Cell Division Phenotype Research Articles

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

Graph metric learning quantifies morphological differences between two genotypes of shoot apical meristem cells inArabidopsis

AbstractWe present a method for learning ‘spectrally descriptive’ edge weights for graphs. We generalize a previously known distance measure on graphs (graph diffusion distance [GDD]), thereby allowing it to be tuned to minimize an arbitrary loss function. Because all steps involved in calculating this modified GDD are differentiable, we demonstrate that it is possible for a small neural network model to learn edge weights which minimize loss. We apply this method to discriminate between graphs constructed from shoot apical meristem images of two genotypes of Arabidopsis thaliana specimens: wild-type and trm678 triple mutants with cell division phenotype. Training edge weights and kernel parameters with contrastive loss produce a learned distance metric with large margins between these graph categories. We demonstrate this by showing improved performance of a simple k-nearest-neighbour classifier on the learned distance matrix. We also demonstrate a further application of this method to biological image analysis. Once trained, we use our model to compute the distance between the biological graphs and a set of graphs output by a cell division simulator. Comparing simulated cell division graphs to biological ones allows us to identify simulation parameter regimes which characterize mutant versus wild-type Arabidopsis cells. We find that trm678 mutant cells are characterized by increased randomness of division planes and decreased ability to avoid previous vertices between cell walls.

Abstract 2630: The LexA-Like Repressor UmuDAb and It's Corepressor DdrR Affect Cell Division Phenotypes in Acinetobacter baumannii

Abstract 2630: The LexA-Like Repressor UmuDAb and It's Corepressor DdrR Affect Cell Division Phenotypes in Acinetobacter baumannii

Molecular oxygen levels regulate Sinorhizobium meliloti cell division through a FixJ-dependent transcription control mechanism

Rhizobia infect the roots of host legumes and induce formation of nitrogen-fixing nodules, where nitrogenase genes are inducibly expressed by micro-aerobic signals. FixL/FixJ is an oxygen signal sensing system that is unique to rhizobia. FixL monitors molecular oxygen levels and phosphorylates the response regulator FixJ, thereby regulating downstream gene expression. The cell division of rhizobia is regulated by a phosphorylation relaying cascade that includes the transcription factors CtrA, GcrA, and DnaA. In Sinorhizobium meliloti the expression of these proteins is regulated by NtrX, which affects cell division. In the present work, by analyzing the cell division phenotypes and gene expression patterns of S. meliloti fixJ and ntrX mutants, we found that S. meliloti cell division is regulated by oxygen gas levels. Under normal conditions, FixJ induced NtrX and DnaA expression, but repressed CtrA and GcrA expression. In contrast, under hypoxic conditions, phosphorylated FixJ specifically bound to gene promoter regions to directly induce CtrA and GcrA expression, but to repress DnaA expression. Our findings reveal that molecular oxygen levels regulate S. meliloti cell division by a FixJ-dependent transcription control mechanism.

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.

Salicylic acid promotes quiescent center cell division through ROS accumulation and down‐regulation of PLT1, PLT2, and WOX5

Salicylic acid (SA) plays a crucial role in plant immunity. However, its function in plant development is poorly understood. The quiescent center (QC), which maintains columella stem cells (CSCs) in the root apical meristem and typically exhibits low levels of cell division, is critical for root growth and development. Here, we show that the Arabidopsis thaliana SA overaccumulation mutant constitutively activated cell death 1 (cad1), which exhibits increased cell division in the QC, is rescued by additional mutations in genes encoding the SA biosynthetic enzyme SALICYLIC ACID INDUCTION DEFFICIENT2 (SID2) or the SA receptor NONEXPRESSER OF PR GENES1 (NPR1), indicating that QC cell division in the cad1 mutant is promoted by the NPR1-dependent SA signaling pathway. The application of exogenous SA also promoted QC cell division in wild-type plants in a dose-dependent manner and largely suppressed the expression of genes involved in QC maintenance, including those encoding the APETALA2 (AP2) transcription factors PLETHORA1 (PLT1) and PLT2, as well as the homeodomain transcription factor WUSCHEL-RELATED HOMEOBOX5 (WOX5). Moreover, we showed that SA promotes reactive oxygen species (ROS) production, which is necessary for the QC cell division phenotype in the cad1 mutant. These results provide insight into the function of SA in QC maintenance.

Differential biosynthesis and cellular permeability explain longitudinal gibberellin gradients in growing roots

Control over cell growth by mobile regulators underlies much of eukaryotic morphogenesis. In plant roots, cell division and elongation are separated into distinct longitudinal zones and both division and elongation are influenced by the growth regulatory hormone gibberellin (GA). Previously, a multicellular mathematical model predicted a GA maximum at the border of the meristematic and elongation zones. However, GA in roots was recently measured using a genetically encoded fluorescent biosensor, nlsGPS1, and found to be low in the meristematic zone grading to a maximum at the end of the elongation zone. Furthermore, the accumulation rate of exogenous GA was also found to be higher in the elongation zone. It was still unknown which biochemical activities were responsible for these mobile small molecule gradients and whether the spatiotemporal correlation between GA levels and cell length is important for root cell division and elongation patterns. Using a mathematical modeling approach in combination with high-resolution GA measurements in vivo, we now show how differentials in several biosynthetic enzyme steps contribute to the endogenous GA gradient and how differential cellular permeability contributes to an accumulation gradient of exogenous GA. We also analyzed the effects of altered GA distribution in roots and did not find significant phenotypes resulting from increased GA levels or signaling. We did find a substantial temporal delay between complementation of GA distribution and cell division and elongation phenotypes in a GA deficient mutant. Together, our results provide models of how GA gradients are directed and in turn direct root growth.

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis.

Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS.IMPORTANCEBacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

Protease‐deficient SOS constitutive cells have RecN‐dependent cell division phenotypes

In Escherichia coli, after DNA damage, the SOS response increases the transcription (and protein levels) of approximately 50 genes. As DNA repair ensues, the level of transcription returns to homeostatic levels. ClpXP and other proteases return the high levels of several SOS proteins to homeostasis. When all SOS genes are constitutively expressed and many SOS proteins are stabilized by the removal of ClpXP, microscopic analysis shows that cells filament, produce mini-cells and have branching protrusions along their length. The only SOS gene required (of 19 tested) for the cell length phenotype is recN. RecN is a member of the Structural Maintenance of Chromosome (SMC) class of proteins. It can hold pieces of DNA together and is important for double-strand break repair (DSBR). RecN is degraded by ClpXP. Overexpression of recN+ in the absence of ClpXP or recN4174 (A552S, A553V), a mutant not recognized by ClpXP, produce filamentous cells with nucleoid partitioning defects. It is hypothesized that when produced at high levels during the SOS response, RecN interferes with nucleoid partitioning and Z-Ring function by holding together sections of the nucleoid, or sister nucleoids, providing another way to inhibit cell division.

Madurahydroxylactone, an Inhibitor of Staphylococcus aureus FtsZ from Nonomuraea sp. AN100570.

FtsZ, a bacterial cell-division protein, is an attractive antibacterial target. In the screening for an inhibitor of Staphylococcus aureus FtsZ, madurahydroxylactone (1) and its related derivatives 2-5 were isolated from Nonomuraea sp. AN100570. Compound 1 inhibited S. aureus FtsZ with an IC50 of 53.4 μM and showed potent antibacterial activity against S. aureus and MRSA with an MIC of 1 μg/ml, whereas 2-5 were weak or inactive. Importantly, 1 induced cell elongation in the cell division phenotype assay, whereas 2-5 did not. It indicates that 1 exhibits its potent antibacterial activity via inhibition of FtsZ, and the hydroxyl group and hydroxylactone ring of 1 are critical for the activity. Thus, madurahydroxylactone is a new type of inhibitor of FtsZ.

The nucleoid occlusion factor Noc controls DNA replication initiation in Staphylococcus aureus.

Successive division events in the spherically shaped bacterium Staphylococcus aureus are oriented in three alternating perpendicular planes. The mechanisms that underlie this relatively unique pattern of division and coordinate it with chromosome segregation remain largely unknown. Thus far, the only known spatial regulator of division in this organism is the nucleoid occlusion protein Noc that inhibits assembly of the cytokinetic ring over the chromosome. However, Noc is not essential in S. aureus, indicating that additional regulators are likely to exist. To search for these factors, we screened for mutants that are synthetic lethal with Noc inactivation. Our characterization of these mutants led to the discovery that S. aureus Noc also controls the initiation of DNA replication. We show that cells lacking Noc over-initiate and mutations in the initiator gene dnaA suppress this defect. Importantly, these dnaA mutations also partially suppress the division problems associated with Δnoc. Reciprocally, we show that over-expression of DnaA enhances the over-initiation and cell division phenotypes of the Δnoc mutant. Thus, a single factor both blocks cell division over chromosomes and helps to ensure that new rounds of DNA replication are not initiated prematurely. This degree of economy in coordinating key cell biological processes has not been observed in rod-shaped bacteria and may reflect the challenges posed by the reduced cell volume and complicated division pattern of this spherical pathogen.

Abstract 3459: BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells

Abstract BRCA1 deficiency alters the relative proportions of progenitor cells in preneoplastic mammary tissue, and typically associates with breast cancers characterized by genomic instability and a basal-like cell phenotype. Oriented division of progenitor cells is one mechanism these cells use to maintain tissue homeostasis, and to suppress tumor formation. We now show that shRNA-mediated reduction of BRCA1 levels in non-tumorigenic and immortalized or freshly isolated, normal human mammary cells alters their plane of division with graded consequences that include the induction of aneuploidy in progeny cells, perturbation of polarity in spheroid cultures, and inhibition of clonal growth with favored expression of basal features. We also demonstrate a requirement for BRCA1 in establishing cortical asymmetry of NUMA-dynein complexes. Mutation of a single BRCA1 allele (BRCA1 185delAG/+) altered the division axis of isolated cells but their deficient spindle positioning was supervised by CDH1-positive adherens, which sustained oriented divisions and produced colonies with luminal features. These findings reveal a previously unrecognized consequence of mutant BRCA1 on the cell division axis, post-mitotic integrity and phenotype control in normal human mammary epithelial cells. Note: This abstract was not presented at the meeting. Citation Format: Zhengcheng He, Oksana Nemirovsky, Nagarajan Kannan, Connie Eaves, Christopher A. Maxwell. BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary 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 3459. doi:10.1158/1538-7445.AM2017-3459

Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division.

Agrobacterium tumefaciens is a rod-shaped Gram-negative bacterium that elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a nongrowing old pole, and the division site creates new growth poles in sibling cells. The A. tumefaciens homolog of the Caulobacter crescentus polar organizing protein PopZ localizes specifically to growth poles. In contrast, the A. tumefaciens homolog of the C. crescentus polar organelle development protein PodJ localizes to the old pole early in the cell cycle and accumulates at the growth pole as the cell cycle proceeds. FtsA and FtsZ also localize to the growth pole for most of the cell cycle prior to Z-ring formation. To further characterize the function of polar localizing proteins, we created a deletion of A. tumefaciens podJ (podJAt). ΔpodJAt cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAt cells, A. tumefaciens PopZ-green fluorescent protein (PopZAt-GFP) persists at nontransitioning growth poles postdivision and also localizes to ectopic growth poles, as expected for a growth-pole-specific factor. Even though GFP-PodJAt does not localize to the midcell in the wild type, deletion of podJAt impacts localization, stability, and function of Z-rings as assayed by localization of FtsA-GFP and FtsZ-GFP. Z-ring defects are further evidenced by minicell production. Together, these data indicate that PodJAt is a critical factor for polar growth and that ΔpodJAt cells display a cell division phenotype, likely because the growth pole cannot transition to an old pole. How rod-shaped prokaryotes develop and maintain shape is complicated by the fact that at least two distinct species-specific growth modes exist: uniform sidewall insertion of cell envelope material, characterized in model organisms such as Escherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, including Agrobacterium tumefaciens Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene, podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAt is a critical factor in normal polar growth and impacts cell division in A. tumefaciens.

Local 3D matrix confinement determines division axis through cell shape

How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Targeting mitosis-regulating genes in cisplatin-sensitive and -resistant melanoma cells: A live-cell RNAi screen displays differential nucleus-derived phenotypes.

Chemoresistance in malignant melanoma remains an unresolved clinical issue. In the search for novel molecular targets, a live-cell high-content RNAi screen based on gene expression data was performed in cisplatin-sensitive and cisplatin-resistant MeWo melanoma cells, Mel-28 cells and a melanocyte cell line. Cells were exposed to 91 siRNAs and distinct nucleus-derived phenotypes such as cell division, cell death and migration phenotypes were detected by time-lapse microscopy over 60 h. Using this approach, cisplatin-sensitive and cisplatin-resistant melanoma cells were compared by automated image analysis and visual inspection. In cisplatin-sensitive MeWo melanoma cells, 14 genes were identified that showed distinct phenotype abnormalities after exposure to gene-specific siRNAs. In cisplatin-resistant MeWo cells, five genes were detected. Nine genes were detected whose knock-down led to differential nuclear phenotypes in cisplatin-sensitive and -resistant cells. In Mel-28 cells, nine genes were identified which induced nuclear phenotypes including all eight genes which were identified in cisplatin-resistant MeWo cells. An analogous RNAi screen on melanocytes revealed no detectable phenotype abnormalities after RNAi. Pathway analysis showed in cisplatin-sensitive MeWo cells and Mel-28 cells an enrichment of at least three genes in major mitotic pathways. We hereby show that siRNA screening may help to identify tumor-specific genes leading to phenotype abnormalities. These genes may serve as potential therapeutic targets in the treatment of melanoma.

The special case of hepatocytes: unique tissue architecture calls for a distinct mode of cell division.

Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase RhoA.

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA–IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer.

Par1b links lumen polarity with LGN–NuMA positioning for distinct epithelial cell division phenotypes

Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal domains and their cleavage furrow rarely bifurcates the luminal domains. We determine that the serine/threonine kinase Par1b defines lumen position in concert with the position of the astral microtubule anchoring complex LGN-NuMA to yield the distinct epithelial division phenotypes. Par1b signaling via the extracellular matrix (ECM) in polarizing cells determined RhoA/Rho-kinase activity at cell-cell contact sites. Columnar MDCK and Par1b-depleted hepatocytic HepG2 cells featured high RhoA activity that correlated with robust LGN-NuMA recruitment to the metaphase cortex, spindle alignment with the substratum, and columnar organization. Reduced RhoA activity at the metaphase cortex in HepG2 cells and Par1b-overexpressing MDCK cells correlated with a single or no LGN-NuMA crescent, tilted spindles, and the development of lateral lumen polarity.

Investigating the Ustilago maydis/Zea mays pathosystem: Transcriptional responses and novel functional aspects of a fungal calcineurin regulatory B subunit

The sustainable control of basidiomycete biotrophic plant pathogenesis requires an understanding of host responses to infection, as well as the identification and functional analysis of fungal genes involved in disease development. The creation and analysis of a suppressive subtractive hybridization (SSH) cDNA library from Ustilago maydis-infected Zea mays seedlings enabled the identification of fungal and plant genes expressed during disease development, and uncovered new insights into the interactions of this model system. Candidate U. maydis pathogenesis genes were identified by using the current SSH cDNA library analysis, and by knowledge generated from previous cDNA microarray and comparative genomic analyses. These identifications were supported by the independent determination of transcript level changes in different cell-types and during pathogenic development. The basidiomycete specific um01632, the highly in planta expressed um03046 (zig1), and the calcineurin regulatory B subunit (um10226, cnb1), were chosen for deletion experiments. um01632 and zig1 mutants showed no difference in morphology and did not have a statistically significant impact on pathogenesis. cnb1 mutants had a distinct cell division phenotype and reduced virulence in seedling assays. Infections with reciprocal wild-type×Δcnb1 haploid strain crosses revealed that the wild-type allele was unable to fully compensate for the lack of a second cnb1 allele. This haploinsufficiency was undetected in other fungal cnb1 mutational analyses. The reported data improves U. maydis genome annotation and expands on the current understanding of pathogenesis genes in this model basidiomycete.