1. by the rods and cones containing high



Melanosomes are lysosome
related organelles that contain high densities of the chemical compound,
melanin (Orlow, 1995). Melanin is the collective name for a family of molecules
carrying out similar functions, but differing in chemical structure (Solano,
2014). The entire role of melanin is still under investigation. Currently, it
is widely accepted that it, plays a large role in selective absorption of the
visible and ultra violet light spectra resulting in pigmentation and
photoprotection (Ito & Wkamatsu, 2003; Kollias, 1994), possesses several
antioxidant properties, scavenges free radicals and also removes toxic singlet
oxygen species (Sarna et al., 1985; Ró?anowska
et al., 2011). Melanin can also protect against the
generation of oxidising species by isolating multivalent transition metal ions
(Pilas et al., 1998). The most
abundant forms of melanin are eumelanin and pheomelanin.

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The melanin content within
melanosomes is responsible for the pigmentation patterns we observe across the
animal kingdom (Ito & Wakamtasu, 2003).Melanosomes biosynthesis occurs within
melanophores of fish and amphibians, the iris, choroid, and the Retinal Pigment
Epithelium (RPE) cells (Prota et al., 2012).

The RPE is a monolayer of
cells lying between the photoreceptors and the choroid. The RPE cells have
apical processes which are finger like extensions which surround the
photoreceptors of the retina (Futter et
al., 2004). The RPE provides nutrients from the choriocapillaries to the photoreceptor
cells of the retina, and reduces photooxidative damage (Strauss, 2005). They
phagocytose and dispose of segments shed by the rods and cones containing high
levels of toxic oxidative products. (Kevany & Palczewski, 2010). The RPE cells
are also heavily pigmented with melanosomes. Vertebrates have two distinct
shapes of melanosomes present in their RPE (Clements et al., 2016). Spherically shaped melanosomes are present in a
layer below longer elongated cylindrical shaped melanosomes which are present
at the base of, and within, the apical processes (Kim & Choi, 1998; Liu et al., 2005, Pelkonen et al., 2016).

Cylindrical melanosomes have
been observed to advance and retreat along the apical processes during light
adaptation and dark adaptation respectively. Burnside et al. (1983) exposed RPE cells of the blue striped grunt (Haemulon sciurus) to high intensities of
light and observed the movement of melanosomes into the apical processes at a
speed of 3.4-3.5 microns/min. This has also been observed occurring in
zebrafish (Danio
rerio) by Burgoyne et al. (2015). This phenomenon is thought
to be limited to in fish and amphibian species (Arey, 1915; Burnside &
Laties, 1979), though more recent evidence from Futter et al., (2004) has revealed a modest but significant movement of
melanosomes in the RPE of mice. Spherically shaped melanosomes have never been
observed entering the apical processes and instead lie in a layer below those
that are cylindrical in shape (Kim & Choi, 1998).

Epidermal melanocytes also
manufacture melanosomes in a wide range of morphologies (Sakurai et al., 1975; Liu et al., 2005; Li et al.,
2010). Analysis by Inazu & Mishima, (1993) and Liu et al. (2005) into the melanin content of differently shaped epidermal
melanosomes found that it differed. Melanosomes containing purely eumelanin are
cylindrical shape, with reduced densities of eumelanin seen in ellipsoidal
melanosomes (Prota, 2012). Melanosomes containing largely eumelanin are called
eumelanosomes. Melanosomes termed pheomelanosomes
contain largely pheomelanin and are present in more spherical shapes (Prota,
2012). As the ratio of pheomelanin to eumelanin increases, the colour the
melanosomes reflect moves from dark black and brown colours to lighter brown
and yellow or reddish hues (McGraw & Wakamatsu, 2004).

Following the results from
the analyses of epidermal melanosomes, one could suggest then that the
spherical and cylindrical melanosomes of the RPE contain different melanin
compositions. However, most studies analysing melanin content of the RPE have
concluded that it contain largely, if not solely, eumelanin regardless of their
morphology (Jimbow et al., 1979; Prota
et al. 1998; Sarna, 2003; Liu et al., 2005).  To clarify if this is the case, one would have
to successfully separate the RPE melanosomes based on their morphology. We hypothesised
that once isolated from the RPE cellular content, differing characteristic
features of the spherical and cylindrical RPE melanosomes could be used to
separate them by their morphology. Once separated, the fractions could then be
chemically analysed to identify any differences in their chemical components.
This would further our understanding of the presence of either shape within the


Materials and Methods


2.1 Preparation of Tissues


Three different species were
used in our investigation; the Domesticated pig (Sus
scrofa domesticus), the Atlantic
Mackerel (Scomber scombrus) and the Common Pheasant
colchicus). 6 porcine
eyes were sourced from the supplier Prestige Pork, Orchard Farm, and obtained
from Ruby and Whites, Bristol. The mackerel eyes were sourced from a wild
population in the English Channel at Looe coast and obtained from The Fish
Shop, Bristol, and the pheasant eyes were sourced from the wild population in
Somerset, England, and obtained from Ruby and Whites, Bristol. Prior to
extracting the RPE from the eyeball, the eyes of the mackerel and pheasant were
removed from the cranium by severing the extraocular muscles and the optical
nerve. Once the eyes were isolated, they were cut transversally around the
equator just below the ora serrata. Next, the anterior section of the eye was
removed exposing the lens and vitreous which were then removed. Tweezers were
used to remove the RPE from the back of the eye and the optical nerve was cut
to free it. The RPE from each porcine eye was placed into separate Eppendorf
tubes. Both RPEs from the fish were placed into one Eppendorf tube, and the
same was done for the pheasant totalling 4 samples. All Eppendorf tubes were
labelled with their content.


2.2 Preparation of Isolated Melanosomes


To isolate the melanosomes
from the RPE tissue, a step by step extraction protocol was carried out. The
same cycle of incubation was used after each step (24 hrs at 38°C and 200 rpm).
Each washing step involved vortexing the sample before centrifuging and removing
the supernatant each time. The procedure went as follows:-

To begin, samples were
washed 3 times in ethanol and a solution of 10mg of dithiothreitol (DTT)
dissolved in 1ml of phosphate-buffered saline (PBS) was added before the
samples were incubated. After incubation, the samples were centrifuged and the
supernatant removed using a pipette. A second solution of 10mg of DTT, 0.4 mg
of proteinase K and 1ml of PBS was added and the samples were incubated. After
incubation, the samples were centrifuged and the supernatant was removed. This
step was repeated until a pellet formed at the bottom on the Eppendorf tube. Next,
the pellet was washed 6 times in biomolecular grade water, and a solution of 5
mg DTT, 1mg Papain and 1ml of PBS was added. The samples were then incubated. After
incubation, the sample was centrifuged and the supernatant was removed. The
sample was then washed 6 times in biomolecular grade water before a solution of
2mg DT, 0.4 mg proteinase K and 1ml PBS was added. The samples were incubated.
After incubation, the supernatant was removed and 2% Triton X-100 solution was
added and the samples were stirred for 4 hours. When complete, the 2% triton
X-100 was removed. Ethanol was used once to wash the samples prior to being
washed 8 times in biomolecular grade water. Another solution of 2mg DTT, 0.4 mg
proteinase K and 1 ml PBS was added before the samples were incubated. After incubation,
the samples were centrifuged and the supernatant removed. Finally, the samples
were washed 3 times in biomolecular grade water and left to dry in the
Eppendorf tubes. A small amount of each sample was removed and placed on electrically
conductive carbon double-sided adhesive tape on a specimen tub and left to dry overnight.


2.3 Scanning Electron


The 3 tissue sample types
prepared as described above were then analysed using an electron microscope. The
samples on the specimen stubs were sputter-coated in 10?m of gold to be viewed under a scanning electron microscope.
The backscattered electron detector microscope was used with a scanning speed
of 6 and working distance of 9 mm. To improve the microscope’s resolution, the
probe current was also altered.


2.4 Gel Electrophoresis


For gel electrophoresis, small quantities of
the sample used. Different gel concentrations and sample concentrations were
used along with different levels of voltage to investigate if separation of the
melanosomes based on their shape was possible using this method. The details of
this are outlined as follows below.


2.4.1 Preparation of Gels using TAE Buffer


The buffer solution used
contained a mixture of Tris base, acetic acid and EDTA (TAE). The first gels
were made at two different concentrations. For a 1% and 0.6% agarose solution,
250mg of agarose was dissolved in 25ml of TAE and 150mg of agarose was
dissolved in 25ml of TAE respectively. The gels were left to cool before being
poured into a gel casting tray to allow them to set. These were then placed in
the electrophoresis chamber and submerged in a buffer solution made up of 10x
concentration of TAE in biomolecular grade water. The melanosome samples were
diluted down to 50% of their original concentration and 25% of their original
concentration using distilled water DNA gel loading dye. 15ml, of both
concentrations of sample, were placed in individual wells for each of the pig,
pheasant and fish samples. Both gels were run using a voltage of 120mV for as long
as possible before the gels melted. 


2.4.2 Preparation of Gels
using SB Buffer


The buffer solution
contained sodium borate (SB) dissolved in biomolecular grade water. Gels formed
from this buffer are able to withstand a higher voltage without melting. Two
concentrations of gels were made by dissolving 150mg of agarose in 25ml of SB
buffer to make a 0.6% gel, and 75mg of agarose in 25ml of SB buffer to make a
0.3% gel. The gels were cooled in gel casting trays before being fully
submerged in a solution made up of 10x concentration of SB buffer within the
electrophoresis chamber. The samples were not diluted with distilled water,
only gel loading dye was added as a 6th of the total concentration
of sample i.e. 6ml of dye was added to 30ml of sample. 15ml of dyed sample was loaded
into a well in both concentrations of gels. Both gels were run for up to 6
hours at 200mV.


3.      Results


Scanning Electron Microscopy


The melanosomes isolated
from the RPE cells were examined using a scanning electron microscope (Figure
1). It showed that across all three species, the melanosomes present in the RPE
are heterogeneously shaped. We observed a wide spectrum of morphologies ranging
from spherical melanosomes to cylindrically shaped melanosomes. The lengths of
the cylindrical melanosomes ranged from approximately 1 µm – 3 µm. The longest
cylindrical melanosomes were observed in the fish sample (Figure 1f). The more
spherical melanosomes measured between approximately 0.3 µm and 1 µm in length.
However, it was evident from the images that the extraction protocol we
followed had not yielded a pure melanosome sample. Cellular debris could be
seen largely in our porcine and pheasant sample. The isolation of mackerel
melanosomes was most successful as the sample had much less tissue content
remaining in the sample compared to the pig and pheasant samples (Figure 1e and

Agarose Gel Electrophoresis


Separation of the
melanosomes based on their shape using agarose gel electrophoresis was
unsuccessful. The first gels made with TAE buffer began to melt approximately
30 minutes after we began their run time. We noted that in this time the
melanosomes in all samples had been attracted towards the anode but were not
entering the agarose gel. The use of SB buffer in further electrophoresis
experiments allowed a greater voltage to be used for at least 6 hours, as well
as a low concentration gel able to withstand the increased voltage. Within this
time we observed the dye being pulled through the gel towards the anode, but the
melanosomes did not travel further than the wells throughout the entire
experiment (Figure 2a). The melanosomes instead could be seen aligned along the
side of the well closest to the anode (Figure 2b). Agarose gels with a
concentration lower than 0.3% could not be used in the experiment as removal of
the comb was too damaging to the gel. The aggregation of the melanosomes
indicated that it was not that the voltage used in the experiment was too low
for the organelles to be carried towards the anode, but it was in fact the
viscosity of the gels.




To analyse the chemical composition
of the melanosomes, high quantities of either shape of the melanosomes would
have to be obtained. The protocol we followed as described above yielded a high
abundance of intact melanosomes, but it did not provide them in pure isolation.
The protocol had been originally tailored to extract melanosomes from feathers,
which contain high levels keratin (Woodin, 1954). Our experiment required the
breakdown of a much higher abundance of structural lipids present in the RPE
cells. To achieve that lipid removal, I therefore suggest that the protocol could
have perhaps been modified to include additional cycles of 2% Triton X-100,
however this should be used in caution as it could otherwise lead to the
breakdown of the melanosome membranes (Orlow et al., 1993). Alternatively, Kushimoto et al. (2001) successfully isolated stage I and stage II
melanosomes from human melanoma cells. They firstly homogenised the cells in
0.25 M sucrose before using separate steps of centrifugation to yield a
fraction containing pure melanosomes, a process that is outlined in Seiji et al. (1963). This process has been
revised and improved by Pelkonen et al. (2016).
This method may therefore be considered as an alternative approach in the
future for melanosome isolation.

The contamination of our
sample with choroidal melanosomes must also be considered. The choroid tissue
runs alongside the RPE around the back of the eye and there is a chance that it
may have been dissected from the eye along with the RPE. Choroidal melanosomes are
spherical in shape (Orlow & Brilliant, 1999; Liu et al., 2005). The origin of the melanosomes within our samples cannot
be identified from SEM imaging as the morphology of spherical melanosomes
within the RPE and those within the choroid is identical. To reduce the chances
of this occurring, more accurate dissecting practises may be required in future

After obtaining our sample
from the extraction protocol, the further separation of spherical and
cylindrical melanosomes could not be carried out using agarose gel
electrophoresis based on our results. The pores of even very low gel
concentrations were too small to facilitate the movement of melanosomes.
However, it allowed us to confirm that melanosomes do have a negative charge. Melanin
contains carboxylic groups which are negatively charged (Ito, 1986). However,
by investigating the electrophoretic velocity of melanosomes extracted from
melanophores for the African leaf frog (Xenopus
laevis), Testorf et al., (2001)
suggested that the melanin they contain is not the main source of the negative
charge of the organelle and that instead, properties such as membrane bound
proteins and associated motor proteins, do. RPE melanosomes move throughout the
cell via the cytoskeleton in a similar fashion to that of melanosomes found
within melanophores (Gibbs et al.,
2004; Klomp et al., 2007). The
negative charge of RPE melanosomes may therefore originate from similar features
of the organelle, however this has never been investigated. Burgoyne et al., (2015) concluded that
cylindrical melanosomes had the same volume as spherical melanosomes, but a
greater surface area. The possibility of using solely charge to separate either
shape of melanosome is opened up based on this research, assuming that the
number of membrane proteins is different between spherical and cylindrical
melanosomes. A suitable method would free flow electrophoresis. The method is
outlined by DanieláLilly
(2009) and has been used by Kushimoto et al.
(2001) to successfully separate stage I and stage II melanosomes retrieved from
human MNT1 melanoma cells.

The presence of melanosomes
within the RPE is important to the survival of the retina. They provide photoprotection
as melanin absorbs much of the visible and ultraviolet light spectra (Riley,
1997). Mondragón
& Frixione, (1989) provided the following evidence for the role of
cylindrical melanosomes within the RPE: during levels of high light intensity, cylindrical
melanosomes are transported up the apical process of the epithelial cells to
surround the photoreceptors. They are returned back to the base of the apical
processes during dark adaptation. This has also been observed in many fish and
amphibians (Bäck
et al., 1965; Burnside et al. 1983; Burgoyne et al., 2015), however evidence for this
occurring within mammals is limited. The transport of melanosomes up and down
the apical processes reduces the extent of accumulative photooxidative damage
by absorpbing excess light (Sarna, 1992) and provides protection against
bleaching of the photoreceptors (Bäck et
al., 1965). These studies provide evidence of the adaptive benefits
cylindrical melanosomes within the RPE, however evidence explaining the presence
of spherically shaped melanosomes within the RPE is limited.

Similarities between RPE
melanosomes and epidermal melansomes have been reported in their melanogenesis,
structure and morphology (Jimbow et al.,
1979). However, the RPE cells and melanocytes originate from different lines of
cells during foetal development. The melanocytes are derived from the neural
crest cells whilst the RPE cells originate from neuroepithelial cells (Sarna et al., 1992). There is also an
important difference in melanosome turnover in either cell over the lifetime of
an individual. Melanogenesis occurs continuously within epidermal melanocytes
to provide pigmentation to growing feathers, hairs and regenerating skin cells
(Jimbow et al., 1986). The ratio of eumelanin to pheomelanin packaged into the
melanosomes is determined by pigmentation genes (Sturm et al., 2001). Peptide hormones ?-, ?– Melanin Stimulating Hormone (MSH) and
adrenocorticotrophic hormone bind to the melanocortin 1 receptor on the
melanocyte membrane (Bertagna, 1994). Activation of this receptor results in
melanocyte maturation or switching of melanin type deposited within the
melanosomes (Mountjoy et al., 1992).
It alters the concentration of compounds available within the premelanosome
preluding to the deposition of differing ratios of eumelanin to pheomelanin as
the melanosome matures (Ito & Wakamatsu, 2000; 2008). Ultimately, the
morphology of a melanosome is dependent on an internal glycoprotein matrix of
the premelanosome built during melanogenesis (Seiji et al., 1963). Eumelanosomes are formed with a striated fibril
structure with additional cross linkages which receive high densities of
eumelanin deposition (Brumbaugh, 1968). Pheomelanosomes have reduced uniformity in their internal structure and
contain vesiculoglobular bodies onto which melanin is deposited (Jimbow et al., 1983).

Investigations into
melanogenesis within RPE cells has revealed that it terminates before birth in
animals (Feeney-Burns, 1980) and thus, the feature of continuous melanogenesis
cannot be extended to RPE melanosomes (Carr & Siegel, 1979; Simon et al., 2008). Kim & Choi (1998)
investigated the morphologies of melanosomes present in rabbit RPE. They
produced electron micrograph images that cylindrical melanosomes are based in
or near the apical processes and spherical melanosomes are generally found
below them. From their findings, they suggested that all melanosomes present
within the RPE are synthesised simultaneously into spherical morphologies
during foetal development. They argued that the shape of the RPE melanosome is
not under genetic control, and instead they adapt their shape to the region
that they are relocated to after they mature. This was further supported by
Burgoyne et al. (2015). They
investigated melanogenesis within the RPE of Zebrafish and revealed that
premelanosomes of both cylindrical and spherically shaped melanosomes are
produced at the same time. At 2 days post-fertilisation melanosomes appear
similar in morphology with equal volumes and it is not obvious what morphology
preludes to which shape. However, by the 5th day, some of the
melanosomes had matured into elongated cylindrical melanosomes and were present
within and at the base of the apical processes of the RPE cells.

Liu et al., (2005) also compared the melanosome morphology between RPE
cells of newborn and mature bovine. They showed that the ratio of elongated to
spherical melanosomes decreased between the newborn and older specimens. They
then went on to measure melanin content of the bovine RPE melanosomes finding
very insignificant levels of pheomelanin leading them to the conclusion that
both shapes of bovine RPE melansomes are eumelanosomes. This conclusion was
also found by Sarna et al., (2003)
who used Electron Spin Resonance (ESR) spectroscopy to show that there was only
eumelanin within the RPE of humans and bovine.


5.      Conclusion

The studies discussed above
provide us with good evidence that the morphology of melanosomes present is not
representative of their melanin composition and that RPE melanosomes are synthesised
as spherical eumelanosomes during foetal development. The development of
cylindrical melanosomes then follows in areas of the RPE cell adjacent to the
apical processes. The presence of solely eumelanin within the RPE is adaptive
as eumelanin has a greater absorbance spectra and pheomelanin is generally
thought to be phototoxic (Brenner & Hearing 2008). However, there is yet to
be a study which can determine if both cylindrical and spherical melanosomes
containing solely eumelanin.

To answer this question,
future investigations should use different methodologies to the ones we practised
in our experiment. It is apparent from Pelkonen et al. (2016) and Kushimoto et
al. (2001) that sucrose density centrifugation is an appropriate method to
extract melanosomes from cells. Free flow electrophoresis would be an extremely
promising method to use to separate the RPE melanosomes by their shape as has
been shown by Kushimoto et al. (2001)
with epidermal melanosomes.

Once a suitable method of
separating cylindrical and spherical RPE melanosomes has been identified,
further experiments need to be conducted to find an accurate method of
quantifying their melanin content. Methods could include chemical degradation
analysis used by Liu et al . (2005)
or more recently Del Bino et al.
(2015) investigating human epidermal cells. Or less destructive methods such as
ESR spectroscopy could be used (Sarna et
al., 2003).

If the chemical composition
of either shape of RPE melanosome is shown to be identical, then many questions
still need to be addressed. If the process of melanogenesis is the same in
melanocytes as it is in RPE cells (Jimbow et
al., 1979), then other factors affecting the shape of melanosomes need to
be investigated. If RPE melanosomes do slowly adjust their shape over time as
suggested by Kim & Choi (1998) and observed by Burgoyne et al., (2015), what aspects of the RPE
cells control this? By perfecting a process of isolating melanosomes based on
shape to allow research into their shape and composition, the process could
also be used to facilitate further investigation into melanosomes or other
lysosomal organelles.