brian covello: neurite outgrowth science report
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Brian Covello 01/15/13
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Summary of: “Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome”
Racchetti, G., Lorusso, A., Schulte, C., Gavello, D., Carabelli, V., D’Alessandro, R., and Meldolesi, J. 2009. Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome. Journal of Cell Science. 123(2): 165-‐170. doi: 10.1242/jcs.059634
Database: Discoveroux The complexity of the world contained within the leaflets of the plasma membrane
is only beginning to reveal itself to scientists. It is a world in which proteins, lipids, nucleic
acids, organic, and inorganic substances are constantly interacting with one another in
myriad dynamic processes. Indeed, the term “fluid-‐mosaic model” is appropriate, for not
only is the membrane in constant flux, but the membrane is consistently being recycled
through coupling of endocytosis and exocytosis (Cocucci, 2007). Regulated exocytosis is a
process by which vesicles from within the cell fuse to the plasma membrane upon some
stimulation such as photolysis of a calcium cage or other intracellular activators
(Borgonovo, 2002). For many decades, scientists believed the process of regulated
exocytosis was reserved for neurons, endocrine, and exocrine cells, the primary culprits for
secretions (Meldolesi, 2011). The electrophysiological patch-‐clamp technique can record
the resistance of a membrane by using a micropette tip capable of maintaining a constant
membrane potential to the membrane (Lindau, 2012). The linear correlation between
capacitance and membrane area allows scientists to monitor the surface area of a cell
(Cocucci, 2007). Through this technique, scientists discovered that the role of regulated
exocytosis is critical not only for secretions but also for changes to the plasma membrane
including, expansion of the surface, change of lipid composition, and insertion of proteins
(Cocucci, 2007).
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Such classical exocytotic vesicles responsible for membrane expansion include
acidic secretory lysosome vesicles and other additional dense vesicles from neurosecretory
cells (Borgonovo, 2002). These processes are best studied in vitro through investigation of
rat pheochromocytoma PC12 cells, as these cells serve as an appropriate model for study of
exocytosis and neurosecretions (Cocucci, 2008). Classically regulated exocytotic vesicles
are acidic, clear, dense, and rely on a distinct class of SNARE proteins, which are inhibited
upon administration of tetanus toxin (Borgonovo, 2002). Thus, in 2002, when scientists
administered tetanus toxin to a PC12-‐27 cell line, a derivation of PC12 defective in
neurosecretions, they were appalled to find an increase in capacitance of the membrane, as
this indicated expansion of the plasma membrane through exocytotic activity (Borgonovo,
2002). They had discovered a rapid, regulated, exocytotic system, composed of vesicles
that were 75-‐115nm in length, non-‐acidic, tetanus toxin insensitive, and packed with
cholesterol and sphingomyelin (Borgonovo, 2002). It was approximated that there were
around 9650 vesicles within a given cell. Combined, these vesicles had the capability to
enlarge the membrane by 169μm (Borgonovo, 2002). Through vesicle marker Ahnak, they
proved the existence of a distinct class of organelles that were responsible for plasma
membrane enlargement (Borgonovo, 2002). They called these vesicles “enlargeosomes”.
Surface expansion is especially important in neuronal development, where neuronal
differentiation leads to axon and dendrite formation (Meldolesi, 2011). Taken together
with the older and slower Ti-‐VAMP vesicles, which can be released through stimulation by
nerve growth factor (NGF), researchers hypothesized that enlargeosomes were distinctly
responsible for a newly discovered form of neurite outgrowth in embryonic (PC12-‐27) and
neonatal (SH-‐SY5Y) neurons (Racchetti, 2009). Understanding of such a mechanism is
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critical for comprehension of neuronal development, for this mechanism may possibly have
medical applications that lead to drugs capable of membrane repair.
The criterion to prove this hypothesis was voluminous. First, one must show that
rapid neurite outgrowth occurs in the cell lines rich in enlargeosomes, PC12-‐27 and SH-‐
SY5Y, while slow outgrowth occurs in cell lines lacking enlargeosomes, wtPC12 (Racchetti,
2009). All cell lines were treated with Y27632, a drug known to induce rapid neurite
outgrowth (Racchetti, 2009). Neurite visualization occurred through DIC-‐time lapse video
(0-‐90 minutes) and phase contrast (at 0,1,6,48 hours after treatment) (Racchetti, 2009).
Analytical cell surface expansion was measured through patch-‐clamp capacitance assays
0,1,3, and 6 hours after treatment (Racchetti, 2009).
To determine whether another mechanism existed for rapid neurite outgrowth, the
team immunolabeled and ran a western blot on the protein marker for enlargeosomes,
Ahnak, and immunolabeling of enlargeosomes were visualized by immunofluorescence
microscopy (Racchetti, 2009). Translocation of this marker would indicate insertion of
enlargeosomes into the membrane. As a condition of the hypothesis, the team needed to
prove that enlargeosomes were a separate and distinct mechanism for rapid neurite
outgrowth. To this end, they individually inhibited Golgi vesicles and endosomes known to
cause slow neurite outgrowth through brefeldin A and LY294002 respectively (Racchetti,
2009). Endosomal activity was further inhibited through an insertion of a dominant
negative construct of ARF6, a protein essential in endosome development (Racchetti,
2009). Lastly, for the purpose of visualizing and quantifying enlargeosome activity in vivo,
the team analyzed western blot data and immunolabeled Ahnak distribution through
immunofluorescence microscopy from rat embryonic neurons (Racchetti, 2009).
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DIC-‐time lapse video and phase contrast microscopy indicated rapid neurite
outgrowth in PC12-‐27 and SH-‐SY5Y within one hour of treatment by Y27632 (Racchetti,
2009). Patch clamp capacitance analysis for PC12-‐27 showed an increased plasma
membrane surface area of 500μm at 1 hour (Racchetti, 2009). In contrast, wtPC12 neurite
outgrowth could be visualized at 6 hours, with surface expansion of 100μm at 1 hour
(Racchetti, 2009). Ahnak levels remained constant throughout the 48 hours, yet strong
translocation of Ahnak to the outermembrane was depicted through immunofluorescence
microscopy (Racchetti, 2009). Rapid neurite outgrowth occurred in PC12-‐27 cells, even in
the presence of brefeldin A, LY294002, and dominant negative ARF6 (Racchetti, 2009).
Western blot data from rat embryonic neurons indicated the highest levels of Ahnak in
embryonic neurons and the lowest levels in adult neurons (Racchetti, 2009).
Immunofluorescence of rat neurons indicated intense outgrowth of neurites in the location
of Ahnak staining (Racchetti, 2009).
Through results analysis, one may determine the speed of neurite growth in PC12-‐
27, SHSY5Y, and wtPC12 cell lines with corresponding analytical measurement of plasma
membrane surface expansion. In addition, Ahnak labeling allows for translocation
visualization and implicates enlargeosomes involvement, while concomitant inhibition of
the classical pathway and proof of enlargeosome involvement may show a distinct
mechanism for rapid neurite outgrowth. Finally, in vivo analysis determines the direction
these results may have in pathological treatments involving neuroplasticity, neuronal
development and membrane repair.
Several clear conclusions may be drawn from the aforementioned results. The DIC-‐
time lapse video and corresponding phase contrast microscopy indicates that upon
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treatment with Y27632, cells that lack enlargeosomes contain a much slower neurite
outgrowth than cells with enlargeosomes. In fact, the cells containing enlargeosomes
experienced membrane expansion at 5 times the rate of the control within the first hour.
Proof that the enlargeosomes were causing this expansion was acquired by showing the
translocation of Ahnak upon treatment with Y27632. Even though western blot analysis of
Ahnak during this time period indicated constant protein levels, this does not necessitate a
weakening of the hypothesis, for plasma membrane expansion by enlargeosomes does not
require a change of protein levels, but merely a change in location.
Although the research indicates that enlargeosome activity was responsible for
rapid neurite outgrowth, the exact mechanism remained vague. The researchers
hypothesized that enlargeosome surface expansion was distinct from classical surface
expansion by Golgi vesicles and endosomes. Indeed, inhibition of these vesicles failed to
slow the neurite outgrowth (Racchetti, 2009). Additionally, inhibition of gene transcription
and translation of the classical pathway failed to stop rapid neurite outgrowth (Racchetti,
2009). With this data, it became clear that enlargeosomes were directly and distinctly
responsible for providing a new mechanism of rapid neurite outgrowth.
These results most likely motivated the final part of this experiment, in which a
transition to in vivo studies occurred. The data shows that enlargeosomes occur in regions
of intense neurite outgrowth in rat neurons, opening the door to many future studies
(Racchetti, 2009). The appearance of high levels of enlargeosomes in embryonic rat
neurons and a linear decrease of enlargeosomes with respect to progressive neuronal
development may indicate a physiological role for enlargeosomes in neuronal development
(Racchetti, 2009). This remains to be proven. The research contained several weaknesses.
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First, an unexplained decrease of membrane expansion occurred in wtPC12 cells at three
hours. Also, the research failed to indicate the number of trials, and the data was not
supported with statistical analysis. Regardless, the researchers had proven the hypothesis.
Enlargeosomes are distinct vesicles responsible for an entirely new form a rapid neurite
outgrowth. Within 10 years, the scientific understanding of the plasma membrane,
neuronal outgrowth, and regulated exocytosis has changed dramatically. Only as the
scientific horizon widens, may one begin to see the vastness of empty space left to be
fulfilled.
There are several intricate and beautiful ties between this research and the
knowledge acquired in a classroom. First, this research helped me gain a deeper insight and
appreciation into the term “fluid-‐mosaic model”. The term itself truly fails to give justice to
the complexity of the plasma membrane. The amount of details surrounding one small
vesicle’s involvement in membrane recycling was enormous. It is only in this context that I
began to understand how little we know. One item not mentioned previously is that the
cholesterol and sphingomyelin composition of enlargeosomes give rise to enhanced
detergent resistance in the plasma membrane (Borgonovo, 2002). I hypothesize that,
similar to caveolae, enlargeosomes not only serve the purpose of enlarging the membrane,
but also play a functional role as well. This provides an enticing future avenue for research.
Interestingly, it was not the search for articles concerning plasma membranes that
led me to this research. Instead, I discovered this article while researching patch-‐clamp
technique, for this technique, which has been in use since 1978, was entirely responsible
for the discovery of the enlargeosome in 2002 (Borgonovo, 2002). Perhaps the most
amazing aspect of this article transcends beyond the tiny realm of biochemistry, for the
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patch-‐clamp technique was only made possible by a conglomeration of physicists and
mathematicians. The discovery of the enlargeosome represents the pinnacle of science,
where an interconnected group of diverse scientists collide into discovery.
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Sources
Borgonovo, B., Cocucci, E., Racchetti, G., Podini, P., Bachi, A., and Meldolesi, J. 2002. Regulated exocytosis: a novel, widely expressed system. Nature Cell Biology. 4:955-‐ 962. doi: 10.1038/ncb888 Cocucci, E., Racchetti, G., Podini, P., Meldolesi, J. 2007. Enlargeosome traffic: Exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both. Traffic. 8:742-‐757. doi: 10.1111/j.1600-‐0854.2007.00566.x Cocucci, E., Racchetti, G., Rupnik, M., Meldolesi, J. 2008. The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4. Journal of Cell Science. 121(18):2983-‐2991. doi: 10.1242/jcs.032029 Lindau, M. 2012. High resolution electrophysiological techniques for the study of calcium-‐ activated exocytosis. Biochimica et Biophysica Acta. 1820:1234-‐1242. doi: 10.1016/j.bbagen.2011.12.011 Meldolesi, J. 2011. Neurite outgrowth: this process, first discovered by Santiago Ramon y Cajal, is sustained by the exocytosis of two distinct types of vesicles. Brain Research Reviews. 66:246-‐255. doi: 10.1016/jbrainresrev.2010.06.004 Racchetti, G., Lorusso, A., Schulte, C., Gavello, D., Carabelli, V., D’Alessandro, R., and
Meldolesi, J. 2009. Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome. Journal of Cell Science. 123(2): 165-‐170. doi: 10.1242/jcs.059634
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