Identifying Ciliary Proteins

Identifying Ciliary Proteins in Mammalian Retinas using a Gentle Extraction Method

Adeline Fredrick

Department of Biochemistry, University of Illinois at Chicago

Drew Lab

November 29, 2023

Abstract:

The retina, a complex structure in the

internal layer of the ey

e that

converts light signals

to neural impulse

to the brain. The structure consists of a junction between photoreceptors

called the inner segment (IS)/outer segment (OS) line. This line has highly modified cilia that

develop the primary cilia of the retina and connect the IS to the OS. The primary cil

ia structure is

important because defects in ciliary proteins are associated with diseases such as retinitis

pigmentosa (RP), a hereditary retinal degeneration.

Currently, we lack an understanding of

protein complexes involved in forming and maintaining pr

imary cilia of the retina. We lack

optimized protocols to obtain samples with primary cilia from retinal photoreceptors to use in

high

throughput proteomic studies (

Wheway et al., 2014)

Here, bovine eyes were used to

conduct the research on ciliary proteins in the retina due to their availability and size which

allows a high yield of proteins harvested per retina. The eyes were dissected in a darkroom to

avoid light stimulation, a Mammali

an ringer solution was used to gently release the retina from

the bac

k of the eye and optic nerve (Panfoli et al., 2022). A lysis buffer was used with a vortex to

break the retina cells. The lysed cells were centrifuged leaving a supernatant. Once the proteins

were retrieved, detergent compatible protein assay was used foll

owed by mass spectrometry to

identify proteins present. Researching the retina allows an in depth look at the structure and

function of a highly modified and specialized primary cilia. Future plans to extend this work

include a co

fractionation mass spectr

ometry pipeline to identify protein complexes in retinal

photoreceptors; this will allow us to map the molecular network of disease relevant cells.

Introduction:

The most important organ of our senses are the eyes. They are responsible for 80% of all

impressions by means of our sight (Why

ood

ision is so

mportant, 2021). Our eyes are our

windows into the world however, according to O’Neal & Luther “1 in 4,000 Americans suffer

from retinitis pigmentosa (RP) and there are no cures” (O’Neal & Luther, 2022). RP is a

hereditary disease that is caused by defects in the primary cilia. Within the

human body there are

two types of cilia, motile and non

motile. Motile cilia are projections on the outside of cells that

help the cell move or help move debris through the body by using a whip

like motion. The

structure of motile cilia comprises of dynein

ATPase motor microtubules that cause the cilia to

move. Non

motile cilia, on the other hand, lack this motor and have a different structure

altogether (

Ishikawa, 2017

). Non

motile cilia, also known as primary cilia, consist of a unique

structure of an axoneme and a basal body. Primary cilia are concentrated in the retina.

The retina

is a thin piece of tissue lining the back of the eye. It is responsible for conducting visuals from

the eye to the brain. With any damage to the retina most vision would be los

t. Primary cilia are

an important part of the retina. These cilia are part of the outer and inner segments of the retina

(Figure 1)

. The outer segment role is to absorb light and transduce the signal into a nerve signal.

The inner segment responsibility is to regulate these nerve signals and keep the outer segment

attached to the retina. (

Baker & Kerov, 2013

). With damage to the outer/inner segment the

information that your eyes intake would never reach your brain and therefore, you would n

ot be

able to

und

erstand

what you were looking at.

My research is to study the primary cilia and the proteins that are responsible for keeping

these cilia viable. To

understand the proteins involved we need

to construct a gentle extraction

method

that can

keep the cilia intact to attempt to keep our sample as densely rich with primary

cilia and proteins.

Currently

there is a lack of understanding of protein complexes involved in

forming and maintaining primary cilia of the retina as well as protocols to obtain samples with

primary cilia from retinal photoreceptors to use in high

throughput proteomic studies. Our go

al is

to determine which protein complexes in primary cilia are present and to characterize them in

disease

relevant tissues by

creating

a new gentle extraction protocol.

Methods:

The bovine eyes were obtained from Innovative Research’s website. The eyes were US

Origin Bovine eye dark adapted retina (

Innovative Grade US Origin Bovine Eye Dark Adapted

Retina

, 2023).

Thirty

ovine eyes were delivered to the lab and the dissection began 40 minutes after

delivery

, however, the sooner the eyes are dissected after delivery the better

. We turned off the

overhead lights and closed blinds to all windows in the room to reduce light exposure to the

retinas. The lens is cut off, and the vitreous humor is discarded to expose the back of the eye and

the retina. Mammalian Ringer solution is ad

ded to a basin with the half

cut backside of the eye.

The eyes were covered with foil and gently shaken with an orbital shaker for 20 minutes at 90

speed with no sign of retinas detaching. We increased the speed to 125 for 30 minutes. The basin

was taped d

own to avoid spillage. After incubation, we used tweezers to gently release the thin

retinal tissue from the back of the eye.

(

Panfoli et al., 2022

Once the retina is released, the optic nerve is cut, and 15 retinas are placed in a 50 mL

centrifuge grade tube and the other 15 retinas are placed in another 50mL centrifuge grade tube.

To the tubes, 500uL of Buffer A (

10 m

M

Pipes, pH 7.0, 5 m

M

MgCl2, and 1× proteinase

inhibitor mixture

) with 50% (w/v) sucrose is added. Both tubes were vortexed to dislodge the

cilia. The tubes were centrifuged for 20 minutes at 13,000 g, 4C.

Once the 20 minutes were finished, we gently removed the tubes to not disturbed the

pellet at the bottom of the tubes. The supernatant was a dark red color and was removed in thirds

and put in a new test tube. We diluted the sample with a 1:1 ratio of Buffer A without sucrose.

We g

ently pipette

up and down to mix the sample.

A sucrose cushion is gently added to the bottom of the tube with a blunt edge needle.

There should be a clear phase separation. Before placing the two samples in the centrifuge again,

weigh the tubes and carefully add Buffer A to even out the weights

. Our tubes weighed

approximately

400 uL each.

We c

entrifuge

both tubes again for 20 minutes at 13,000 g, 4C.

Carefully remove tubes from the centrifuge to not disrupt the phase separation. The cilia

are in the interphase separation zone and are recovered using a micropipette. We added the two

interphases into one tube (about 7mL total). Dilute sample into a 1:1 r

atio with Buffer A (no

sucrose) and centrifuge for 1 hour, 13,000g, 4C.

After centrifugation, there is a pellet at the

bottom of the tube which is incubated in Buffer B (

10 m

M

Pipes, pH 7.0, 5 m

M

MgCl2, 1%

Triton X

100, 1 m

M

DTT, and 1× proteinase inhibitor mixture

). The sample is centrifuged again,

and the supernatant is filtered and saved (Lui et at., 2007). The supernatant contains proteins that

are found in primary cilia.

sample was injected into a machine called High

performance Liquid

Chromatography (HPLC). HPLC separates the components of the sample down its column and

divides them into fractions from large to small (

Wan et al., 2015)

. Once the HPLC identifies the

sample constituent and collects the sample data, we estimate

the size of proteins and compare

it to previous protein standards. The full protocol is found in

Appendix A.

Results:

The goal of this research was to find a gentle extraction

method

and identify

protein

complexes within primary cilia. Our research started with dissection then led to HPLC and Mass

Spec runs (Figure 2). Our research was run twice, once in January 2023 and once in May 2023.

During our first run, we did not run a successful HPCL sample, however, we sent two replicate

samples to Mass

Spec and received a list of ~1,200 proteins within both samples. To analyze this

data, we compared our data with the Syscilia database. Syscilia’s

database records all cilia within

the human proteasome. After merging our data with the syscilia database we found 23 genes that

our bovine sample had

in common with humans

With the data from the two replicates and the syscilia database we created a bar graph to

indicate which genes were

similar

and which genes were the most abundant (Figure 4). The most

common gene DPYSL2 with an abundance of ~80 psm is associated with neuronal development

and axon growth. Within the 23 genes found,

three

of them were directly linked to human

ciliopathies. Genes CNGB1 and STX3 are associated with

etinitis

igmentosa which is a disease

that affects how the retina responds to light. Both genes

had an abundance of roughly 13 psm.

CNGB1 is a coactivator for transcription, cell adhesion, and neurogenesis. Gene STX3 is

important for docking synaptic vesicles and for membrane fusion. The other gene linked to

human ciliopathies is LZTFL1. This gene c

orresponds with Bardet

Biedl Syndrome (BBS). BBS

is a ciliopathy that affects rod and cone development in the retina, renal formation, mental

development, and obesity (

Elawad et al., 2022

). LZTFL1 has an abundance of ~5 psm.

Using the Go Enrichment database, we looked at the top 40 most abundant genes in our Mass

Spec data and found that they were enriched with non

motile cilium, cilium

structure, and

photoreceptor annotations. As a result, we know that our 40 most abundant genes are associated

with the structure of primary cilia or photoreceptors in the retina.

Using the bovine data identified by mass

spec,

I looked into a paper that conducted a

similar experiment with mouse eyes

authored by Liu and colleagues

. Looking at their raw data I

formulated a graph of their mouse retina protein data to our bovine retina protein data and

created a scatter plot (Figure 3).

The scatter plot places the abundance of bovine proteins from

our raw Mass

Spec data on the y axis and the abundance of L

and colleagues’ paper on the x

axis. I made the graph coordinated as logs

because

the raw plot was congested within the 1

psm regions. The Spearman correlation was 0.394 showing that our data is correlated with Liu’s

however

it is not as correlated as I predicted. This could be due to

the use of

different

species

differences in sample preparatio

or the use of

different

mass spec

machines which hinders the

chances of having perfect correlation. When investigating the highest abundant proteins in both

datasets I found that there are eight proteins that are similar in abundance with both Lui et al.

data and our bovine data. The prote

in with the highest abundance of both species is a spectrum

beta chain known as Fodrin which plays a role in the central nervous system and cell secretion.

This confirms that our gentle extraction method produced protei

ns found in both bovine and

mouse specimens.

During our first run we had issues with our HPLC plot. Our data was not consistent with

our control group. However, during our second run, our data formed distinct peaks that

correlated with proteins. For our HPLC run we tested two replicate samples split

into 51

fractions that we combined into

nine

larger fractions. We found the highest peak to be between

45 fractions or within the fifth fraction out of nine with a normalized value of over 1. The

second highest peak in our HPLC data was within the secon

d fraction where we observed a

normalized value of 0.4. The rest of the nine larger fractions showed normalized values around

0.25.

With this information, we decided to perform a protein assay to test how much protein

actually obtained in the nine fractions. We used BIORAD DC Assay which is a detergent

compatible protein assay that is colorimetric to protein concentration. It is meant

to mimic the

Lowry assay with a faster incubation period. Figure 6 indicates the concentration of protein in

each of the 9 fractions. Fraction three contained the highest protein concentration of around

14.35ng. Our HPLC data did not correlate with this data as our hig

hest peak of protein

absorbance

was in fraction 3 but our highest HPLC peak was in fraction 5

. We calculated our

total protein concentration and concluded that we did not have enough protein to accurately run a

Mass

Spec session. We found that we had betwe

en 4.12ng and 14.35 ng of protein concentration

in our second run. We needed each fraction in protein assay to be 5ug

therefore, we decided to

not send our second experimental run through a mass

spec analysis.

Discussion:

In this

study

we wanted to find a gentle extraction method to detach primary cilia from

the

retina due

the gap in the proteomics field of

understanding protein complexes involved in

forming and maintaining primary cilia of the retina

and

because

lack optimized protocols to

obtain samples with primary cilia from retinal photoreceptors to use in high

throughput

proteomic studies. To shrink this gap,

we created a new protocol based on Panfoli and

colleagues’

protocol.

We used buffers, centrifugation,

ass

spec, and HPLC to

obtain a high yield of proteins

and

dentify them. During run 2 we took a small sample of

aw bovine retina to view the primary

cilia’s original form

under a microscope

, however, we could not find the structure. This could be

because we did not use enough retina to be able to view the primary cilia or

that

the retina we

used did not have the number of primary cilia we expected it to have. This could also be the

reason why we did not have enough protein

concentration in run 2 to use the mass

spec machine.

This

lack of proteins

was not what we were expecting because in run 2 we tripled the number of

bovine eyes from 10

used in

the first run to 30 in the second run

yet

we still had extremely low

protein concentration. In run 1 we pooled all fractions together to be able to obtain 5 microliters

of protein concentration to run the mass

spec machine and identify protein within the sample

(Figure 4). Based on the results, we found 23 bovine proteins a

ssociated with human proteins

however, since we pooled all purified fractions together, we did not have data on individual

protein abundance in each fraction. In run

, due to the lack of protein concentration in the 9

pooled fractions, we focused on getti

ng an accurate HPLC sample. We pooled fractions 12

into 9 fractions. Fraction 1 pooled fractions 12

18, fraction 2 pooled 19

25, 26

33 fraction were

pooled for fraction 3, fraction 4 included 34

40, fraction 5 was 41

45, fraction 6 had 46

50,

fraction 7

included 51

55, fraction 8 pooled 56

59, and fraction 9 included 60

63. We observed

that pooled fraction 5 containing fractions 41

45 had the tallest peak in both runs. This indicates

that there were high protein levels there (Figure 5).

We compared our data to previous work from Li

and colleagues who studied mouse

retinas. We took the ~1200 proteins we identified in our mass

spec data and L

’s data to create

a scatter plot based on abundance. The plot exhibited many proteins in both data sets with low

abundance being

similar

. However, the most abundant protein in both mouse and bovine is a

spectrum beta chain also known as

odrin. Fodrin plays a role in the development of the central

nervous system and cell secretion. Fodrin being the mo

st abundant in both species is

understandable because of the large rol

retinas hav

in the central nervous system. The most

abundant protein in the mouse sample is a cytoplasmic dynein heavy chain 1 which is an ATPase

and a motor protein for vesicles and organelles along microtubules. One of primary

cilia’s main

functions in the retina is to convert light to images and to this many vesicles are used. Having an

abundance of this protein in the mouse sample is consistent with the cilia’s function. The most

bundant bovine protein is a pyruvate kinase which regulates cell metabolism

(Israelsen &

Vander Heiden, 2015). Comparing bovine proteins to mouse proteins, the protein with the

highest abundance in bovine and lowest abundance in the mouse sample is a brain acid soluble

protein I

functions

as an interference of oncogenic capacity of MYC

. MYC

is a disease

protein coder (Hartl et al., 2020). On the other hand, the highest protein abundance in mice and

lowest in the bovine sample is a rootletin

which

is a major component of ciliary rootlets that

aids

ciliogenesis. It is unclear as to why th

e bovine sample ha

such a low abundance of an

important ciliary protein. It may be due to how we prepared our sample for the mass

spec run.

The lack of rootlin protein

could also be due to the way we harvested the retina. The rootlin is

connected to the inner segment of the primary cilia. When we dissected the eye to obtain the

retina,

we may have left the inner segment still attached to the back of the eye. Another probable

cause could be our detergents are too strong and denatured this specific protein.

We identified

more unique proteins with 1,653 total proteins greater than two peptide spectral matches than the

Liu data set with

only

1,059 total proteins greater than two spectral matches (Liu et al., 2007).

Interestingly, the three proteins found in Figure 4

, CNGB1, STX3, and LZTFL1,

are

correlated with human ciliopathy diseases are not associated with the bovine proteins that are in

relation with Liu and colleagues mouse data.

In future experiments, we will discuss why our protein concentration has been so low

regardless of the

number of eyes used. We will look into the company that supplied the eyes and

perhaps try to reach out to a

local

farm to obtain fresh cow eyes as suggested in the Panfoli et al

paper. The fresh eyes may decrease the deformation of primary cilia and proteins as well. We

may also need to increase our cow eye sample from 30 to 50

100 eyes.

However, t

his increase

causes an issue of needing to quickly and eff

iciently dissection the eyes to avoid degradation of

proteins. Another change we could make in the methods is to decrease the use of buffers to

obtain a more concentrated sample. This may increase the protein concentration as well.

In sum, this research is important to understand the proteomics of primary cilia

, to

procure

a gentle extraction method to understand what constitutes these cilia

and to be able to

research diseases associated with proteins within the cilia. With this information, we hope to aid

disease

research in

areas such as

retinitis pigmentosa and

ardet

iedl syndrome that deal with

primary cilia structure and proteins.

References

Baker, S. A., & Kerov, V. (2013). Photoreceptor inner and outer segments.

Current topics in

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, 231

–

265.

https://doi.org/10.1016/B978

417027

8.00007

Elawad, O. A. M. A., Dafallah, M. A., Ahmed, M. M. M., Albashir, A. A. D., Abdalla, S. M. A.,

Yousif, H. H. M., Daw Elbait

, A. A. E., Mohammed, M. E., Ali, H. I. H., Ahmed, M. M.

M., Mohammed, N. F. N., Osman, F. H. M., Mohammed, M. A. Y., & Abu Shama, E. A.

E. (2022). Bardet

Biedl syndrome: a case series.

Journal of medical case reports

(1),

169.

https://doi.org/10.1186/s13256

022

03396

Hartl, M., Puglisi, K., Nist, A., Raffeiner, P., & Bister, K. (2020). The brain acid‐soluble protein

1 (BASP1) interferes with the oncogenic capacity of MYC and its binding to calmodulin.

Molecular Oncology

(3), 625

–

644. https://doi.org/10.1002/1878

0261.12636

Innovative Grade US Origin Bovine Eye Dark Adapted Retina

. (2023). Innovative Research.

https://www.innov

research.com/products/id691

innovative

grade

origin

bovine

eye

dark

adapted

retina?variant=31766895296592

Ishikawa T. (2017). Axoneme Structure from Motile Cilia.

Cold Spring Harbor perspectives in

biology

(1), a028076.

https://doi.org/10.1101/cshperspect.a028076

Israelsen, W. J., & Vander Heiden, M. G. (2015). Pyruvate kinase: Function, regulation and role

in cancer.

Seminars in Cell & Developmental Biology

(43), 43

–

51.

https://doi.org/10.1016/j.semcdb.2015.08.004

Liu, Q., Tan, G., Levenkova, N., Li, T., Pugh, E. N., Jr, Rux, J. J., Speicher, D. W., & Pierce, E.

A. (2007). The proteome of the mouse photoreceptor sensory cilium complex.

Molecular

& cellular proteomics : MCP

(8), 1299

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1317.

https://doi.org/10.1074/mcp.M700054

MCP200

Panfoli,I etal.(2022).Maximizing the Rod Outer Segment Yield in Retinas Extracted from Cattle

Eyes.Bio

protocol12(14):e4474.DOI:10.21769/BioProtoc.4474.

O'Neal TB, Luther EE. Retinitis Pigmentosa. (2022). Retinitis Pigmentosa.

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Medicine

https://www.ncbi.nlm.nih.gov/books/NBK519518/

Wan, C., Borgeson, B., Phanse, S.

et al.

(2015). Panorama of ancient metazoan macromolecular

complexes.

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344 (2015).

https://doi.org/10.1038/nature14877

Wheway, G., Parry, D. A., & Johnson, C. A. (2014). The role of primary cilia in the development

and disease of the retina.

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(1), 69

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https://doi.org/10.4161/org.26710

Why good vision is so important

. ZEISS Group. (2021, October). https://www.zeiss.com/vision

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vision

important.html#:~:text=By%20far%20the%20most%20important,best%20protect%20us

%20from%20danger.

Figure 1:

A diagram of the structure of a primary cilium

from Lui et al.

The outer segment

(OS)

is where

the primary cilia is located

. The OS initiates visual signal

transductions.

The G

protein coupled receptor Rhodopsin is arrayed in disks in

the outer

segment of the rod cells. Light activates rhodopsin thereby hyperpolarizing the rod cell and

sending a signal to downstream on

center bipolar cells.

The inner segment (IS) contains the

mitochondria and is where metabolism and regulation of membrane potential take place. The

basal

body

is the speciGic name for the centrosome in the primary cilia. The axoneme consists of

nine doublet microtubules. It lacks the central pair of microtubules, radial spokes, and out/inner

dynein that are seen in motile

cilia. These

differences in

the structure make the primary cilia

non

motile.

Figure 2:

Infographic of Methods

schematic of the methods used to conduct this experiment. The bovine eye was dissected

using tools such as scalpels and forceps.

the ROS/Primary cilia are separated from the retinal

tissue using centrifugation techniques.

the puriCied samples are ran in the HPLC to

fractionate

the sample to identify the protein interactions

the proteins in the centrifuged samples are

identiCied using a mass

spec machine.

Cre

ated by

BioRender

Figure 3:

Scatter plot of bovine data vs. Lui et. al mouse data

A depiction of our bovine protein abundance data compared to a study by Liu et al. who

used a

similar puri=ication process with mouse retinas. Both samples contained similar

proteins at a low abundance and few proteins

correspond

with a high abundance in each

sample.

Mouse Abundance

(psm)

Bovine Abundance

(psm)

Spearman correlation=0.394

Gene Name

Abundance (psm)

Rep. 1

Rep. 2

Associated with

Retinitis Pigmentosa

Associated with

Bardet

Biedl Syndrome

Figure 4:

Mass

Spectrometry Data

rom our MS analysis we identi6ied ~1200 bovine proteins; using GO enrichment

analysis the 40 most abundant were enriched with non

motile cilium, cilium

structure, and photoreceptors annotations. We cross referenced the proteins in our

sample with the Sysci

lia database gold standard and found 23 proteins associated

with cilia in our sample. The graph shows the abundance of 23 ciliary proteins

within replicate samples.

The genes CNGB1 and STX3 are associated with the

hereditary disease called Retinitis pigmen

tosa that causes the retina to degenerate.

The gene LZTFL1 is associated with Bardet

Biedl Syndrome with affects how cilia

are structured. This disease causes blindness, obesity, and low mental capacity.

Figure 5

HPLC

Data

In the second run of the experiment, we used HPLC to identify the fractions

with the highest protein concentration.

Two replicated samples were in the

HPLC to determine if there were differences in the sample.

The sample

contained 12

63 wells that were pooled into nine fractions as seen above. The

fraction with the highest peak and protein concertation was number Iive with

wells 41

45 pooled

together.

Figure 6:

Protein Assay Data

In the second run of the experiment protein assay was used to indicate that pooled

fraction with the highest protein concentration. Based off of this data fraction three

comprised of wells 26

33 contained the highest protein concentration. However, this

action did not depict the highest peak in Figure 5.

The protein concentration in each

fraction should have been within the range of ~5ug. This sample contain only 4

14 ng

of protein. This run was not analyzed by mass spec due to the lack of protein

concent

ration in each well.

Appendix

This procedure will be performed in 3 parts on the same day. Part 1 is retina dissection/extraction. Part 2 is

ciliary complex purification. Part 3 is the HPLC protocol, this is detailed in a separate document. Preparation of

buffers/reagents, tools, and w

orkspace should be done the day before.

*Eye specimen will arrive at approximately 10AM

Buffers

Mammalian Ringer (MR) 1L

Mix the following into 500mL MQ water, add solution to 1000mL graduated cylinder and bring to volume. pH

to 6.9

●

157 mL 1M NaCl

●

.5 mL 1M KCl

●

3.5 mL 200mM Na2HPO4

●

4 mL 200mM NaH2PO4

●

.05 mL 1M MgCl2

●

.05mL 1M CaCl2

●

2mM glucose (0.36g)

Aliquot enough MR solution for experiment.

●

add protease inhibitor (cOmplete

™

, Mini, EDTA

free Protease Inhibitor Cocktail) 1 tablet per

10mL buffer

Filter sterilize solution.

Buffer A 1L

●

10mM Pipes

●

5mM MgCl2

●

Protease inhibitor

●

pH 7

Buffer B 1L

●

10 mM Pipes,

●

pH 7.0,

●

5 mm MgCl2,

●

1% NP

40,

●

1× protease inhibitor mixture

Part 1 Separating the Eye Semi

cup from the lens and vitreous humor

based on

Panfoli, I. et al. (2022). Maximizing the Rod Outer Segment Yield in Retinas Extracted from Cattle

Eyes. Bioprotocol 12(14): e4474. DOI: 10.21769/BioProtoc.4474.

All steps for Part 1 must be carried out at room temperature in a dark room.

Be sure to don proper PPE before beginning, lab coat, gloves, cut resistant gloves, and eye protection. Cut

resistant gloves go UNDER nitrile gloves.

Starting with whole bovine eyes, remove excess tissue with dissecting scissors. A lot of tissue

and muscle will be present.

This step should be done ASAP upon receipt of bovine eye specimen

Be sure to work slowly, so as not to squirt vitreous humor (eye goop) outside the dissection tray.

Puncture the eye just below the lens with a scalpel. Then use scissors to cut a larger hole in the

eye below the lens. Used forceps to hold the eye and sciss

ors to cut. The retina is a blueish white

opalescent layer. This will leave you with the lens and the "semi

cup" containing the retina.

refer to the image below taken from the bioprotocol

This step should be done in a nalgene tub or dissection tray lined with aluminum

foil for easier cleanup

Place the eye semi

cups in a secondary container for retina detachment with MR

Fill the secondary container from step 3 with enough MR to completely cover the eye semi cups.

Incubate the eyes w/ MR for 10

15 min, cover tray/tub with aluminum foil. Gently shake the

container every couple of minutes, or place on an orbital shaker at low speed if available. You

will know when it's done as the retina can be seen freely floating in

side the eye "semi

cup", it

will only be attached by the optic nerve.

Once the retina is detached ( it will just be hanging on by the optic nerve) quickly invert the eye

"semi

cup" , dispose of the MR and use scissors to cut the optic nerve, fully releasing the retina.

Invert the eye “semi

cup” over a waster container to catch the unwanted MR

solution

Once inverted, move the specimen over the 50mL conical tube for collection and

cut the optic nerve

All retinas should be placed in 50mL centrifuge tubes. Split the number of retinas between two

tubes for centrifuge balancing

Part 2 Isolation of Cilia

based on

Liu et al. 2007

https://pubmed.ncbi.nlm.nih.gov/17494944/

Precool the Sorvall centrifuge. Put a sticker on the centrifuge that you need for this machine.

Turn on machine: login 4109, pass 1234. If it doesn’t work, login and pass are on a fridge in our

lab. Put an empty rotor in a machine, tighten the screws and s

elect it via the ‘Rotor’ button. You

need 4°C, so select the temperature and spin the empty rotor for 15mins, at low rpm (1.5

2k).

Starting with (2) 50mL conical tubes containing the retinas, add 500uL of Buffer A (w/ 50%

(w/v) sucrose) per retina.

Vigorously vortex the tubes from step 1, to dislodge cilia.

Centrifuge the tubes from step 2 for 20 minutes, 13,000 g, 4C

Use the Sorvall centrifuge, the rotors are in the cold room

Gently remove the tubes from the centrifuge so as not to disturb the pellet

Place the 50 mL tube in a single tube rack

There will appear to be multiple layers in the supernatant, but treat all layers the

same.

Remove the supernatant in thirds starting from the top, and put in a new tube

Dilute the collected sample from step 5 1:1 in Buffer A (without sucrose)

Mix by gently pipetting up and down

In each tube from step 6 add an equal volume of 50% sucrose solution(50%w/v sucrose in buffer

A).

This is done carefully with a long blunt edge needle and syringe. The syringe tip

is placed near the bottom of the tube and the plunger is depressed to slowly fill

the bottom with the sucrose cushion. There should be clear phase separation ( a

line at the

interface of the sample and sucrose cushion)

Weigh to ensure centrifuge will be balanced. Add extra buffer A on top (very

carefully and slowly) to bring to correct mass

Centrifuge Sample from Step 7 for 20 minutes, 13,000 g, 4C.

Carefully remove the tubes from the centrifuge so as not to disturb the phase separation. Our

cilia will be at the interphase zone (around the phase separation line mentioned above)

10.

Use a micropipette to remove the interphase zone resulting from step 9, and place in new tube.

Use smallest tubes possible, see note in step 12 for reason.