Synapsins do not define resting/recycling status of vesicles Based on our observ
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Question
Synapsins do not define resting/recycling status of vesicles
Based on our observation that the synapsins interact specifically
with SVs in the resting pool, it might be expected that deletion of
the synapsins should increase the proportion of recycling pool
vesicles at the expense of the resting pool. This could happen due
to two different reasons: either the synapsins directly define the
status of the SVs as resting or recycling, or a larger fraction of the
resting vesicles is lost into the axon of TKO neurons due to their
higher mobility. To assess this possibility, we first measured the
size of the recycling pool near the active zone using hyperkalemic
loading of FM1–43 (Ryan et al., 1993). The peak FM1–43 inten-
sity of synapses in TKO neurons (n 29/925) was significantly
weaker than in WT ones (n 29/964, p 0.001; Fig. 7A,B)
(Gabriel et al., 2011), much like the decrease in the total vesicle
population (Fig. 1). The extent of FM1–43 destaining was similar
for both groups (WT, n 24/790; TKO, n 21/663; p 0.38;
Fig. 7A,C) (Gabriel et al., 2011), illustrating that loss of the syn-
apsins does not affect the propensity of the recycling vesicles to be
released. Our results using FM1–43 argue against a compensa-
tory increase in the size of the recycling pool. However, because
synapsin may participate in endocytosis (Bloom et al., 2003),
which could affect FM1–43 loading, we also performed experi-
ments to probe the recycling pool in an endocytosis-independent
manner. For this we used the alkaline trapping method (Sankara-
narayanan and Ryan, 2001; Fig. 7D) in neurons expressing Syn-
aptophysin I-2XpHluorin (Granseth et al., 2006). The relative
fraction of the recycling vesicles within the presynaptic terminals
of cultured hippocampal neurons has been estimated to be
0.5
on average (Fernandez-Alfonso and Ryan, 2008; Kim and Ryan,
2010). Our findings reveal a comparable fraction, which was
equivalent for WT (n 11/225) and TKO synapses (n 19/412,
p 0.77; Fig. 7E,F). Our results demonstrate that the synapsins
do not participate in the allocation of vesicles into specific func-
tional pools (but see Akbergenova and Bykhovskaia, 2010), con-
trary to the hypothesis stated above. From this conclusion it
follows that the resting and recycling pools scale in relation to one
another in a synapsin-independent manner.
What do you think of the aim for this experiment and the conclusion? (300 words)
renbuch et al.Synapsin Controls Mobility of Resting Vesides TKO 5 um 60 40 20- 0 01.0-T 0.5 0.0 WT TKO WT TKO D Rest Stim NHCI 1001 WT 0.6 0.4 0.2 O 0 0 50 100 %Recycling WT TKO igure 7. ubdivision of vesides into resting and recxing pools B unaffected by the synapsins. A, Synapses loaded with FM1-43 (top)in WT (left) andsynapsin TKD (right) neurons. Notice the brighter signal in WT neurons. The same fields are shown after unloading (bottom) B. Normalized intensity of FM1-43 intensity in loaded terminals (mean ± SEM), as shown in AC ercentage of FM1-43unloaded byhyperkalemic stimulation D,Representativeimages of synapsesof WT neurons expressing Synapto- hysin -2xpHluorinatrest Oeft),after therecyding pool isrevealed by hyperkalemicstimulation in the presence ofbafilomyin A (midde) and after the total pools visualized by alkalinization of allvesides by NH,CI saline (right)E,Cumulative probability plots of thefration of he recyding pool out of the total population for allsynapses analyzed. The plots overlap. F, Average of the mean values obtained from independent experiments. No difference is observed between WT and TKO neuros. pExplanation / Answer
Aim of the experiment:
To examine whether synapsins cluster synaptic vesicles around the active zone by means of immobilization.
Conclusion of the experiment
Researchers have concluded that synapsins precisely immobilize synaptic vesicle in resting pool . Neurons that do not have synapsins , resting seminal vesicle mobility equal to ones that are recycling. Hence, transport of seminal vesicle into the axon is enriched at the expenditure of retaining of Seminal vesicles inside the terminal. However, there is no change in relative division of Seminal vesicles amongst resting and recycling pools. Hence, researchers have concluded that there is a distinct machinery that control the interconnection of these pools.
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