Mitotic Structures Research Articles

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

ABSTRACTOur knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.

Abstract 3460: TBK1 regulates mitotic progression by modulating spindle assembly checkpoint in cancer cells

Abstract TANK Binding Kinase 1 (TBK1) is a non-canonical IkB kinase that contributes to KRAS-driven lung cancer. It is activated by phosphorylation of Serine-172 by TLR and RIG1 signaling, and this circuit triggers phosphorylation of IRF3 and IRF7, activation of NFκB and the expression of proinflammatory genes and interferons. In addition to its role role in regulating innate immunity, TBK1 also promotes oncogenesis by phosphorylating Akt and enhancing cell survival and by promoting autophagy and mitophagy. TBK1 is also induced under hypoxic conditions and expressed at significant levels in many solid tumors. TBK1 also contributes to prostate cancer dormancy and drug resistance by inhibiting mTOR and to tamoxifen resistance of breast cancer cells by enhancing transcriptional activity of ERα. Recent studies from our lab revealed a novel role for TBK1 in regulating mitosis. It was found that levels of phospho-TBK1 increases and localizes to centrosomes and the mitotic spindles during mitosis. Depletion of TBK1 was shown to trigger defects in spindle apparatus and prevents mitotic progression (Pillai et al., Nature Communications, 2015). TBK1 physically interacts and phosphorylates centrosomal protein CEP170 and mitotic apparatus protein NuMA. At the same time, it is not clear whether TBK1 regulates mitosis my modulating the Spindle Assembly Checkpoint (SAC). Our results show that TBK1 colocalizes with Cdc20 on the centrosomes. Additionally, TBK1-inhibited cells showed an increase in the colocalization of BUBR1 and Cdc20, with enhanced recruitment of BUBR1 to kinetochores. To further study how TBK1 affects SAC, lung and breast cancer cells were depleted of TBK1 by CRISPR/Cas9 mediated genome editing. Cells depleted of TBK1 showed very few cells in the mitotic stage; those that entered mitosis had residual levels of TBK1 and showed multipolarity and unusually stable and bundled microtubules. TBK1 knockout cells not only showed aberrant mitotic structures and had elevated levels of SAC components including BUBR1 and Cdc20. Surprisingly, level of mitotic Cyclin B1 remained unchanged in spite of elevated levels of Cdc20 indicating a possible inactivation of Anaphase Promoting complex (APC/C). Also, percentage of Cyclin B1 positive cells was significantly high in mitotic cells enriched using double thymidine block in the presence of TBK1 inhibitor BX795 (R9+BX) and MRT67307 (R9+MRT) as compared to untreated mitotic cells (R9). Further, double thymidine blocked released cells displayed elevated levels of SAC components upon treatment with TBK1 inhibitors. All these findings suggest that TBK1 facilitates mitotic progression through satisfying SAC. Citation Format: Neha Jaiswal, Smitha Pillai, Namrata BoraSinghal, Srikumar Chellappan. TBK1 regulates mitotic progression by modulating spindle assembly checkpoint in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3460. doi:10.1158/1538-7445.AM2017-3460

Abstract 2050: Polyploid tumor cells: a chemoresistant cell type in triple-negative breast cancer

Abstract Background: Triple-negative breast cancers (TNBC) are associated with an extremely poor prognosis due to their aggressive behavior and rapid resistance to chemotherapy. Chemotherapy may select for resistant subclones and lead to tumor recurrence. Although, DNA aneuploidy has been a long known prognostic biomarker in breast cancer, the biological role of aneuploid or polyploid cancer cells in tumorigenesis and chemoresistance is largely undefined. Polyploid cells arise due to repeated rounds of DNA duplication in the absence of mitosis. Chemotherapeutic DNA-damaging agents or mitotic inhibitors can also induce formation of polyploid cells and subsequent senescence. However, recent reports indicate that polyploid cells represent a viable and proliferating subpopulation within a tumor that may escape chemotherapy and contribute to tumor recurrence. Methods: In this study, we examined the prevalence and functional significance of polyploidy occurring de novo or induced by chemotherapy in TNBC cell lines. DNA ploidy was analyzed in Hoechst 33342 stained cells using FACS. Cells with greater than 4N DNA content were defined as polyploid. Live-cell imaging was done to observe cell division patters in polyploid cells using IncuCyte Zoom. Cell proliferation was assessed in the absence and presence of chemotherapy drugs with distinct modes of action (docetaxel and paclitaxel: mitotic inhibitors, cisplatin: DNA cross-linking agent, and etoposide or VP16: topoisomerase inhibitor). RNAseq was performed to compare gene expression profiles between treatment-naive diploid and polyploid cells. Results: Hoechst 33342 staining determined the prevalence of polyploid cells in HCC1395(6.7%) and HCC1937 (5.8%) cells lines. Live-cell imaging in polyploid cells showed formation of mitotic structures suggesting multi-polar cell division. Cell proliferation assay revealed that polyploid cells grow slower than diploid cells and show reduced sensitivity to all four chemotherapy drugs. Docetaxel treatment resulted in induction of polyploidy and drug-induced senescence in parental TNBC cells. Drug-induced polyploid cells were resistant to subsequent docetaxel treatment. Time-lapse imaging showed budding of small daughter cells from polyploid senescent cells. Majority of senescent cells died, some cells survived and regenerated the diploid and polyploid subpopulations similar to those present in parental cells. RNAseq identified differentially expressed genes involved in G1-S and G2-M checkpoint pathways (BRCA1/2, RAD51), cell proliferation (Aurora kinase A/B) and apoptosis (BIM, BIRC3). Conclusions: Above findings indicate that there are molecular and functional differences between diploid and polyploid cells that are both naturally occurring or induced by chemotherapy. In our future studies, we will examine combinatorial approaches to overcome polyploidy-associated chemoresistance. Citation Format: Rekha Gyanchandani, Adrian Lee. Polyploid tumor cells: a chemoresistant cell type in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2017-2050

Cell-cycle dynamics of chromosomal organization at single-cell resolution.

SummaryChromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures facilitating chromosome segregation, and decondensed interphase structures accommodating transcription, gene silencing and DNA replication. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Whole-genome 3D structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data thereby allow for re-interpretation of chromosome conformation maps through the prism of the cell cycle.

DETERMINATION OF THE CYTOTOXICITY OF NEW NANOCOMPOSITES BASED ON SAPONITE

Development of new nanomaterials is a relevant area of research. However, the specific physical and chemical properties of nanomaterials compared to conventional microparticles can carry unexpected risks to the ecosystem. Therefore, the newly created research of nanomaterials currently is a very urgent task. The article deals with the cytotoxicity of new Nb-containing nanocomposites and nanomaterials SiO2, which is included in their composition. HR-TEM micrographs show that all Nb (V) containing nanocomposites consist of relatively small, sheet-like particles at nanometer scale. Nanocomposites Nb-Saponite (Et) samples are composed of particles with size is below 100 nm, smaller than the Nb-Saponite (Cl) sample. This difference in a crystal size could be related to different procedure adopted to prepare the solids. Photomicrographs obtained at higher magnification showed basal plane lattice strips of nanocomposites, their interlayer space was about 1.3 nm. The study has found that new nanocomposites show no toxicity, unlike their part – nanosized materials SiO2. The results of studies on the cytotoxic effect on cells of A. cepa L. have shown that nanosized materials SiO2 leads to a decrease in mitotic index and chromosomal disorders structures, while not causing nanocomposites toxic effect. The research has also revealed that the action of nanosized materials SiO2 concentrations in the range of 450-600 mg/l changed the duration of mitosis phase, growth of prophase unit was observed while ana-phase and telophase units decreased. Thus, we should conclude that the cytotoxicity of nanosized materials due to their size, which cause significant chemical activity and high capacity for penetration of nanoparticles into plant organisms.

CHK2 overexpression and mislocalisation within mitotic structures enhances chromosomal instability and hepatocellular carcinoma progression

ObjectiveChromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is...

The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer.

Chromatin assembly factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but promotes the localization of specific loci (nucleolar-associated domains [NADs]) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the sumoylation-interacting motif within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.

Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA-/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

The RNA-binding protein ATX-2 regulates cytokinesis through PAR-5 and ZEN-4.

The spindle midzone harbors both microtubules and proteins necessary for furrow formation and the completion of cytokinesis. However, the mechanisms that mediate the temporal and spatial recruitment of cell division factors to the spindle midzone and midbody remain unclear. Here we describe a mechanism governed by the conserved RNA-binding protein ATX-2/Ataxin-2, which targets and maintains ZEN-4 at the spindle midzone. ATX-2 does this by regulating the amount of PAR-5 at mitotic structures, particularly the spindle, centrosomes, and midbody. Preventing ATX-2 function leads to elevated levels of PAR-5, enhanced chromatin and centrosome localization of PAR-5-GFP, and ultimately a reduction of ZEN-4-GFP at the spindle midzone. Codepletion of ATX-2 and PAR-5 rescued the localization of ZEN-4 at the spindle midzone, indicating that ATX-2 mediates the localization of ZEN-4 upstream of PAR-5. We provide the first direct evidence that ATX-2 is necessary for cytokinesis and suggest a model in which ATX-2 facilitates the targeting of ZEN-4 to the spindle midzone by mediating the posttranscriptional regulation of PAR-5.

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression

The Runt-related transcription factors (RUNX) are master regulators of development and major players in tumorigenesis. Interestingly, unlike most transcription factors, RUNX proteins are detected on the mitotic chromatin and apparatus, suggesting that they are functionally active in mitosis. Here, we identify key sites of RUNX phosphorylation in mitosis. We show that the phosphorylation of threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts RUNX DNA binding activity during mitotic entry to facilitate the recruitment of RUNX proteins to mitotic structures. Moreover, knockdown of RUNX3 delays mitotic entry. RUNX3 phosphorylation is therefore a regulatory mechanism for mitotic entry. Cancer-associated mutations of RUNX3 T173 and its equivalent in RUNX1 further corroborate the role of RUNX phosphorylation in regulating proper mitotic progression and genomic integrity.

Mitotic spindle formation in Triparma laevis NIES-2565(Parmales, Heterokontophyta).

The parmalean algae possess a siliceous wall and represent the sister lineage of diatoms; they are thought to be a key group for understanding the evolution of diatoms. Diatoms possess well-characterized and unique mitotic structures, but the mitotic apparatus of Parmales is still unknown. We observed the microtubule (MT) array during interphase and mitosis in Triparma laevis using TEM. The interphase cells had four or five centrioles (∼80nm in length), from which MTs emanated toward the cytoplasm. In prophase, the bundle of MTs arose at an extranuclear site. The position of centrioles with respect to an MT bundle changed during its elongation. Centrioles were observed on the lateral side of a shorter MT bundle (∼590nm) and on either side of an extended MT bundle (∼700nm). In metaphase, the spindle consisted of two types of MTs-MT bundle that passed through a cytoplasmic tunnel in the center of the nucleus and single MTs (possibly kinetochore MTs) that extended from the poles into the nucleus. The nuclear envelope disappeared only at the regions where the kinetochore MTs penetrated. In telophase, daughter chromosomes migrated toward opposite poles, and the MT bundle was observed between segregating chromosomes. These observations showed that MT nucleation does not always occur at the periphery of centrioles through cell cycle and that the spindle of T. laevis has a similar configuration to that of diatoms.

Early replication stress leads to abnormal mitosis and genome rearrangement

Yeast cell cycle genetics suggests that cells under replication stress generate damage signals that are sufficient to activate checkpoints and restrain mitosis. Yet in mammalian cancer cells, replication stress is not necessarily sufficient to block division. This can lead to abnormal mitotic structures including chromosome bridges, lagging chromosomes, ultrafine anaphase bridges, and micronuclei. This abnormal mitosis is a source for increased mutations including dramatic chromosome rearrangements including chromothripsis, resulting in characteristic genome instability. We have examined the response to different forms of replication stress in the fission yeast, S. pombe, employing visual methods and single‐cell live‐cell analysis.First, we observe distinct patterns of single‐strand binding protein RPA and homologous recombination protein Rad52 depending on the timing and type of stress we induce. This creates a fingerprint of different S phase‐specific stress responses which we can correlate to distinct outcomes.Second, we identify a novel stress‐associated phenotype in a temperature sensitive mutation of the Mcm4 helicase subunit. Despite under‐replicated DNA, the cells fail to arrest during temperature shift, and following release. Biochemical analysis suggests that they evade the checkpoint. Our dynamic pedigrees reveal surprising examples of division under stress including all the mitotic structures described above, including ultrafine anaphase bridges and apparent micronuclei. These abnormal mitoses are associated with genome instability, mutations, and chromosome rearrangement in the surviving cells.Our study shows that a subset of cells within a whole population can continue to divide, despite significant negative effects on genome integrity, and establishes a genetic model to study mechanisms that allow division to occur despite stress.Support or Funding InformationNIH R01 GM081418 &GM111040

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence.

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.

Quantifying the Effect of Electric Fields in the Frequency Range of 100-500 khz on Mitotic Spindle Structures

It is well established that electric fields can influence cells: Low frequency electric fields (f 1 MHz) cause tissue heating. More recently, it was shown that exposing dividing cells to low intensity (∼1 V/cm) electric fields in the frequency range of 100 - 500 kHz leads to disruption of the mitotic process, and ultimately cell death. This discovery motivated the development of Tumor Treating Fields (TTFields) therapy: a non-invasive treatment that utilizes electric fields in the intermediate frequency range to treat solid tumors. TTFields have been approved by the US Food and Drug Administration for the treatment of recurrent glioblastoma multiforme since 2011.It is thought that TTFields exert their effect on cells by inhibiting tubulin polymerization through interactions of the electric fields with the highly polarized tubulin dimers. However, only few studies have been performed that quantify the intracellular intensity of electric fields, in the frequency range of 100-500 kHz, and how these fields disrupt tubulin polymerization. Here we present the results of computational simulations and in-vitro studies that address these issues. The simulations show that the frequency range of 100-500 kHz marks a transition region in which the magnitude of the electric field inside cells increases significantly, and that the threshold at which this increase occurs depends on the dielectric properties of the cell's membrane and cytoplasm. The experiments show that exposure of cell cultures to TTFields leads to a 10-20% increase in the amount of unpolymerized tubulin dimers within the cells, suggesting that TTFields influence tubulin polymerization dynamics. These studies provide new insight into the biophysical mechanisms that underlie TTFields therapy.

Abstract B48: Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer

Abstract Background: Triple negative breast cancers (TNBC) are aggressive, heterogeneous tumors, and currently lack targeted therapies. Chemotherapy is the mainstay treatment, but tumor recurrence is a major cause of mortality and morbidity. Chemotherapy may select for resistant subclones that have a survival advantage over the bulk population of cancer cells and cause tumor relapse. DNA aneuploidy (abnormal number of chromosomes) is a common genetic feature in breast cancer and has been frequently associated with poor prognosis. Although, aneuploid or polyploid cancer cells can contribute to intratumor ploidy heterogeneity, their functional role in tumorigenesis and chemoresistance is largely undefined. Traditionally, polyploid cells were thought to arise due to defects in cell division and were considered to be senescent. However, recent reports have shown that polyploid cells represent a viable and proliferating subpopulation within a tumor and may have a direct role in regulating chemoresistance. Methods: In this study, we isolated and functionally characterized the diploid and polyploid sub-populations derived from TNBC cell lines. DNA ploidy was assessed in Hoechst 33342 stained cells using flow cytometry. Cells with greater than 4N DNA content were considered as polyploid. Next, we analyzed cell proliferation in HCC1937 cells using CFSE and DNA labeling. Live-cell imaging was performed on flow-sorted diploid and polyploid cells using IncuCyte Zoom to monitor cell proliferation in the absence and presence of mitotic inhibitor (docetaxel). Results: DNA ploidy analysis identified presence of polyploid cells in HCC1395 and HCC1937 cell lines at a frequency of 6.7% and 5.8% respectively. Polyploid cells have higher forward-scatter than diploid cells suggesting that they are larger in size. In addition, nuclear staining with Hoechst 33342 showed that giant polyploid cells contain enlarged and/or multiple nuclei. Time-lapse microscopy in polyploid cells revealed mitotic structures suggestive of multipolar cell division. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeling showed that polyploid cycle slower than the predominant diploid population. Further, the IncuCyte cell proliferation assay also confirmed that sorted polyploid cells divide slower than the diploid cells and displayed resistance to docetaxel. Concusions: These findings indicate that there are functional differences between diploid and polyploid sub-populations. In our future studies, we will examine the isolated diploid and polyploid cells for differences in their tumorigenic potential and role in tumor recurrence. Citation Format: Rekha Gyanchandani, Adrian Lee. Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B48.

A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

The Drosophila S2 tissue culture cells are a widely used system for studies on mitosis. S2 cells are particularly sensitive to gene silencing by RNA interference (RNAi), allowing targeted inactivation of mitotic genes. S2 cells are also well suited for high-resolution light microscopy analysis of mitosis in fixed cells, and can be easily immunostained to detect mitotic components. In addition, S2 cells are amenable to transformation with plasmid encoding fluorescently tagged mitotic proteins, allowing in vivo analysis of their behavior throughout cell division. However, S2 cells have not been widely used for transmission electron microscopy (TEM) analysis, which provides ultrastructural details on the morphology of the mitotic apparatus that cannot be obtained with high-resolution confocal microscopy. Here, we describe a simple method for the ultrastructural analysis of mitosis in Drosophila S2 cells.•Our method, which involves fixation and sectioning of a cell pellet, provides excellent preservation of mitotic structures and allows analysis of a higher number of mitotic divisions per sample, compared to correlative light-electron microscopy.•Dividing cells are randomly oriented within the pellet and are sectioned along different planes, providing all-around information on the structure of the mitotic apparatus.

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

ORC proteins in the mammalian zygote.

The origin recognition complex (ORC) proteins, ORC1-6, are the first known proteins that bind DNA replication origins to mark the competency for the initiation of DNA synthesis. These proteins have complex mechanisms of assembly into the ORC complex and unexpected localizations in the mitotic chromosomes, cytoplasm, and nuclear structures. The mammalian zygote is a potentially important model that may contribute to our understanding of the mechanisms and features influencing origin establishment and in the identification of other functions of the ORC proteins. Together with expected localizations to the chromatin during G1, we found an unexpected distribution in the cytoplasm that appeared to accumulate ORC proteins suggesting potential roles for ORC subunits in mitosis and chromatin segregation. ORC1, 2, 3, and 5 all localize to the area between the separating maternal chromosomes shortly after fertilization. ORC4 forms a cage around the set of chromosomes that will be extruded during polar body formation before it binds to the chromatin shortly before zygotic DNA replication. These data suggest that the ORC proteins may also play roles in preparing the cell for DNA replication in addition to their direct role in establishing functional replication origins.

Critical waves and the length problem of biology

It is pointed out that the mystery of how biological systems measure their lengths vanishes away if one premises that they have discovered a way to generate linear waves analogous to compressional sound. These can be used to detect length at either large or small scales using echo timing and fringe counting. It is shown that suitable linear chemical potential waves can, in fact, be manufactured by tuning to criticality conventional reaction-diffusion with a small number substance. Min oscillations in Escherichia coli are cited as precedent resonant length measurement using chemical potential waves analogous to laser detection. Mitotic structures in eukaryotes are identified as candidates for such an effect at higher frequency. The engineering principle is shown to be very general and functionally the same as that used by hearing organs.

CG10126, A Calcium‐Binding Microtubule‐Associated Protein, Promotes Mitosis during Drosophila Development

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that regulates signaling pathways critical for development and proliferation in Drosophila. EGFR activity is also highly upregulated in a number of human cancers, making identification of its downstream targets important for understanding both basic cellular processes and disease states. We have identified CG10126, a Drosophila homolog of the human protein Calcyphosine‐like, as a transcriptional target of EGFR signaling. Calcyphosine encodes a calcium‐binding protein regulated in several human cancers, but little is known of its function. Here we examine the role of CG10126 during Drosophila development. We expressed RNA interference constructs at various points in development. Reducing the level of CG10126 in eye imaginal discs produced adults with small eyes, while reducing its level ubiquitously in embryos was generally lethal. To further examine the role of CG10126, we used mass spectrometry to identify the binding partners of CG10126 in the presence or absence of calcium. Specifically in the presence of calcium, CG10126 bound α‐ and β‐tubulin and several spindle assembly proteins, suggesting that it may influence formation of the mitotic spindle. Consistent with this, embryos with reduced CG10126 show a diminished number of mitotic nuclei and abnormal spindle structures. Calcium is indispensable for cell cycle regulation; our results suggest that calcium waves present during mitosis may enable CG10126 to influence microtubule dynamics and spindle assembly. Together, our studies suggest that CG10126, a downstream target of EGFR signaling, is a microtubule‐associated protein that affects cell proliferation by regulating mitotic spindle formation.