best suited for a given experimental condition
- Amphiphatic styryl dyes such as FM1-43. These dyes have an apolar side which results in their binding to membranes, and a polar side which prevents the molecules to pass through membranes. These molecules are phluorescent, allowing their visualization, and the phluorescence intensity can be quantified. Incubation of cultured cells, Drosophila NMJs or other synaptic systems with FM1-43 results in binding of FM1-43 to membranes (including presynaptic membranes). Stimulation of vesicle exocytosis, e.g. by incubation with high potassium buffers, results in exocytosis and subsequent endocytosis of synaptic vesicles, resulting in uptake of FM1-43 in synaptic vesicles. After a subsequent washing step, only FM1-43 that has been taken up in SVs will remain, and the fluorscence intensity of presynatpic terminals is a proxy for the number of endocytosed SVs.
- SynaptopHluorin: a fusion protein of pHluorin to the lumenal portion of synaptic
vesicle proteins (typically synaptobrevin). pHluorin is a GFP variant (pKa = 7.1), of
which the fluorescence is quenched at the acidic pH of SVs, and recovered on SV fusion and exposure to the near-neutral extracellular pH. Thus, stimulation of exocytosis, e.g. by high-frequency stimulation of cultured neurons, will result in an increase of pHluorin fluorescence, followed by a decrease which is due to SV recycling by endocytosis, followed by re-acidification.
Application of bafilomycin, a membrane-permeable blocker of the V-type ATPase that is required for vesicle re-acidification, can be used to trap vesicles at neutral pH after endocytosis, thus allowing synaptopHluorin to remain fluorescent even after endocytosis. Thus, comparing the change in fluorescence intensity in presynaptic terminals upon HFS in the presence and the absence of bafilomycin allows to estimate total endocytosis (difference between the two traces). - electrophysiology: endocytic recovery of the increase in surface area produced by a secretory burst at the calyx of Held giant nerve terminal can be monitored by measurement of membrane capacitance (Cm).
- Imaging methods, e.g. internalization of antibodies against luminal domains of SV proteins. Antibodies can either be fluorescently labeled or coupled with the pH-sensitive cyanine dye derivate Cy-pHer5E.
5.3. [endocytosis] discuss the sequential steps of clathrin-mediated endocytosis (a), the different classes of molecules involved, and give examples of molecules (b).
draw and then check figure on slide.
It starts with the presynaptic membrane, which has synaptic vesicle proteins and PIP2.
The clathrin adaptors will bind to the SVs and the PIP2.
The adaptors recruit clathrin.
There’s an interaction with the actin cytoskeleton to stabilise the vesicle.
N-Bar proteins (such as endophilin and amphiphysin) interact and generate/maintain the membrane curvature.
Synaptojanin dephosphorylates PIP2 and starts shedding the clathrin coat.
Dynamin induces the fission of the endocytic vesicle.
The vesicle is released.
The clathrin coat is disassembled by Hsc70 and auxilin.
A
Clathrin-mediated endocytosis
> Major pathway of endocytosis, in particular at rest and during modest activity
> Steps in the nucleation of clathrin-coated pits:
step1. interaction of clathrin adaptors and/or a subset of their accessory factors with the lipid bilayer and with membrane proteins
step2. interactors of adaptors with each other, with other accessory clathrin
factors, with cargo proteins, and with clathrin adaptor leads to the rapid growth of the coat in a feed-forward, cooperative fashion.
step3. a deeply invaginated clathrin-coated bud with a narrow neck is formed; fission of this neck in a reaction that requires the GTPase dynamin leads to a free vesicle that rapidly uncoats.
> Unique properties of clathrin-mediated SV endocytosis:
* highly homogeneous small size of the vesicles (there must be molecules that make sure vesicles have the right diameter when endocitosed)
* specificity of the cargo: all needed membrane proteins have to be included into the nascent vesicle in the appropriate stochiometry
* speed of the process (15-20 sec)
> Molecular mechanisms underlying this specificity: poorly understood
> The clathrin lattice is a chicken-wire-like structure, likely involved in generation/maintenance of membrane curvature. Clathrin also functions as a scaffold for the clustering of the adaptors and thus of the membrane cargo to be internalized.
> adaptors bind to:
1. cytoplasmically exposed domains or endocytic “motifs” of vesicle membrane proteins, as well as to PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane
2. clathrin heavy chain and/or other adaptors and endocytic factors
B
Major endocytic proteins involved in sv recycling include
clathrin coat components (Clathrin, AP-2, epsin - PI(4,5)P2, stonin)
BAR domain- containing components (endophilin, amphiphysin)
phosphoinositide metabolism (pipk1 gamma, synaptojanin)
scaffolding (intersecting, eps15)
membrane fission (dynamil)
clathrin lattice dissembly (hsc70 auxilin)
- Interactions are important to recruit and concentrating all the synaptic vesicles proteins that need to be recycled and bring them closely together
The adapter proteins interact, not only with the synaptic vesicles proteins (eg AP 2 with synaptojanin) but also with other proteins (eg PI (4,5) P2)
Bar domain containing proteins
A. ,generate/sense bilayer curvature
B. interact with plasma membrane which generates membrane curvature because it likes curved membranes
Clathrin coated pits are typically observed at the outer margins of the active zones (=endocytoc or periactive zone)
5.4. [endocytosis] how do membrane fission and uncoating occur?
- membrane fission requires the GTPase dynamin: dynamin oligomerizes into spirals at bud necks –> GTP-dependent dimerization of the GTPase module –> GTP hydrolysis –> conformational change of neighboring domains –> constriction the dynamin spiral and the underlying tubular bud neck
- Uncoating (clathrin shedding) is ATP-dependent and requires Hsc70
ATPase and its cofactor auxilin - Shedding of adaptors is dependent on
PI(4,5)P2 hydrolysis by synaptojanin
