Measuring Endocytosis And Exocytosis Flashcards
Who suggested CME is responsible for endocytosis of vesicles?
Heuser and Reese 1973
Who suggested kiss and run as the mechanism of endocytosis?
Cecerelli et al 1973
Why can endocytosis not be CME alone?
Hippocampal neurons take 40s for CME
Therefore, is neuron contains 40 vesicles the. Ax firing rate could only be 1Hz
Actual firing rate can be up to 20Hz for 2 seconds
What are the 4 ways we can measure neurotransmitter release?
Amperometry
Capacitance measurements
Styryl (FM) dyes
Synapto-pHluorin
What types of cells can you conduct amperometry on?
Chrommaffin/ mast cells
Those which contain neurotransmitters which can be oxidised
What transmitters can be oxidised?
Catechloamines
Therefore cannot be used in glutamatergic or GABA neurons
Why can’t amperometry be conducted in the CNS?
Carbon fibre electrode too large to fit in cleft
Explain how amperometry works
Patch pipette chromaffin cell and voltage step it (or apply extracellular KCl)
Causes NT release
Positivity charged carbon fibre will oxidise (take an electron) from the NT
conduct a current which is proportional to the amount of oxidation, and therefore neurotransmitter release
What types do signal does amperometry produce?
Foot signal then spike
Sometimes only a foot signal exists
What does a foot signal in amperometry suggest?
Incomplete collapse of vesicle into plasma membrane - kiss and run?
Foot signals have flickers within them…what do these signify?
Fusion pore flickers last about 400(micro)seconds
Fusion pore opens and closes very rapidly
What is capacitance?
The storage of charge which is a property of the lipid bilayer (myelinAtion decreases this)
What is the equation for capacitance?
Cm=Q/V
Q = amount of charge which is moved across a membrane V = change in voltage
Capacitance is how much charge can be stored per unit voltage
What is capacitance proportional to? Why is this?
The surface area of cell
In a larger cell more charge will have to move across membrane for a given change in voltage
There will be a smaller membrane potential difference in a larger cell for a fixed movement of charge as there will be a smaller density of charge difference in a larger cell
With change in voltage (V) on the x axis and Q on the y what is the relationship?
Linear y=mx+c y=q x=v m=capacitance c=0
As the gradient is capacitance larger cells will have a steeper gradient
Units of capacitance?
Farads
Why can change in capacitance be used to measure exocytosis?
As vesicles fuse with membrane capacitance will increase (measured via patch clamp amplifier)
What is a massive disadvantage for capacitance measurement?
Works well for large vesicles as large change on capacitance is observable
E.g. In chromaffins cell vesicles are 200nm in diameter so you can detect individual vesicle fusion in a step wise manner (2 fFarads each)
Neuronal vesicles too small and are below the limit of detection
How does capacitance measurements support kiss and run?
In a study by sun et al 2002 it was observed that in the Calyx of held synapse there was a 65aF change in capacitance 1ms before a miniature EPSP
Capacitance decayed in 50ms
Too quick for CME
What are some chemical characteristics of styryl FM dyes?
Amiphipatic molecules
Net charge of +2 (prevents them from crossing membrane
Hydrophobic carbon dye can have different lengths depending on dye
Fluoresce in lipid membranes only
What is the dissociation rate from the membrane proportional to?
Length of hydrophobic carbon tail
E.g. Short length (FM2-10) dissociate quicker than short length (FM1-84)
How do FM dyes allow measurements of endocytosis and exocytosis?
Apply dye to outside of memebrane
Exocytosed vesicles will take up dye when incorporated into membrane
when vesicles endocytosed will take due with them into cell
Wash off dye on external membrane
Restimulate to cause exocytosis of dye covered vesicles
Fluorescence will disappear as dye is released
How can dye departition rate be used to kiss and run evidence?
Different dyes are released by membrane at different rates, therefore decrease in fluorescence on restimulation will vary between dyes.
When vesicle dyes fuse with plasma membrane dye will lateral diffuse on plasma membrane (Tdiff = 100ms) and/or dissociated from membrane (Toff=3seconds)
If vesicle completely fuses with plasma membrane dye will lateral diffuse and come off from the membrane (Tdiff>Toff therefore will diffuse about 3-4(micro)M before being released)
If kiss and run fusion pore limits lateral diffusion, therefore rate of destaining is determined only by the departition rate of dye
Why do we need to compared two FM dyes for the evidence of kiss and run?
Lateral diffusion rate will be about the same for both dyes by Toff will vary depending on hydrophobic tail length
If you use one you do not know if rate of defluorescence is due to lateral diffusion then Toff or just Toff alone. However, with two any difference in the decrease in fluorescence is due to the T off. Those with a quicker Toff will decrease in Fluorescence quicker as more dye will be released through the temporary fusion pore. Therefore if there is a difference in F decrease it must be kiss and run
Disadvantage of FM dyes
Detailed kinetics not possible
Measurements in deep tissue difficult
What pattern does destaining of a single vesicle follow?
A stepwise decreases with a dead time of 23seconds
Indicates fusion pores rapidly cycle between open and closed state on continual AP firing
What is a synapto-pHuolin dye?
pH sensitive the which fluoresces when exposed to alkaline pH and lose it when exposed to acidic pH
How can pHuolins be used?
Attach to intracellular synaptobrevin (pH sensitive GFP)
Will fluoresce when fuse with plasma membrane
Will decrease in fluorescence when internalised and restored with vesicle
What does the upstroke on the fluorescent time graph with phluorins represent? (Without bafilomycin)
Exocytosis (as increase fluorescence)
However, there will be concurrent endocytosis and reacidification which acts to decrease fluorescence
How does bafilomycin work?
Blocks vesicular atpase
Therefore, endocytised vesicles remain fluorescent
What is the advantage of using bafilomycin?
Measure the full extent of exocytosis
Known as alkaline trapping
What does the difference if fluorescent for the upstroke with and without bafilomycin represent?
The extent of endocytosis
Control = exo - (endocytosis + reacidifcation)
Baf = exocytosis
Baf-control = endocytosis
What does the decrease in fluorescence using phluolin dyes represent?
Endocytosis and reacidifcation
What is the rate limiting step in the decrease in fluorescence using phluorin dyes? Why?
Endocytosis
Addition of extracellular acid on downslope attenuates fluorescence
This means the phluoin is still on the membrane
Decay due to endocytosis
What effects the rate of endocytosis?
Rate quicker on neuron stimulation
Quicker in the presence of more extracellular calcium
Who conducted the study that found that endocytosis involves 3 processes?
Gandhi and Stevens (2003)
What did Gandhi and Steven s 2003 find?
Using synapto phluolin dyes
44% of endocytosis occurred fast (800ms)
20% occurred have time course of 10 seconds
36% have time course of >45 seconds
What does the 44% with time course of 800ms in gandhi and Stevens study represent?
Kiss and run
What does the 20% with time course of 10a in gandhi and Stevens study represent?
Compensatory mechanisms
What does the 36% with time course of >45S in gandhi and Stevens study represent?
CME
