Wilhelm_Science_2014

<p>title: &nbsp;Compositon of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins.</p>

<p>

authors: &nbsp;Benjamin Wilhelm, Sunit Mandad, et al &nbsp;senior author &nbsp;Silvio Rizzoli</p>

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work done at <b>Gottingen</b> Med Center.</p>

<p>

Synaptic vesicle recycling has long served as a model for the general mechanisms

of cellular trafficking. We used an integrative approach, combining<b> quantitative</b>

<b>immunoblotting</b> and mass spectrometry to determine protein numbers; electron

microscopy to measure organelle numbers, sizes, and positions; and super-resolution

fluorescence microscopy to localize the proteins. Using these data, we generated a

three-dimensional model of an“average”synapse, displaying 300,000 proteins in atomic

detail. The copy numbers of proteins involved in the same step of synaptic vesicle

recycling correlated closely. In contrast, copy numbers varied over more than three orders

of magnitude between steps, from about <b>150 copies for the endosomal fusion protein</b>s

to more than <b>20,000 for the exocytotic ones</b>.</p>

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<b>Exocytosis</b> (wiki) = cell directs contents of secretory vesicles OUT of the cell membrane

and into the extracellular space. &nbsp;(Note: above: &nbsp;that process requires 100X more protein

than does ENDOcytosis.) &nbsp;</p>

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&nbsp;

May 30, 2014 12:11 PM &nbsp;Silvio sent me the article pdf. &nbsp;v prompt!</p>

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they display 300,000 proteins in atomic detail.</p>

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<b>synaptic bouton occupies its OWN compartment</b>!!!&nbsp;

&nbsp;(recall my synaptosome studies in 1971)</p>

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They created synaptosomes from &nbsp;cortex and cerebellum of rats. &nbsp;Got &nbsp;58.5% &nbsp;resealed

vesicle loaded synaptosomes. &nbsp;&nbsp;did EM: &nbsp;got spatial params (size, surface, volume)</p>

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page 1024: &nbsp;our quantification got 23% of total protein in the preparation.

Synaptosome make up 58% of the preparation; &nbsp;thus we addressed 40.5% of total protein in synaptosome.</p>

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All quantitated proteins (using iBAQ and immunoblot) added up to 88.4% of protein weight.</p>

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quant immumnoblotting: &nbsp;&nbsp;for 62 synap proteins:</p>

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total # of particles in the synaptosome prep = 17 million and 58% are

synaptosomes. &nbsp;&nbsp;about &nbsp;10M synaptosomes per microgram.</p>

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volume = 0.37 micron3 &nbsp;&nbsp;&nbsp;volume = 4/3 &nbsp;pi r3; = 4* r3 =&nbsp;

so, if volume = 0.4 &nbsp;then &nbsp;r3 = 0.1; &nbsp;&nbsp;<b>so radius is about 200 nm </b>!!!!</p>

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note: surface area = 2.3 micron2 &nbsp;= &nbsp;4 pi r2 = &nbsp;12 * r2

so, &nbsp;2.4 /12 = .2 &nbsp;and sqrt of .2 gives a radius 400 nm&nbsp;</p>

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so, avg radius is prob about 300 nm &nbsp;(avg). &nbsp;and diam = 600 nm.</p>

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of vesicles = 384

vesicle volume = .015 &nbsp;(total volume) &nbsp;must divide by number

interves disatnce = 60 nm.</p>

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volume of mitochondria = .12 &nbsp;so, radius &nbsp;may be about 150 nm. &nbsp;cubed = .003 &nbsp;* 4 = .12 .</p>

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they state in article &nbsp;(see caption of figure 3) &nbsp;display <b>synap vesicles have a diameter of 42 nm</b> &nbsp;!!!</p>

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Figure 3... mind blower !!! &nbsp;&nbsp;look at cast of characters...</p>

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vATPase = vesicular ATP ase;</p>

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exocytic fusion proteins &nbsp;are SNAREs &nbsp;abundance matched by actin and tubulin;</p>

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&nbsp;</p>