Isolation and ultrastructural characterization of squid synaptic vesicles

Author Posting. © Marine Biological Laboratory, 2011. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 220 (2011): 89-96.Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.JAD is supported by the RI-INBRE program award # P20RR016457-10 from the National Center for Research Resources (NCRR), NIH

Spatial control of neuronal metabolism through glucose-mediated mitochondrial transport regulation.

Eukaryotic cells modulate their metabolism by organizing metabolic components in response to varying nutrient availability and energy demands. In rat axons, mitochondria respond to glucose levels by halting active transport in high glucose regions. We employ quantitative modeling to explore physical limits on spatial organization of mitochondria and localized metabolic enhancement through regulated stopping of processive motion. We delineate the role of key parameters, including cellular glucose uptake and consumption rates, that are expected to modulate mitochondrial distribution and metabolic response in spatially varying glucose conditions. Our estimates indicate that physiological brain glucose levels fall within the limited range necessary for metabolic enhancement. Hence mitochondrial localization is shown to be a plausible regulatory mechanism for neuronal metabolic flexibility in the presence of spatially heterogeneous glucose, as may occur in long processes of projection neurons. These findings provide a framework for the control of cellular bioenergetics through organelle trafficking

Spatial control of neuronal metabolism through glucose-mediated mitochondrial transport regulation.

Eukaryotic cells modulate their metabolism by organizing metabolic components in response to varying nutrient availability and energy demands. In rat axons, mitochondria respond to glucose levels by halting active transport in high glucose regions. We employ quantitative modeling to explore physical limits on spatial organization of mitochondria and localized metabolic enhancement through regulated stopping of processive motion. We delineate the role of key parameters, including cellular glucose uptake and consumption rates, that are expected to modulate mitochondrial distribution and metabolic response in spatially varying glucose conditions. Our estimates indicate that physiological brain glucose levels fall within the limited range necessary for metabolic enhancement. Hence mitochondrial localization is shown to be a plausible regulatory mechanism for neuronal metabolic flexibility in the presence of spatially heterogeneous glucose, as may occur in long processes of projection neurons. These findings provide a framework for the control of cellular bioenergetics through organelle trafficking

Mitochondrial transport is increased in regenerating axons co-deleted for PTEN and SOCS3.

<p>(A) Representative kymographs from live imaging of mitochondria in regenerating axons from PTEN^f/f; SOCS3^f/f and PTEN^f/f; SOCS3^f/f; SynCre neurons 20 h post injury. Consecutive line scans of the axon were arrayed top to bottom so that the y-axis of the kymograph represents the time and the x axis the position of the object studied. Stationary objects therefore appear as vertical lines and motile ones as diagonals. (B, C) Box plot showing the moving frequency of motile mitochondria (B) and their distance travelled (C) in regenerating axons of the indicated genotypes. Co-localization between MitoDsRed and Mitotracker confirmed that the vast majority of the mitochondria in the transfected cells were labeled (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184672#pone.0184672.s001" target="_blank">S1 Fig</a>). Mann-Whitney <i>U</i> test on the number of mitochondria. n = 136–223 mitochondria from 18–22 axons and 4–6 individually cultured embryos from 2 independent litters. (D-F) Representative kymographs (D) and quantification of the moving frequency (E) and distance travelled (F) from live imaging of EYFP-synaptophysin-positive synaptic vesicle precursors in regenerating axons of the indicated genotypes. Mann-Whitney <i>U</i> test. n = 153–226 synaptic vesicles, 12–17 axons, 4–5 individually cultured embryos from 2 independents experiments. (G-I) Representative kymographs (G) and box plots of moving frequency (H) and total distance travelled (I) from live imaging of Rab7-GFP positive endosomes in regenerating axons of indicated genotype. (Mann-Whitney <i>U</i> test. n = 69–105 late endosomes, 13–14 axons, 5–4 individually cultured embryos from 2 independents experiments. (J and K) Retrograde <i>(J)</i> (toward cell body) and anterograde <i>(K)</i> (toward axon’s tip) moving frequencies of the mitochondria analyzed in (A). Mann-Whitney <i>U</i> test. (L) Mitochondrial densities in the axons analyzed in (A). Two tailed Student’s Unpaired <i>t</i>-test. Data in all the box plots are represented with a box that delimitates the lower (Q1) and the upper quartile (Q3) of the distribution. Horizontal line indicates the median (Q2) and whiskers indicate the maximum and minimum of the distribution.</p

A high mitochondrial transport rate characterizes CNS neurons with high axonal regeneration capacity

<div><p>Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. DRG neurons globally increase axonal transport during regeneration. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. Here, using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes) in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration.</p></div

Characterization of Synapsin Cre in neurons cultured in microfluidic chambers.

<p>(A) Immunohistochemistry of cortical neurons (DIV6) isolated from Synapsin Cre; stop^f/f TdTomato transgenic mice. Anti Tuj1 antibody was used as a neuronal marker. TdTomato (magenta in the merged image) is present in almost all the neuronal cell bodies. Scale bar = 50μm. (B) Immunohistochemistry using Tuj1 (axonal marker, magenta), MAP2 (dendrite marker, green first row) or GFAP (glial cell marker, green second row) antibodies on E18 mouse cortical neurons culture (DIV7) in microfluidic chambers. Higher magnifications images of somal and axonal compartments are shown in the second and third columns. Neurons were plated in the chamber on the left and their axons grew through the grooves in the center section to emerge in the axonal chamber at the right. 450 μm microgrooves allows a complete isolation of axons from dendrites and glia as indicated by the absence of those markers from the right-hand chamber. Antibodies typically did not reach inside the microgrooves unless explicitly caused to enter (not shown), which is why grooves remain largely dark. Scale bar = 100μm (C) Tuj1 immunohistochemistry of E18 mouse cortical neurons cultures (DIV7) in microfluidic chambers: No Injury (left), immediately after injury (center) and 20 h after injury (right). (D) E18 mouse cortical neurons cultures from Synapsin Cre; stop^f/f TdTomato embryo culture fixed 20 h post axonal injury.</p

PTEN and SOCS3 co-deletion increased mitochondrial transport in cultured neurons whose axons were not severed.

<p>(A-C) Representative kymographs (A) and quantification of moving frequency (B) and distance travelled (C) from live imaging of mitochondria in PTEN^f/f; SOCS3^f/f and PTEN^f/f; SOCS3^f/f; SynCre in intact axons. Mann-Whitney <i>U</i> test on the number of mitochondria. n = 117–118 mitochondria from 13–14 axons and 4–5 individually cultured embryos from 2 independent experiments. (D-F) Representative kymographs (D) and quantification of moving frequency (E) and distance travelled (F) from live imaging of synaptophysin-positive synaptic vesicle in intact axons of indicated genotype. Mann-Whitney <i>U</i> test. n = 123–212 synaptophysin-positive synaptic vesicles from 13–14 axons and 4–5 individually cultured embryos from 2 independent experiments.</p

Deletion of PTEN and SOCS3 improves axonal regeneration of E18 cortical neurons.

<p>(A) Schematic of the <i>in vitro</i> platform to study the axonal transport in regenerating PTEN^-/-; SOCS3^-/- cortical neurons. PTEN^-/-; SOCS3^-/- E18 cortical neurons were obtained by breeding PTEN^f/f; SOCS3^f/f mice with the PTEN^f/f; SOCS3^f/f; Synapsin Cre (SynCre) mice. The cortex of each embryo was processed individually so that each microfluidic chamber was seeded with neurons from a single embryo. Thereby PTEN^f/f; SOCS3^f/f; SynCre neurons were compared to PTEN^f/f; SOCS3^f/f from littermate embryos. All neurons were cotransfected with MitoDsred2 and EYFP-Synaptophysin or Rab7-GFP. (B) Quantification of <i>in vitro</i> axonal regeneration of PTEN^-/-; SOCS3^-/- and control cortical neurons 20h post injury. n = 9–11 microfluidic cultures of individual embryos from 5 independent experiments. Two tailed Student’s Unpaired <i>t</i>-test.</p