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The Research on Mitosis and Cell Reconstruction

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Mitosis is the cell division which results in the two-daughter cell that have the same number of the chromosome as the parents have. I have used three research paper for my review paper they are multivalent binding of PWWP2AtoH2A.Z regulates mitosis and neural crest differentiation, VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis And Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis. All these papers are interrelated to each other.

For the first one Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis, Hela cell which is the oldest and commonly used immortal cell which is human cell line. In this research article Hela cell is under 3D context in all cell cycle. The change in the 3D ultrastructure of Hela cell were based on the functional aspect.

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The methods and material used in this research are cell culture where Hela Kyoto and mouse C2C12 myoblast cell were cultured in Thermo Fisher Scientific. The cell was grown at 37 °C. LM of HeLa cells and mouse C2C12 myoblast cells were stained by the DAPI.. ( Manja Luckner, 2018)

The chance of loosing cell could be during cell handling. EM preparation was done after the fix gum and coverslip was removed and the sample was washed three times with dd water. Then thin embedding and ultra-thin embedding was done respectively. The ultra-thin embedding results in milling and direct excess to the target cell. The conductive coating was conducted in the platinum. Then the high-resolution SEM which is used to show the potential for CLEM. The Hela and the mouse C2C12 myoblast were watched in an Auriga 40 SEM. ( Manja Luckner, 2018)

The 3D reconstruction was done were the dataset were aligned and image stacks from CLSM. The result obtained were Locating target cells in routine. The position of the LM was figured out by the EM through SEM. The one way for re localization of selected ROIs is by milling of FIB or SEM of a target cell. The precise alignment is due to the cross section of the glass slide. The DNA methyltransferase is used for the CLEM of Dnmt in mouse C2C12 myoblast cell. There are ultrastructural changes during mitosis. The varying of the interphase shape that includes nuclear tunnels, nuclear pores. The first visible part is outer nuclear of the ring complex. Prophase is the in position where nuclear envelope opens on both centrosomes. The anaphase of the chromosome, a disc shaped mass with rim region which shows both spindle fiber and cell interior. The formation of midzone and midbody with FEM, Kinetochores is recognized by tripartite appearance with length 40. The golgi ribbon is not a ribbon, golgi network is cup shaped that can form 3D. Dictyosomes forms the cluster which can be randomly distributed in interphase cell whereas the bundle of the actin is present in all stage of the mitosis. There is the network of the sheets and tubules which are formed in the ER. The interlaces bulk of the cell is in the interphase of the nucleus.

The nuclear envelope perforates in the region while facing the centrosomes. During the mitosis, chromosome separate from cytoplasmic constituents.. (Manja Luckner, 2018)

In anaphase, the maximum compaction of the chromatin is reached. There is the uneven chromosome at the center of the chromosome. In anaphase, the sensitive dye is used for LM that provide LM level. The pulling force of the MT-movement is the strands withdrawn from the chromosomes. Duplication of a single cell element is important before cell division.

Saturated chamber of Acetone can be used for the resin layer to be thick. High precision can be used for the milling plane. Ramp are made needless with the direct access to target cell. The topography of the target cell is visible all the time.

2nd For the cell division, chromosomal remodeling is important which requires histone modification due to the sequential change in chromatin during mitosis. Chromatin kinases like cyclin dependent, polo-like and Aurora kinases that control cell cycle that involves multiple sequential.

The methods and material used in this research are cell lines where medium was grown in the medium. HEK293T and HeLa cell were used. The U2OS is used for cell cycle which were grown. Cell cycle analysis is done by ethanol, phosphate buffered, and centrifugation was done and stained with the solution of the propidium iodide, RNAase and PBS. Plasmid was done by Murine VRK1 plasmid. The cell transfections is used for the expression of the protein. The VRKI is suppressed by siRNA. The cell was transfected by the reagent Lipofectamine and then specific experiment was performed. The histone phosphorylation defect is determined by the murine. The transfected agent used for U2OS is siVRK1-02. The cell is cultured and scraped and centrifuged. Then again cell was resuspended with trizol and was kept for incubation for about 5 minutes. The chloroform is used as the homogenous mixture for the cell. The tube is centrifuged and RNA phase was separated to new Eppendorf and 2-propanol again the mixture was centrifuged and the floating matter was discarded. Then ethanol is added and centrifuged and pellet was resuspended in RNasefree water. Then they purified the RNA by lysis buffer. The primers used for qRT-PCR were: sur-vivin (forward: 5′-AGG ACC ACC GCA TCT CTA CAT-3′, and reverse: 5′-AAG TCT GGC TCG TTC TCA GTG-3′) and GAPDH (forward: 5′-GGT CTT ACT CCT TGG AGG CCATGT G-3′ and reverse: 5′-ACC TAA CTA CAT GGT TTA CATGTT -3′) as an internal control. The reactions was per-formed with the kit iScript™ One-Step RT-PCR Kit With SYBR®Green (Bio-Rad) in a total volume of 25 μL in a iCycler thermocycler (Bio-Rad). Data were analyzed using the Bio-Rad iQ5 software (Bio-Rad).( David S. Moura,2018)

The various antibodies are used some are AURKB, cyclin DI, Flag-tag, GST-Tag, HA-Tag, histone H3, Myc-Tag, V5-Tag, VRK1. The resin glutathione is used for the expression and purification of glutathione S-transferase. The expression and purification of the protein was performed using the E. coli Bl21DE3 strain that was trans-formed with a pGEX–4T–GST plasmid containing the pro-tein to express (pGEX4T–VRK1).( David S. Moura,2018)The protein were extracts from the Suave lysis buffer which is with the phosphates inhibitors and proteases inhibitors. The acid extraction was done with histone by HeLa cell, which was washed, lyses, centrifuged and trichloroacetic acid was obtained. The protein was separated by the SDS-PAGE by their size, denaturing condition. Ip was performed by 0.5 to 0.2 mg of the 1ml of total The protein interactions from this publication have been submitted to the IMEx (http ://www.imex cons orti consortium through IntAct [73] and assigned the identifier IM-25676. (David S. Moura,2018)

The result obtained from the experiment was VRK1 can form a protein complex with AURKB which are variable that depends on the cell cycle phase. VRK1 and AURKB localization and interaction in cell cycle progression where VRK1 is always found in the all the phase of the mitosis cell division. The phosphorylation is affected by the complex between VRK1 and AURKB. H3 residue was found in the kinase. VRK1 and AURKB interaction inhibits phosphorylation of histone H3 in vivo that is required by chromosome condensation. There is the depletion of VRK1 alters the centromeric localization of AURKB in nocodazole treated cells. The phosphorylation of histone H3 in Thr3 is by VRK1 that is required for centromere formation. In mitosis-arrested cells, the loss of VRK1 resulted in a redistribution of AURKB, instead of having the granular aspect of centromeres in prophase. Depletion of VRK1 resulted in a diffuse redistribution of AURKB on chromatin. (David S. Moura,2018) The VRK1 helps to protect protein from degradation.

The histone modification is the way for the cell cycle that can determine the proteins. There is the change in the chromatin reorganization.

The third article is Multivalent binding of PWWP2AtoH2A.Z regulates mitosis and neural crest differentiation. The various biological series is observed such as transcriptional regulation, cell cycle control, DNA repair.

The methods and the materials used in this research are cell culture and transfection where HeLa Kyoto cell which was grown in the DMEM medium. The following primary antibodies were used in this study:a-PWWP2A (Novus (Acris), NBP2-13833),a-H2A.Z (Abcam, ab4174),a-H2A (Abcam, ab13923),a-H3 (Abcam, ab1791),a-H3K4me3(Diagenode, C15410003),a-H3K9me3 (Diagenode, C15410056),a-H3K36me3 (Active Motif, 61101),a-H3S10ph (Active Motif, 39253and 39636),a-GST (clone 6G9; gift from E. Kremmer, HelmholtzMunich),a-GFP (Roche, 11814460001),a-CENP-E (Active Motif,39620),a-ZNHIT1 (Sigma, HPA019043),a-alpha-Tubulin (ActiveMotif, 39527),a-Flag (Sigma, F1804). (Sebastian Pünzeler,2017)

The cloning of the RNA was isolated. Also the mononucleuosome for IP were isolated and solubilized with the buffer. The data analysis is done by Quantitative mass spectrometry,nCIP-seq,RNA-seq and data analysis Recombinant expression and purification of GST proteins and in vitro binding studies, Fluorescence recovery after photobleaching and live cell imaging, Electrophoretic mobility shift assay, Modeling of PWWP domain structure, Xenopus experiments The cell cycle and the proliferation analysis is done by the following double-stranded siRNAs were used: Luciferase, 50-CUUACGCUGAGUACUUCGA-30; ctrlII, 50-UAAGGCUAUGAAGAGAUACTT-30; PWWP2A#1, 50-GGACAGAAGUCAAGUGUGAUUTT-30; PWWP2A#2, 50-GCUAUUAAACUACGACCCAUUTT-30; cells were transfected with siRNA using oligofectamine (Invitrogen) according to the manufacturer’s instructions. (Sebastian Pünzeler,2017)

They identified that PWWP2A as novel H2A.Z-nucleosome interactor, the experiment in the H2A.Z and HeLa Kyoto showed that the protein binding and how to determine cell type. Multivalent binding mode of PWWP2A enables H2A.Z-nucleosomebinding specificity as well as strong chromatin interaction where it pulled down the tagged and untagged H2A.Z with nucleosomes. The pull down of the GST from HeLa cell shows the right size of GST. Schematic representation of recombinant GST-tagged PWWP2A and PWWP domain deletion (DPWWP) and PWWP domain only (PWWP)-containing constructs (top)used in cell-derived mono-IPs followed by Coomassie staining and immunoblotting (bottom). * indicates respective GST proteins. Notice that both the PWWP domain alone as well as the PWWP-deletion protein are able to interact with nucleosomes, indicating at least two independent nucleosome binding sites within PWWP2A. (Sebastian Pünzeler,2017)

The result of the IP is with mononucleosis’s which is from HeLa cell that express GFP-H2A and GFP-H2A.Z isoforms detected with anti-GFP antibody. FRAP quantification curves of average GFP signal relative to fluorescence signal prior to bleaching from transiently expressed GFP (negative control), GFP-tagged histones, and GFP-tagged PWWP2A mutants (n=5–14). See also Appendix Fig S2B for FRAP experiments comparing recovery signals of different chromatin binding proteins to GFP-PWWP2A, as well as Appendix Fig S2C–E for FRAP analyses of stable and transient GFP and GFP-tagged proteins expressing HeLa cells. (Sebastian Pünzeler,2017)

PWWP2A predominantly localizes to the promoter region of highly transcribed gene, PWWP2A depletion leads to deregulation of gene expression and results in a proliferation defect caused by a metaphase-anaphase block where, PWWP2A is active transcribed gene. PWWP2A does not influence H2A.Z occupancy, while H2A.Z’sC-terminal tail contributes to PWWP2A chromatin recruitment where there is strong cell cycle due to PWWP2A loss but the H2A.Z is not affected due to its it’s loss. PWWP2A expression analysis by qPCR2days after knockdown with two independent siRNAs (PW#1, PW#2) using two different primer pairs (PW_2: recognizes main splice product; PW_all: recognizes all predicted splice forms). Luciferase siRNA (Luci) and non-transfected wild-type (wt) cells were used as controls. All data were normalized to HPRT1/HMBSS expression and depicted as % of luci transfectants. Error bars indicate SEM of four biological replicates. (Sebastian Pünzeler, 2017)

There is three different domains confer H2A.Z variant-specificity, nucleosome interaction, and DNA, PWWP2A binds chromatin do confer H2A.Z. They propose that PWWP2A modulates expression of manyH2A.Z-containing genes and speculate that the combined deregulation of metabolism and morphogenesis causes defects in mitotic spindle pulling and sliding as well as neural crest stem cell migration and differentiation. The latter alterations are hallmarks of human cranio-facial diseases, such as neurocristopathies (Zhanget al, 2014) affecting facial features and include eye abnormalities, as seen in DiGeorge, Treacher-Collins, or Waardenburg syndromes (reviewed in Mayor & Theveneau, 2013). (Sebastian Pünzeler, 2017)


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