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Oh Jun Wei, Jessica Lim Jia YingJurong Junior College, Anglo-Chinese Junior College

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Exploring keratin 14 filament reorganization during cell cycle progression

Oh Jun Wei and Jessica Lim Jia Ying

1. Background and Purpose of Research Area

Intermediate Filaments (IF) form a complex protein network together with actins and tubulins.

Type I and Type II IF comprises of keratins that are acidic and basic respectively [8]. Keratins do

not exist as a single monomer but instead form heterodimers with their associated IF partner, i.e.

K5 and K14, which assemble to form filamentous heteropolymers in epithelial cells [9]. While all

keratin filaments serve to provide mechanical support, distinct keratin pairs have shown to have

specific functions[10-11]. Keratin 14 and 5 has also been linked to human diseases including

Epidermolysis Bullosa Simplex (EBS) [12].

Cytoskeletons are regulated by several post-translational modifications [1]. Phosphorylation is the

main regulatory process of IFs, whereas microtubules and microfilaments are regulated by

associated protein partners [2]. Phosphorylation of IF gives rise to its dynamic nature of being

able to respond to cell stress and mitosis by equilibrating the solubility of the filaments [3]. The

phosphorylation sites on IF can (usually) be found at the head and tail domain of the structure as

they are more accessible to enzymes [4]. Many have reported the importance of phosphorylation

in IF re-organisation, especially under stressful conditions and cell cycle progression. [5].

Reversible actions of various kinases have been documented to induce the breakdown of IF

network to soluble aggregates[6,7}. The purpose of our research is to elucidate novel roles of

phosphorylation of keratin 14 and assess the significance of phosphorylation and de-

Fig 1: The skin comprised of two distinct layers: epidermis and the dermis. Keratinocytes are the skin cells present in the stratified epidermis. Keratin 14 and 5 are predominantly found in the proliferating basal keratinocytes.

Day 3Day 1

Adapted from http://cmdi.medicine.dal.ca/Human_Histology


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phosphorylation of keratin 14 in cell cycle progression. In this study we utilized the method of

site-directed mutagenesis to investigate the effect of a constitutive phosphorylated IF in vitro.

2. Hypothesis of Research

The hypothesis of our research falls upon the phosphorylated states of K14. It is observed in

other IF that phosphorylation into soluble aggregates occurs prior to cell division. Hence, if K14

is in a constant phospho-null state (in Ser>Ala mutant), complete segregation of the cells would

not be likely to occur as the insoluble filament network still encloses the cells. Furthermore,

since dephosphorylation is observed to occur after cell division, it is a hypothesis that if K14 is in

a constant phospho-mimic state (in Ser>Glu mutant), the resulting cells will have abnormal

phenotype, as the filament network is absent to maintain the structural integrity of the cells.

3. Aim of Research

The aim of the investigation is to determine the effects of phosphorylation and

dephosphorylation of the various sites in K14 and its implication on cytokinesis.

4. Materials and Methods

4.1 Cell culture and cell lines

Two K14-null keratinocytes cell lines namely KF5 and KX were used in this study. Due to time

constraints however, the data for KF5 were not tabulated. The cell lines were cultured in

complete RM+ media containing 75% DMEM (Dulbecco’s modified Eagle’s medium), 25%

Ham’s F12 medium, 1% L-glutamine, 1% penicillin/streptomycin, 10% fetal bovine serum

(FBS), hydrocortisone (0.4µg/ml), transferrin (5µg/ml), lyothyronine (2 x 10^-11 M), adenine

(1.9 x10^-4 M), insulin (5µg/ml) and epidermal growth factor (EGF) (10ng/ml). Cells were

passaged every 3 days.

4.2 Site-directed mutagensis

Phosphorylation mutants of K14 were generated using QuikChange Site-directed Mutagenesis

kit (Aligent Technologies, CA, USA) according to the manufacturer’s instructions.

4.2.1. Polymerase Chain Reaction (PCR)

Forward and reverse primers (short oligonucleotides) were designed using keratin 14 (K14)

cDNA sequence as the reference to generate the following mutations: S32E, S33E, S32A, S33A


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and double mutant S32A33A. PCR was performed using 50 ng of template DNA (K14 cDNA

fused with a Green Fluorescent Protein (GFP) in a plasmid vector, pAc-GFP) and 1 uM of

primers. The cycle conditions were: 95 oC for 50 seconds for denaturation, 68 oC for 50 seconds

for annealing, 68 oC for 7 minutes for elongation with 18 cycles. The reaction was then allowed

to go to completion at 68 oC for 10minutes and cooled at 16 oC.

4.2.2. Bacterial transformationAfter completion of PCR, 1µl of Dpn 1 restriction enzyme was added to the reaction tube and

incubated at 37 oC for 3 hours. This enzyme recognizes and cuts the methylated template DNA,

but uncut the unmethylated PCR product. The plasmids were then added individually to 50 ul of

XL-1 Blue supercompetent cells and kept in ice for 1 hour. These cells were then immediate

subjected to heat shock at 37 oC for 42 seconds. 100µl of SOC was added to the shocked bacteria

suspension and left to incubate at 37 oC for 1hour. The bacterial cells were plated onto LB plates

containing ampicillin and kanamycin. Individual clones were picked and inoculated in 3ml of LB

overnight.

4.2.3 Plasmid DNA preparations and sequencingBacteria cultures were transferred into eppendorf tubes and were spun @14800 rpm for 4

minutes. Supernanant was discarded & replaced with 250µl of Buffer P1. 250µl of Buffer P2 and

350µl of Buffer N3 were then added step-wise and mixed. The tube was then left to stand for

5minutes and centrifuged for 12 minutes @14800rpm. The supernatant was then poured into a

QIAprep spin column and centrifuged again for 90seconds @14800rpm. After discarding the

washings, 750µl of Buffer PE was added and the tube was centrifuged again for 90seconds

@14800rpm. The washings were then discarded again and the column was centrifuged again for

4 minutes @14800rpm. The attached filter was then placed onto a new eppendorf tube and the

DNA was eluted with 50µl of Buffer EB. The tube was then subjected to centrifugation for 90

seconds @ 14800rpm. All of the plasmid DNA samples were sequenced with K14 specific

primers using Big Dye sequencing mixture. PCR was performed as follows: 96 oC for 1.5

minutes, 96 oC for 10 seconds, 50 oC for 55 seconds and 60 oC for 2minutes. Step 2-4 were

repeated for 35 cycles and the reactions were then allowed to cool at 16 oC. Samples were set to

the DNA sequencing facility at the Institute of Molecular and Cell Biology for purification and


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to capillary electrophoresis on the ABI PRISM 3730xl DNA Analyzer. Sequencing data were

analyzed with SeqMan, a Lasergene sequence assembly software.

4.3 Transient transfection

KX and KF5 cells were seeded on 12 mm coverslips and were transfected with wild type (WT)

and mutant (S32E, S33E, S32A33A) GFP-tagged K14 constructs using Neon Transfection

system from Invitrogen (set at 2 pulses of 1150 V for 30 ms) and Effectene Transfection system

from Qiagen, alternatively. Transfection was performed according to the manual; the efficiency

was estimated based on the GFP expressing cells.

4.4 Immunocytochemistry and Microscopy

Transiently transfected cells were fixed in 10% PFA for 10 minutes on coverslips. Following, the

cells were washed with PBS twice and kept in sodium azide in the 4 oC refrigerator. Nuclear

staining was carried out with 4',6-diamidino-2-phenylindole (DAPI) 200 µl of DAPI (diluted

1:2000) mix was added to each well in a 24-well flask and subsequently left to sit for 10 minutes

wrapped in an aluminium foil. The wells were then washed 2 times with PBS and the coverslips

with the cells were mounted onto microscope slides. Images were taken using a Photometrics

CCD camera (CoolSNAP HQ2) that was installed on a Z stage (Applied precision, USA)

equipped inverted Deltavision epifluoresence microscope (Applied Precision) together with an

Olympus UApo/340 40x (N.A. 1.35) oil immersion objective lens.

5. Results and Discussion

The transfected cells were fixed at 2 days and 4 days post-transfection respectively, and at least

150 GFP positive cells were used for the statistics for each construct. Unfortunately, due to the

short 5 week period we did not attempt both transfection methods for 2 days and 4 days. The

transfection efficiency of the Neon Transfection System and Qiagen Effectene Transfection

System were compared by calculating the percentage of GFP tagged positive cells as an

indicator. The Qiagen Effectene Transfection System showed a higher number of GFP-positive

cells [Fig 2A], probably due low viability of keratinocytes exposed to electroporation. It is

therefore recommended to use the Qiagen Effectene Transfection System. However, an

exception is observed with the construct S32A. A higher transfection efficiency of 38.2% was

obtained when the Neon Transfection System was used as compared to 17.5% when the

Qiagen Effectene Transfection System was used.


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Microscopic images were screened for abnormal phenotype based on four criteria that were

easily distinguished: i) intermediate filament (IF) bridges ii) multi-nucleated cells iii) keratin

aggregates and iv) abnormal morphology.

IF bridges occurred at a low frequency [Fig 2B]. The plasmids that were transfected into the cells

probably affected the mitotic rate of these cells adversely, leading to a low frequency of IF

bridges. At 4 days, S33A had 3x more IF than WT. There were more IF bridges observed in

S33A and the double mutant, S32-S33A after 4 days as compared to the wild type and the E

constructs. This indicates that IF bridges are more prevalent when K14 cannot be

phosphorylated, supporting the hypothesis that cells are not able to undergo complete cytokinesis

when they are in a constant phospho-null state. There were no IF bridges observed in S32A

which suggests that Ser32 in K14 may not be a significant site of phosphorylation as compared

to S33A. Interestingly, the E constructs also showed IF bridges, which might indicate that more

than one phosphorylation sites are involved in the disassembly of the entire filamentous network.

Multinucleated cells were of a higher frequency after 4 days as the cells underwent more mitotic

cell cycles [Fig 2C]. All constructs had more multinucleated cells as compared to the wild type

after 4 days, implying that both phosphorylation and dephosphorylation of the filament network

are essential in ensuring complete cytokinesis. The multinucleated cells were also observed to be

larger. Together with the low frequency of IF bridges observed in these mutants, a probable

cause for this phenotype could be due a higher tendency of poorly separated daughter cells to

merge after unsuccessful cytokinesis, forming a giant filamentous structure rather than two

separate cells with a connecting IF bridge. This would explain that S33A has a higher frequency

of IF bridges but relatively lower frequency of multinucleated cells compared to the null

frequency of IF bridges but relatively much higher frequency of multinucleated cells for S32A. It

might also be possible that different constructs may have the ability to change the nature of these

keratinocytes which is one limitation that we have not explored yet. Notably, both S32E and

S33E have similar frequencies of IF bridges but S33E have a higher frequency of multinucleated

cells after 4 days. Therefore, Ser33 may be a more significant site of dephosphoryation for

complete mitosis than Ser32.


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It is observed that a greater number of aggregates were found in those Effectene transfected cells

[Fig 2D]. This was probably due to the high copy number of plasmids that were uptake by the

cells. Both S32E and S33E had more aggregates than S32A and S33A, which further supports

that the filament network is phosphorylated into soluble aggregates prior to cell division.

However, contradicting this same hypothesis is the double mutant construct which has the

highest number of aggregates, almost four times the number of aggregates observed in the wild

type. This suggests the possibility of a drastic effect brought about by the double mutated sites or

the possibility that there might be another significant site of phosphorylation triggered in K14

other than Ser32 and Ser33, leading to the high frequency of aggregates and the double mutant

could have activated some stressed pathway to breakdown the filament network into aggregates

to remove the faulty K14. The observation that S32A has more aggregates than the wild type

further supports this hypothesis.

Due to time constraints, transient transfection was only performed which may result in originally

transfected cells losing the effects brought about by the transfected plasmids which would

explain some discrepancies in our observations. The effects of these plasmids were also

unknown, whether they affect the rate or nature of mitosis in these cells remains to be seen. It is

also unclear whether different transfection methods will affect the results other than the

transfection efficiency.

WT S32A S33A S32A-S33A S32E S33E0

204060 GFP Cells

2d Effectene 4d Neon

Perc

enta

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A K14-WT

Figure 2: Transfection efficiency of Neon Transfection System and Qiagen Effectene Transfection System and occurrence of abnormalities in K14 phosphorylated mutants

K14-WTK14-WTK14-WTK14-WTK14-WTK14-WTK14-WTK14-WT


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6. Conclusion

Phosphorylation and dephosphorylation play essential roles in the mitotic cell cycle though the

exact effects are yet to be determined. Our results reinforce the hypothesis that the filament

network of K14 will be phosphorylated to its soluble form – aggregates prior to mitosis and

dephosphorylated near the resulting stage of mitosis. More studies should be done to discover the

potential kinases which play a role in phosphorylation of S32 and S33 sites of K14. Stable

transfection methods should also be used to ensure that most cells retain the effects of the

mutated sites for further observation and recording purposes.


10203040 Aggregates


Perc

enta

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10152025 Multinucleated Cells


Perc

enta

ge/%

WT S32A S33A S32A-S33A S32E S33E01234 IF Bridges


Perc

enta

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B

C

D

K14-S32AK14-S32A-S33A

K14-S33EK14-WT

K14-S32AK14-S32A-S33A

K14-S33EK14-WT

K14-S33AK14-S33E

K14-WT K14-S32A-S33A

Figure 2: Transfection efficiency of Neon Transfection System and Qiagen Effectene Transfection System and occurrence of abnormalities in K14 phosphorylated mutants


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7. Bibliography

[1] Herrmann, H. & Harris, J. (1998). Subcellular biochemistry. New York: Plenum.

[2] Omary, M., Ku, N., Tao, G., Toivola, D. & Liao, J. (2006). ‘Heads and tails’ of intermediate

filament phosphorylation: multiple sites and functional insights. Trends in biochemical sciences,

31 (7), 383--394.

[3] Tsujimura, K., Ogawara, M., Takeuchi, Y., Imajoh-Ohmi, S., Ha, M. & Inagaki, M. (1994).

Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis.. Journal

of Biological Chemistry, 269 (49), 31097--31106.

[4] Omary, M., Ku, N., Tao, G., Toivola, D. & Liao, J. (2006). ‘Heads and tails’ of intermediate

filament phosphorylation: multiple sites and functional insights. Trends in biochemical sciences,

31 (7), 383--394.

[5] Omary, M., Ku, N., Liao, J. & Price, D. (1997). Keratin modifications and solubility

properties in epithelial cells and in vitro.. Sub-cellular biochemistry, 31 105--140.

[6] Miller, R., Khuon, S. & Goldman, R. (1993). Dynamics of keratin assembly: exogenous type

I keratin rapidly associates with type II keratin in vivo. The Journal of cell biology, 122 (1),

123--135.

[7] Ku, N., Liao, J., Chou, C. & Omary, M. (1996). Implications of intermediate filament protein

phosphorylation. Cancer and Metastasis Reviews, 15 (4), 429--444.

[8] Herrmann, H., B\"Ar, H., Kreplak, L., Strelkov, S. & Aebi, U. (2007). Intermediate

filaments: from cell architecture to nanomechanics. Nature Reviews Molecular Cell Biology, 8

(7), 562--573.


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[9] Hatzfeld, M. & Burba, M. (1994). Function of type I and type II keratin head domains: their

role in dimer, tetramer and filament formation. Journal of cell science, 107 (7), 1959--1972.

[10] Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000.

[11] Kreplak, L. & Fudge, D. (2007). Biomechanical properties of intermediate filaments: from

tissues to single filaments and back. Bioessays, 29 (1), 26--35.

[12] Wagner, M., Trost, A., Hintner, H., Bauer, J. & Onder, K. (2013). Imbalance of

intermediate filament component keratin 14 contributes to increased stress signalling in

epidermolysis bullosa simplex. Experimental dermatology, 22 (4), 292—294


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Supplementary Notes

Constructs Name Primers

S33E K14-S33E-F CAGCCGCATCTCCgaaGTCCTGGCCGGAG

K14-S33E-R CTCCGGCCAGGACttcGGAGATGCGGCTG

S32A-S33A 2A K14-S32A-2A-F CTCCAGCCGCATCgCCgccGTCCTGG

K14-S32A-2A-R CCAGGACggcGGcGATGCGGCTGGAG

S32E K14-S32E-F CTCCAGCCGCATCgaaTCCGTCCTGGCC

K14-S32E-R GGCCAGGACGGAttcGATGCGGCTGGAG*S32A and S33A primers were designed before the start of the attachment

Phosphatases

KinasesSoluble K14Insoluble K14

*Not drawn to scale

GFP from jellyfish fused to K14

33SS

32

Keratin 14GFP