Mitotic Stages Research Articles

Mitotic Studies and Genotoxic Assessment of Edible Nigerian Aroids to Selected Oilfield Chemicals

The genotoxic potentials of two water-based oilfield chemicals on edible aroids were investigated. Five accessions of Colocasia eculenta and three accessions of Xanthosoma maffafa were exposed to graded concentrations of sodium azide and potassium chromate; while the accessions without any chemical additive were used as controls. Results revealed that the peak periods of cell division were recorded between 12:00 noon to 4:00 pm in both the treated and control accessions. Metaphase cells increased after prophase cells and continued mostly between 10.00 am and 4.00 pm. Variations observed with the treatments included: shifts of metaphase peaks, high percentages of prophase cells, high intensity of cytoplasmic staining and induction of micronuclei among others. Sodium azide was observed to be a stronger genotoxic substance than potassium chromate. Accession NCe 001 had the highest survival rate while NCe 002 recorded the least rate of survival. Statistical evidence revealed that the difference in various mitotic stages and time of harvest between different accessions and treatments was significant at both 5% and 1%. The study provides useful information that would be used to promote cytogenetic researches as well as the exploitation and improvement of this neglected and underexploited economic plant.

Inhibition of Notch signaling by the p105 and p180 subunits of Drosophila chromatin assembly factor 1 is required for follicle cell proliferation.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.

THE DEPENDENCE OF DOSE-EFFECTS IN HUMAN RADIATION CYTOGENETICS.

To study the frequency and spectrum of chromosome aberrations in human peripheral blood lymphocytes depending on the radiation dose, the stage of the mitotic cycle and the periods of fixation of the cell culture. The test system of blood lymphocytes of donors, the metaphase analysis of chromosome aberrations (a uniform staining of the drugs). The gamma-irradiation was performed on the device with the source 60Co at the dose rate of 0.5 Gy/min, the dose range was 0.25-4.0 Gy. The lymphocyte culture was irradiated after 0, 24, 40 and 48 hours from the beginning of the incubation, which corresponds to G0-, G1-, S-, G2-stages of the mitot- ic cycle. The cells were fixed after 52 hours and 62 hours from the beginning of the incubation. The author's experimental data on the regularities of chromosome aberrations formation during irradiation at the different periods of the mitotic cycle of human lymphocyte culture are presented. The character of the dose dependences of structural damages of chromosomes during the mitotic cycle with an application of the linear, lin- ear-square and parabolic models is analyzed. The greatest yield of exchange-type aberrations is registered at the irradiation in G0- and G1-stages of the mitotic cycle, which submits to the linear-square dependence on an irradia- tion dose. When irradiating cells in the S- and G2-stages, the main contribution to the spectrum of radiation-induced chromosome aberrations is made by deletions, the level of which increases linearly with the dose. This is evidenced by the negative values of the quadratic term in the regression equations for these stages. The analysis of the curves using the parabolic model Y = k · Dn has shown that, at the irradiation of lymphocytes in G0- and G1-stages, the dose dependences approach the quadratic ones, which confirms the reasonableness of the interpretation of the obtained cytogenetic data from the point of view of the «classical» theory of the radiation-induced chromosome aberrations formation. However, the model of the spline regression is more accurate at the approximation of the dependences of the cytogenetic effects in the region of low doses of irradiation. The results obtained by the irradiation of human lymphocyte culture at different stages of the mitot- ic cycle indicate that the same radiation dose induces different qualitative and quantitative cytogenetic effects depending on the physiological state of the cell at the time of irradiation. The combination of such factors as a radi- ation dose rate, a mitotic cycle stage, the post-irradiation conditions, and an individual radiosensitivity are reflect- ed in quantitative and qualitative variations of the cellular radiosensitivity.

Karyotype analysis and report on B-chromosome in Gloriosa superba L. by differential staining

In this investigation Gloriosa superba L. was cytogenetically studied with orcein, CMA and DAPI-staining for authentic characterization. “Complex Chromocenter Type” of interphase nuclei with 3–4 bigger darkly stained heterochromatic regions was found in this species. The prophase chromosomes were “Interstitial Type” with darkly stained region at different interstitial sites of chromosomes. This species had 2n = 22 chromosomes with heterogenous centromeric formulae 14 m + 8 sm. In addition to the regular chromosomes, 1–6 small chromosomes were found in several mitotic pro-metaphase, metaphase and anaphase stage which covered 43.57% of the total cell count of this species suggesting the probable occurrence of B-chromosome. Presence of B-chromosome was probably the first report for this genus. After fluorescent banding, a pair of dot like CMA fluoresced bands were observed whereas no bright band was found in G. superba after DAPI-staining which suggested the coexistence of GC- and AT-base pairs in the genome. Thus, the compilation of the above information will be very useful for cytogenetical characterization of G. superba L. in Bangladesh.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Effect of foliar application of NPK nanoparticle fertilization on yield and genotoxicity in wheat (Triticum aestivum L.)

Two field experiments were conducted at the experimental farm of the Faculty of Agriculture (Saba Basha), Abess Region, Alexandria University, Egypt, over two harvest seasons (2016/2017 and 2017/2018) to study the effect of a nanoparticle fertilizer (Hyper Feed Amino NPs) compound on productivity and genotoxicity in two wheat cultivars compared with conventional mineral fertilizer. Total yield, its components, and yield components were measured. After harvest, grains of both cultivars were collected and germinated to produce root tip. Mitosis division and chromosomal aberrations were observed. Mitotic indexes (%) were obtained from an analysis of about 2000 cells. Root tips of control treatments showed normal mitotic stages, and the mitotic index ranged from 61.67 ± 8.40 to 68.49 ± 11.23%. Although fertilization with NPK NPs produced an increase in yield, and root-tip cells showed various types of chromosomal aberrations such as multinuclei, micronuclei, abnormal metaphase, stickiness, nucleus erosion, nucleus elongation, fragments, c-metaphase, chromosome deletion, c-chromosome, gab chromosome, lagging chromosome, distributed anaphase, bridge, abnormal telophase, telophase with unequal division, cell membrane damage, cell elongation, and increased nuclear content. The 100% NPK NPs treatment produced 35.79 ± 9.32% and 38.98 ± 4.22% abnormal cells in Sids 12 and Al-Rasheed, respectively. Our results demonstrate that wheat root tip cells readily internalize NPK NPs which can interfere with normal cell function.

On the Hypothesis of the Physical Mechanism Underlying the “Magnetism of Living Matter,” an Outstanding Discovery in Biophysics by L.A. Blumenfeld

The experimental data that describe and confirm the validity of the physical event referred to as “the magnetism of living matter,” which was discovered by L.A. Blumenfeld and his team at the end of the 1950s, are discussed and analyzed, as well as the corresponding theoretical debate of the 1960s. This debate did not propose any explanations for the observed effects but rather questioned them; as a result, the discovery was not only forgotten, but even scientifically discredited. A new theoretical explanation (a physical hypothesis) for the experimental dataset was proposed by Blumenfeld et al. in a series of publications in the 1960s to the 1990s. The physical hypothesis on the magnetism of living matter relies on the analysis of the experimental results by Blumenfeld, the theoretical view of the authors on the physical principles underlying the functioning of living matter, and the known and earlier described data on the mitotic stages in eukaryotic cell division, which was the object of Blumenfeld’s experimental studies.

XY oocytes of sex-reversed females with a Sry mutation deviate from the normal developmental process beyond the mitotic stage†.

The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry− mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry− females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry− PGCs was associated with aberrant β-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry− oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as “Swyer syndrome,” in which patients with an XY karyotype present as typical females and are infertile.

Comparison of the Modifying Action of Astaxanthin on the Development of Radiation-Induced Chromosome Instability in Human Blood Lymphocytes Irradiated in vitro at Different Cell Cycle Stages

This study presents the results and comparison of the distinctive features of the modifying effect of astaxanthin (20.0 μg/mL) on γ-radiation-induced chromosomal instability in human peripheral blood lymphocytes that were irradiated in vitro (1.0 Gy) at the G0, S, and G2 stages of the cell cycle. The study showed that the effect of astaxanthin on cells irradiated at cell cycle stages S and G2 did not lead to changes in the mean frequencies and the spectrum of radiation-induced chromosomal aberrations (p > 0.05). A powerful protective effect of astaxanthin on the level of radiation-induced chromosomal instability (p < 0.001) was observed only in cells that were irradiated at the G0 stage. Thus, the dependence of the radioprotective effect of astaxanthin on the mitotic cycle stage at which the cells were exposed to radiation was established.

Chromosome progression and mitotic times behavior are mimicked by an stochastic unstable dynamics

Mitosis is one of the most important processes of living matter. In this paper we analyze the consequences of assuming mitosis as being dictated by an unstable dynamics grounded in an antagonist genetic circuit. Based on this approach main characteristics of chromosome movement behavior in different mitotic stages can be mimicked. We describe the statistical variability of mitotic progression times – an aspect unvisited in previous studies – and find a remarkable relationship between mitosis times, both in healthy and malignant eukaryotic cells. We propose a tentative methodological approach to reconstruct the mitotic dynamical attractor.

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.

Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation.

We isolated isobavachalcone (IBC) from Angelica keiskei (AK) as an anti-obesity component. IBC dose-dependently inhibited 3T3-L1 adipocyte differentiation by down-regulating adipogenic factors. At the mitotic clonal expansion stage (MCE), IBC caused cell cycle arrest in G0/G1 with decreased expression of cell cycle-regulating proteins. IBC also inhibited autophagic flux by inducing intracellular accumulation of LC3B and SQSTM1/p62 proteins while decreasing expression levels of regulating factors for autophagy initiation. In parallel with the inhibition of adipocyte differentiation, IBC decreased intrahepatic fat deposits and rescued the liver steatosis in high fat cholesterol diet-fed zebrafish. In this study, we found that IBC isolated from AK suppresses mitotic clonal expansion and autophagy flux of adipocytes and also shows anti-obesity activity in a high cholesterol-diet zebrafish model by decreasing intrahepatic fat deposits. These results suggest that IBC could be a leading pharmacological compound for the development of anti-obesity drugs.

Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse MFC cells by electron microscopy.

Nucleolus structure alters as the cell cycle is progressing. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase. Fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs) are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly take place. Hitherto, little is known about the cell cycle-dependent reorganization of these structures. In this study, we followed the nucleolus structure during the cell cycle by electron microscopy (EM). We found the nucleolus experienced multiple rounds of structural reorganization within a single cell cycle: (1) when nucleoli are formed during the transition from late M to G1 phase, FCs, DFCs and GCs are constructed, leading to the establishment of tripartite nucleolus; (2) as FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization; (3) at late G1, the reassembly of FC/DFCs results in a structural transition from bipartite nucleolus towards tripartite nucleolus; (4) as cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Of note, FC/DFCs were not observed until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) when cells are at mitotic stage, FC/DFCs disappear before nucleolus structure is disassembled. These results also suggest that bipartite nucleolus can exist in higher eukaryotes at certain period of the cell cycle. As structures are the fundamental basis of diverse cell activities, unveiling the structural reorganization of nucleolar FCs and DFCs may bring insights into the spatial-temporal compartmentalization of relevant cellular functions.

Plasmodium APC3 mediates chromosome condensation and cytokinesis during atypical mitosis in male gametogenesis

The anaphase promoting complex/cyclosome (APC/C) is a highly conserved multi-subunit E3 ubiquitin ligase that controls mitotic division in eukaryotic cells by tagging cell cycle regulators for proteolysis. APC3 is a key component that contributes to APC/C function. Plasmodium, the causative agent of malaria, undergoes atypical mitotic division during its life cycle. Only a small subset of APC/C components has been identified in Plasmodium and their involvement in atypical cell division is not well understood. Here, using reverse genetics we examined the localisation and function of APC3 in Plasmodium berghei. APC3 was observed as a single focus that co-localised with the centriolar plaque during asexual cell division in schizonts, whereas it appeared as multiple foci in male gametocytes. Functional studies using gene disruption and conditional knockdown revealed essential roles of APC3 during these mitotic stages with loss resulting in a lack of chromosome condensation, abnormal cytokinesis and absence of microgamete formation. Overall, our data suggest that Plasmodium utilises unique cell cycle machinery to coordinate various processes during endomitosis, and this warrants further investigation in future studies.

Effect of Chemical Mutagens on Chromosomal behavior of Allium cepa L

Effect of chemical mutagens was studied in mitotic cells of Allium cepa L. For this purpose root tips were treated with saturated solution of peradichloro benzene, 0.2% EMS and 0.2 % freshly prepared colchicines for 4hours to 9hours.The studies revealed a wide range of chromosomal abnormalities such as stickiness, laggards, chromosomal bridge, unequal separation, nuclear budding, multinuclear cells etc.The parentage of chromosomal abnormalities in different mitotic stages were calculated on mitotic index, frequency of phases and percentage of abnormalities in mitosis. The chromosomal abnormalities increase with increase duration of treatment. The results showed that in colchicines and EMS more than 50% abnormalities ware observed after 8 hrs of treatment, while in PDB it takes 6 hours treatment. Minimum MI was recorded as 65% in controlled cells. In colchicines and EMS treated cells 0.9.5%, 20% MI ware observed after 8hrs of treatment. Whereas in PDB maximum 23% MI was recorded in cells treated for 6hrs.

Single-Cell Mass Spectrometry of Subpopulations Selected by Fluorescence Microscopy.

Specific subpopulations in a heterogeneous collection of cells, for example, cancer stem cells in a tumor, are often associated with biological or medical conditions. Fluorescence microscopy, based on biomarkers labeled with fluorescent probes, is a widely used technique for the visualization and selection of such cells. Phenotypic differences for these subpopulations at the molecular level can be identified by their untargeted analysis by single-cell mass spectrometry (MS). Here, we combine capillary microsampling MS with fluorescence microscopy for the analysis of metabolite and lipid levels in single cells to discern the heterogeneity of subpopulations corresponding to mitotic stages. The distributions of ATP, reduced glutathione (GSH), and UDP- N-acetylhexosamine (UDP-HexNAc) levels in mitosis reveal the presence of 2-3 underlying subpopulations. Cellular energy is found to be higher in metaphase compared to prometaphase and slightly declines in anaphase, telophase, and cytokinesis. The [GTP]/[GDP] ratio in cytokinesis is significantly higher than in prometaphase and anaphase. Pairwise correlations between metabolite levels show that some molecules within a group, including certain amino acids and nucleotide sugars, are strongly correlated throughout mitosis, but this is not related to their pathway distances. Correlations are observed between monophosphates (AMP and GMP), diphosphates (ADP and GDP), and triphosphates (ATP and GTP) of different nucleosides. In contrast, there is low correlation between diphosphates and triphosphates of the same nucleoside (ADP and ATP).

Pharmacokinetic-pharmacodynamic modelling of neutrophil response to G-CSF in healthy subjects and patients with chemotherapy-induced neutropenia.

The objective of the present study was to use pharmacokinetic-pharmacodynamic modelling to characterize the effects of chemotherapy on the granulopoietic system and to predict the absolute neutrophil counts (ANCs) for patients with chemotherapy-induced neutropenia treated with filgrastim and pegfilgrastim. Data were extracted from 10 phase I-III studies conducted in 110 healthy adults, and 618 adult and 52 paediatric patients on chemotherapy following administration of filgrastim or pegfilgrastim. The structural model accounted for ANC dynamics and the effects of filgrastim and pegfilgrastim, chemotherapy and corticosteroids. The impact of neutrophils on drug disposition was based on a drug receptor-binding model that assumed quasi-equilibrium and stimulation of the production and maturation of neutrophils upon treatment. The chemotherapy and corticosteroid effects were represented by kinetic-pharmacodynamic-type models, where chemotherapy stimulated elimination of neutrophil precursors at the mitotic stage, and corticosteroids stimulated neutrophil production. The systemic half-lives of filgrastim (2.6h) and pegfilgrastim (10.1h) were as expected. The effective half-life of chemotherapy was 9.6h, with a 2-day killing effect. The rate of receptor elimination from mitotic compartments exhibited extreme interindividual variability (% coefficient of variation >200), suggesting marked differences in sensitivity to chemotherapy effects on ANCs. The stimulatory effects of pegfilgrastim were significantly greater than those of filgrastim. Model qualification confirmed the predictive capability of this model. This qualified model simulates the time course of ANC in the absence or presence of chemotherapy and predicts nadir, time to nadir and time of recovery from different grades of neutropenia upon treatment with filgrastim and pegfilgrastim.

A guide to classifying mitotic stages and mitotic defects in fixed cells.

Cell division is fundamental to life and its perturbation can disrupt organismal development, alter tissue homeostasis, and cause disease. Analysis of mitotic abnormalities provides insight into how certain perturbations affect the fidelity of cell division and how specific cellular structures, molecules, and enzymatic activities contribute to the accuracy of this process. However, accurate classification of mitotic defects is instrumental for correct interpretation of data and formulation of new hypotheses. In this article, we provide guidelines for identifying specific mitotic stages and for classifying normal and deviant mitotic phenotypes. We hope this will clarify confusion about how certain defects are classified and help investigators avoid misnomers, misclassification, and/or misinterpretation, thus leading to a unified and standardized system to classify mitotic defects.

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.