brian covello: neurite outgrowth science report

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