SSEF Report 5

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Jurong Junior College, Anglo-Chinese Junior College

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 progressionOh Jun Wei and Jessica Lim Jia Ying1. 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]. Adapted from http://cmdi.medicine.dal.ca/Human_Histology Day 1Day 3Fig 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.

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-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 ResearchThe 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 ResearchThe 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 Methods4.1 Cell culture and cell linesTwo 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 (Dulbeccos modified Eagles medium), 25% Hams F12 medium, 1% L-glutamine, 1% penicillin/streptomycin, 10% fetal bovine serum (FBS), hydrocortisone (0.4g/ml), transferrin (5g/ml), lyothyronine (2 x 10^-11 M), adenine (1.9 x10^-4 M), insulin (5g/ml) and epidermal growth factor (EGF) (10ng/ml). Cells were passaged every 3 days.4.2 Site-directed mutagensisPhosphorylation mutants of K14 were generated using QuikChange Site-directed Mutagenesis kit (Aligent Technologies, CA, USA) according to the manufacturers 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 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, 1l 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. 100l 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 250l of Buffer P1. 250l of Buffer P2 and 350l 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, 750l 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 50l 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 to capillary electrophoresis on the ABI PRISM 3730xl DNA Analyzer. Sequencing data were analyzed with SeqMan, a Lasergene sequence assembly software. 4.3 Transient transfectionKX 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 MicroscopyTransiently 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 l 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 DiscussionThe 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.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 br