Cytokinetic Defects Research Articles

NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis.

Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.

DNA DAMAGE AND OTHER NUCLEAR ANOMALIES AMONG GYM USERS: A COMPARATIVE STUDY BETWEEN BRAZIL AND SPAIN

Populations from different countries may present different cellular modifications among themselves, and the Buccal Micronucleus Cytome (BMCyt) assay in human buccal mucosal tissue may be a marker to evaluate these modifications. This study evaluated and compared DNA damage and other nuclear anomalies between Brazilian and Spanish gym users. This is a cross-sectional study carried out with gym users of Santa Cruz do Sul/Brazil and Madrid/Spain. The BMCyt assay was performed for biomarkers of DNA damage (micronuclei and/or nuclear buds), cytokinetic defects (binucleated cells), proliferative potential (basal cell frequency) and/or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells) in human buccal mucosal. Of the 228 individuals evaluated, 163 were Brazilian, and 65 were Spanish. Gym users of both countries differed between weight, body mass index, body fat, and muscle mass. The Brazilians presented a significantly higher frequency of micronuclei, nuclear buds, cells with condensed chromatin and karyorrhexis. Spaniards, however presented a significantly higher frequency of karyolytic cells. In conclusion, Brazilian gym users presented significantly higher rates of DNA damage and cell death, while the Spanish presented a higher frequency of advanced stage cell death.

Phosphorylation of RGS regulates MAP kinase localization and promotes completion of cytokinesis

Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.

The Phytochemical Scoulerine Inhibits Aurora Kinase Activity to Induce Mitotic and Cytokinetic Defects.

To identify novel bioactive compounds, an image-based, cell culture screening of natural product extracts was conducted. Specifically, our screen was designed to identify phytochemicals that might phenocopy inhibition of the chromosomal passenger protein complex in eliciting mitotic and cytokinetic defects. A known alkaloid, scoulerine, was identified from the rhizomes of the plant Corydalis decumbens as being able to elicit a transient mitotic arrest followed by either apoptosis induction or polyploidy. In examining the mitotic abnormality further, we observed that scoulerine could elicit supernumerary centrosomes during mitosis, but not earlier in the cell cycle. The localization of NUMA1 at spindle poles was also inhibited, suggesting diminished potential for microtubule recruitment and spindle-pole focusing. Polyploid cells emerged subsequent to cytokinetic failure. The concentration required for scoulerine to elicit all its cell division phenotypes was similar, and an examination of related compounds highlighted the requirement for proper positioning of a hydroxyl and a methoxy group about an aromatic ring for activity. Mechanistically, scoulerine inhibited AURKB activity at concentrations that elicited supernumerary centrosomes and polyploidy. AURKA was only inhibited at higher concentrations, so AURKB inhibition is the likely mechanism by which scoulerine elicited division defects. AURKB inhibition was never complete, so scoulerine may be a suboptimal AURK inhibitor or work upstream of the chromosomal passenger protein complex to reduce AURKB activity. Scoulerine inhibited the viability of a variety of human cancer cell lines. Collectively, these findings uncover a previously unknown activity of scoulerine that could facilitate targeting human cancers. Scoulerine, or a next-generation analogue, may be useful as a nontoxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Abstract 3557: miR-181a is a key driver of genomic instability and ovarian cancer tumorigenesis through the regulation of RB1

Abstract Epithelial ovarian cancer (EOC) is one of the deadliest gynecological cancers currently diagnosed in women with approximately 20,000 new cases and 15,000 deaths per year. Ovarian cancer can be stratified into distinct subtypes of which high-grade serous ovarian cancer (HGSOC) comprises 90% of all cases and has the highest mortality rate. Reducing the high mortality rate associated with HGSOC currently has two primary barriers: 1) most HGSOCs are diagnosed at a post-metastatic stage and 2) up to 70% of patients recur within 5 years. Both of these limitations are due to the fact that the drivers of tumor initiation are poorly understood. In order to change the status quo of the last 30 years, a new approach that focuses on understanding the origins of HGSOC is required. Our lab has recently discovered that miR-181a promotes key hallmarks of HGSOC progression in fallopian tube secretory epithelial cells (FTSECs) which are precursor cells of HGSOC. We show that enhanced expression of miR-181a in FTSECs results in increased proliferation, survival, and anchorage independent growth in vitro. Furthermore, FTSECs with miR-181a overexpression are able to form tumors in vivo whereas FTSECs that overexpress a control scramble miRNA cannot. Using cell cycle analysis, dynamic live-cell imaging, and SNP array analysis, we also found that miR-181a overexpression increases aneuploidy and genomic instability in FTSECs. We show that miR-181a promotes defects in nuclear structure, ruptures in the nuclear envelope, as well as mitotic and cytokinetic defects affecting proper chromosome segregation. A combination of global mRNA expression profiling and target prediction software identified the tumor suppressor retinoblastoma (RB1) as a target for miR-181a mediated FTSEC tumorigenesis. Using an RB1 3’UTR luciferase assay, along with stable expression of an shRNA against RB1 in FTSECs, we are able to show that miR-181a directly targets RB1 and that knockdown of RB1 phenocopies miR-181a mediated tumorigenesis. Taken together, our data shed light on a novel mechanism that promotes genomic instability and tumorigenesis in the early phases of HGSOC development. Our results suggest miR-181a as a promising early detection biomarker as well as a prognostic tool to guide patient treatment. Citation Format: Matthew Knarr, Lily Kwiatkowski, Anil Nagaraj, Ronny Drapkin, Analisa DiFeo. miR-181a is a key driver of genomic instability and ovarian cancer tumorigenesis through the regulation of RB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3557.

PM10-related DNA damage, cytokinetic defects, and cell death in COPD patients from Chiang Dao district, Chiang Mai, Thailand.

Exposure to PM10 generated by biomass burning may reduce lung function and induce cytogenetic effects, especially in chronic obstructive pulmonary disease patients. This study investigated the frequency of DNA-damaged cells, cells with cytokinetic defect, and different types of cell death using a buccal micronucleus cytome assay. The correlations between each biomarker and lung function were investigated. The changes in these biomarkers associated with high pollutant levels (PM10 > 50μg/m3) and low pollutant levels (PM10 < 50μg/m3) were evaluated to explore whether PM10 exposure induced genotoxic damages and cytokinetic defects in COPD patients when the daily average PM10 concentration reached above 50μg/m3. Fifty-eight COPD patients and 26 healthy subjects living in Chiang Dao district, Chiang Mai, Thailand, were recruited in this study. The results revealed that buccal cells with micronuclei (high vs low 1.09 ± 1.95 vs 0.29 ± 0.64 in COPD patients) and binucleated cells (high vs low 11.43 ± 18.68 vs 1.60 ± 1.31 and 7.77 ± 12.76 vs 1.00 ± 1.17 in COPD and healthy subjects, respectively) observed during the high pollutant period were more frequent than in the low pollutant period. Moreover, exposure to PM10 increased the risk of micronucleus induction in COPD patients 295.23-fold.

USP7 Regulates Cytokinesis through FBXO38 and KIF20B

The ubiquitin specific protease 7 (USP7 or HAUSP) is known to regulate a variety of cellular processes by binding and deubiquitylating specific target proteins. To gain a more comprehensive understanding of its interactions and functions, we used affinity purification coupled to mass spectrometry to profile USP7 interactions. This revealed a novel interaction with FBXO38, a poorly characterized F-box protein. We showed that USP7 stabilizes FBXO38 dependent on its catalytic activity by protecting FBXO38 from proteasomal degradation. We used a BioID approach to profile the protein interactions (and putative functions) of FBXO38, revealing an interaction with KIF20B, a Kinesin-6 protein required for efficient cytokinesis. FBXO38 was shown to function independently from an SCF complex to stabilize KIF20B. Consequently, depletion of either FBXO38 or USP7 led to dramatic decreases in KIF20B levels and KIF20B at the midbody, which were manifested in cytokinetic defects. Furthermore, cytokinetic defects associated with USP7 silencing were rescued by restoring FBXO38 or KIF20B. The results indicate a novel mechanism of regulating cytokinesis through USP7 and FBXO38.

Periodontitis: Genomic instability implications and associated risk factors

Periodontitis is a bacterial infection characterized by the presence of a dense inflammatory infiltrate, which may result in increased DNA damage and other nuclear/cellular abnormalities. Therefore, it is important to evaluate the periodontal diseases influence on DNA damage and other nuclear/cellular abnomalies formation as cancer risk markers. Thus, the aim of this study was to evaluate the periodontal diseases effect, according to its severity, on the occurrence of DNA damage and other nuclear/cellular abnormalities. This is a cross-sectional study with 77 subjects from the dentistry clinic of the University of Santa Cruz do Sul, Brazil, divided in control group (26 subjects), moderate periodontal disease group (26 subjects) and severe periodontal disease group (25 subjects). All subjects answered self-referenced questionnaires, underwent periodontal clinical examinations and allowed the collection of oral mucosa cells for the BMCyt. In relation to DNA damage biomarkers (micronuclei (MN) and/or nuclear buds (NBUD)), our results indicated no increase in MN frequencies (p > 0.05), however it indicated significant difference in NBUD frequencies between groups (p < 0.024). This result suggests that the periodontal disease status may influence DNA damage. Regarding the other nuclear/cellular abnormalities, was observed a significant difference in the binucleated (BN) frequencies between groups (p < 0.05). Moreover, the periodontitis severity was associated to an increase in the combined (summed) frequency of cells with different levels of DNA damage (MN and/or NBUD), cytokinetic defects (BN cells) and/or cell death (karyorrhexis, pyknotic and karyolytic cells) (r = 0.235; p = 0.040). Periodontal disease depending on its severity, induces nuclear anomalies in buccal cells.

A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis.

Plant cytokinesis involves membrane trafficking and cytoskeletal rearrangements. Here, we report that the phosphoinositide kinases PI4Kβ1 and PI4Kβ2 integrate these processes in Arabidopsis thaliana (Arabidopsis) roots. Cytokinetic defects of an Arabidopsis pi4kβ1 pi4kβ2 double mutant are accompanied by defects in membrane trafficking. Specifically, we show that trafficking of the proteins KNOLLE and PIN2 at the cell plate, clathrin recruitment, and endocytosis is impaired in pi4kβ1 pi4kβ2 double mutants, accompanied by unfused vesicles at the nascent cell plate and around cell wall stubs. Interestingly, pi4kβ1 pi4kβ2 plants also display ectopic overstabilization of phragmoplast microtubules, which guide membrane trafficking at the cell plate. The overstabilization of phragmoplasts in the double mutant coincides with mislocalization of the microtubule-associated protein 65-3 (MAP65-3), which cross-links microtubules and is a downstream target for inhibition by the MAP kinase MPK4. Based on similar cytokinetic defects of the pi4kβ1 pi4kβ2 and mpk4-2 mutants and genetic and physical interaction of PI4Kβ1 and MPK4, we propose that PI4Kβ and MPK4 influence localization and activity of MAP65-3, respectively, acting synergistically to control phragmoplast dynamics.

Genomic and Immune Profiles of Multiple Myeloma Revealed By Whole Genome and Transcriptome Sequencing

Introduction:We analyzed genomic profile in line with immune checkpoint molecule expression in multiple myeloma using whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) data.Methods:We performed WGS of CD138+ selected tumor and the matched normal from saliva of multiple myeloma patients at 90-60x and 30x respectively from 37 patients. At the same time, we performed WTS using both tumor (CD138+) and immune milieu (CD138-) samples from these patients. Structural variations were called with Delly2 and Manta and the intersect of two call sets were used as the final SV calls. Copy number analysis was done with Sequenza and custom scripts. Somatic single nucleotide variants were identified with Mutect1 and Strelka and filtered against the panel of normal. RNA-seq was aligned using STAR algorithm and gene expression was quantified using RSEM algorithm. We extracted the expression of 21 immune checkpoint-related molecules (LAG3, PD-L1, IDO1, TIM3, CD27, CTLA-4, ICOS, TIGIT, PD-1, PD-L2, VISTA, and their ligands). To compare cell populations within a set of CD138-sorted MM samples, we used CIBERSORT algorithm to estimate predict fractions of leukocyte RNA.Results & Discussion:A total of 1,359 somatic structural variants were identified, with a median of 28 structural variant events per patient. The most common translocation event was t(4;14) (n=6, 16.2%). We identified novel recurrent deletions in CCSER1 (n=5, 13.5%) either deleting exon 10 (n=3) or exon 6 (n=2). CCSER1 has been proposed to a have a role in cell division with defects leading to cytokinetic defects. We observed an enrichment of whole-genome duplicated samples with CCSER1 deleted patients. We propose that CCSER1 deletion may have a role in whole-genome duplication events seen in multiple myeloma. Chromothripsis was also seen in 7 patients (18.9%). A total of 9 chromothripsis events were observed and chromosome 6 was most frequently involved (n=3). Recurrent mutation in IGKV4-1 chr2: 89185484 and 89185485 (p.Ala60) was observed suggesting dysfunctional antigen recognition in myeloma cells.From immune checkpoint perspective, expression of Galectin-9, a ligand of TIM3, was outstandingly high in immune milieu suggesting potential role of TIM3 inhibitor in myeloma. On the other hand, VISTA was highly expressed in tumors. When we matched the transcriptomic profiles with genomic variants, NRAS G61R mutant showed high expression of TIM3/Galectin-9 axis suggesting characteristics immune milieu of RAS mutant myeloma.Conclusions:We could identify novel CCSER1 recurrent deletion and IGKV4-1 mutation in multiple myeloma using WGS. WTS immune milieu analysis suggest potential role of TIM3 inhibitor in myeloma, especially in RAS mutated cases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genotoxic damage in end-stage renal disease

End-stage renal disease (ESRD) patients are at increased risk for cardiovascular events and cancer, possibly due to genomic instability associated with renal disease and/or its therapy. Prognostic biomarkers of genomic instability may prove useful for initiating appropriate intervention strategies. We conducted a case-control study, performing the single-cell gel electrophoresis assay (circulating leukocytes) and the micronucleus cytome assay (buccal epithelial cells). Cases (ESRD patients; n = 55) were on weekly/fortnightly dialysis therapy and controls (n = 39) were healthy adults. The patients had significantly elevated levels of DNA damage and micronucleated cells. DNA damage showed higher validity and sensitivity than did chromosome damage, for discriminating patients from controls. The patient group showed significant increases in cell proliferation, cytokinetic defects, and cell death, and a decrease in repair index. Correlations were seen between genetic damage and both time-on-medication and time-on-dialysis; between condensed chromatin cells and sex; and between pyknotic cells and dietary pattern. Following stratification by age, gender, and dialysis frequency, significantly elevated DNA damage and MN frequency were seen in the fortnightly dialysis patients, perhaps due to accumulated uremic toxicants. DNA and chromosome damage may be useful prognostic biomarkers for initiating timely interventions against co-morbidities in ESRD patients.

The Correlation Between Depression and Hopelessness Scores and Cytological Features of Exfoliated Buccal Cells in Females

Background: Depression is a mental health disorder, which affects all the communities around the world. One of the major components of depression is hopelessness. There is evidence that depression is accompanied by increased reactive oxygen species, which contribute to DNA damage. Objectives: This study aimed at evaluating whether depression and hopelessness are associated with markers of DNA damage, proliferative potential, and cell death. Methods: The sample included 59 healthy females from Fars province, southern Iran. Depression and hopelessness scores were measured using Beck’s depression inventory and Beck’s hopelessness scales, respectively. Smears were taken from the buccal mucosa of participants, and using micronucleus assay, they were analyzed for various cell population and genome damage markers, including cytokinetic defects (binucleated cells), proliferative potential (basal cell frequency), cell death (condensed chromatin, karyorrhectic, pyknotic, and karyolitic cells), and biomarkers of DNA damage (micronuclei and nuclear buds). Results: None of the micronucleus (MN) assay parameters had an association with depression scores. There was a significant negative correlation between the frequency of basal cells and hopelessness scores (r = -0.323, P = 0.012). Other parameters of MN assay showed no association with hopelessness scores. Conclusions: As the basal cells are biomarkers of proliferative potential, the results suggest that cellular regeneration is decreased in buccal mucosa of people with high level of hopelessness. However, more studies with larger sample sizes are required to verify this conclusion.

Assessment of exposure to pesticides in rural workers in southern of Minas Gerais, Brazil

The aim of the study was to assess of occupational exposure to pesticides in rural workers using genotoxicity test, bioindicators and clinical evaluation. Blood, urine and buccal samples from persons, rural workers exposed to a complex mixture of pesticides with organophosphates (n=94) and without organophosphates (n=94) were collected to compare the activities of cholinesterases, the levels of urinary dialkyl phosphates, genotoxicity data, from a cytome assay. Biomarkers were analysed by traditional/published methods Control group consisted of 50 other persons, non- occupationally exposed to pesticides from the city of Alfenas, Minas Gerais, Brazil. All subjects underwent a clinical evaluation. In the group exposed to organophosphates, the activity of acetylcholinesterase, butyrylcholinesterase and total cholinesterase was lower by 63.8%, 12.8%, and 14.8%, respectively, and 92.6% of the group had dialkyl phosphates present in their urine. The cytome assay was used to measure biomarkers of DNA damage (micronuclei and/or elimination of nuclear material by budding), cytokinetic defects (binucleated cells), and proliferative potential (basal cell) and/or cell death (condensed chromatin, karyorrhectic, pyknotic, and karyolytic cells). The group exposed to organophosphates showed significant changes in all these parameters compared to the control group and showed significant changes in budding, condensed chromatin and karyolytic cells compared with the group non-exposed to organophosphates. Data from the clinical evaluation showed significant changes in the central nervous, respiratory and auditory systems. The studied biomarkers are able to distinguish occupational and environmental exposure to pesticides and the data showed hazardous exposure to organophosphates and afforded valuable data to estimate the risk to cancer development.

Investigation of the role of four mitotic septins and chitin synthase 2 for cytokinesis in Kluyveromyces lactis

Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S. cerevisiae counterparts. Fluorophore-tagged versions of the K. lactis septins localized to a ring at the incipient bud site and split into two separate rings at the bud neck later in cytokinesis. One of the key proteins recruited to the bud neck by septins in S. cerevisiae is the chitin synthase Chs2, which synthesizes the primary septum. KlCHS2 was found to be essential and deletions showed cytokinetic defects upon spore germination. KlChs2-GFP also localized to the bud neck and to punctate structures in K. lactis. We conclude that cytokinesis in K. lactis is similar to S. cerevisiae and chimeric septin complexes are fully functional in both yeasts. In contrast to some S. cerevisiae strains, KlChs2 and KlCdc10 were found to be essential.

Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis.

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis.

Effective growth-suppressive activity of maternal embryonic leucine-zipper kinase (MELK) inhibitor against small cell lung cancer.

Maternal embryonic leucine zipper kinase (MELK), that plays a critical role in maintenance of cancer stem cells (CSCs), is predominantly expressed in various types of human cancer including small cell lung cancer (SCLC). SCLC usually acquires resistance to anti-cancer drugs and portends dismal prognosis. We have delineated roles of MELK in development/progression of SCLC and examined anti-tumor efficacy of OTS167, a highly potent MELK inhibitor, against SCLC. MELK expression was highly upregulated in both SCLC cell lines and primary tumors. siRNA-mediated MELK knockdown induced significant growth inhibition in SCLC cell lines. Concordantly, treatment with OTS167 exhibited strong cytotoxicity against eleven SCLC cell lines with IC50 of < 10 nM. As similar to siRNA knockdown, OTS167 treatment induced cytokinetic defects with intercellular bridges, and in some cell lines we observed formation of neuronal protrusions accompanied with increase of a neuronal differentiation marker (CD56), indicating that the compound induced differentiation of cancer cells to neuron-like cells. Furthermore, the MELK inhibition decreased its downstream FOXM1 activity and Akt expression in SCLC cells, and led to apoptotic cell death. OTS167 appeared to be more effective to CSCs as measured by the sphere formation assay, thus MELK inhibition might become a promising treatment modality for SCLC.

GTPase domain driven dimerization of SEPT7 is dispensable for the critical role of septins in fibroblast cytokinesis.

Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts, and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7flox/floxfibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7flox/floxfibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

The X-Linked-Intellectual-Disability-Associated Ubiquitin Ligase Mid2 Interacts with Astrin and Regulates Astrin Levels to Promote Cell Division.

Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409, and is degraded during cytokinesis. Mid2 depletion led to astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant astrin in astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis. These results could help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.

Opposing Effects of Inhibitors of Aurora-A and EGFR in Autosomal-Dominant Polycystic Kidney Disease.

Aurora-A kinase (AURKA) overexpression in numerous tumors induces aneuploidy, in part because of cytokinetic defects. Alisertib and other small-molecule inhibitors targeting AURKA are effective in some patients as monotherapies or combination therapies. Epidermal growth factor receptor (EGFR) pro-proliferative signaling activity is commonly elevated in cancer, and the EGFR inhibitor erlotinib is commonly used as a standard of care agent for cancer. An erlotinib/alisertib combination therapy is currently under assessment in clinical trials, following pre-clinical studies that indicated synergy of these drugs in cancer. We were interested in further exploring the activity of this drug combination. Beyond well-established functions for AURKA in mitotic progression, additional non-mitotic AURKA functions include control of ciliary stability and calcium signaling. Interestingly, alisertib exacerbates the disease phenotype in mouse models for autosomal-dominant polycystic kidney disease (ADPKD), a common inherited syndrome induced by aberrant signaling from PKD1 and PKD2, cilia-localized proteins that have calcium channel activity. EGFR is also more active in ADPKD, making erlotinib also of potential interest in this disease setting. In this study, we have explored the interaction of alisertib and erlotinib in an ADPKD model. These experiments indicated erlotinib-­restrained cystogenesis, opposing alisertib action. Erlotinib also interacted with alisertib to regulate proliferative signaling proteins, albeit in a complicated manner. Results suggest a nuanced role of AURKA signaling in different pathogenic conditions and inform the clinical use of AURKA inhibitors in cancer patients with comorbidities.