Abnormal Spindle Orientation Research Articles

Rassf7a promotes spinal cord regeneration and controls spindle orientation in neural progenitor cells.

Spinal cord injury (SCI) can cause long-lasting disability in mammals due to the lack of axonal regrowth together with the inability to reinitiate spinal neurogenesis at the injury site. Deciphering the mechanisms that regulate the proliferation and differentiation of neural progenitor cells is critical for understanding spinal neurogenesis after injury. Compared with mammals, zebrafish show a remarkable capability of spinal cord regeneration. Here, we show that Rassf7a, a member of the Ras-association domain family, promotes spinal cord regeneration after injury. Zebrafish larvae harboring a rassf7a mutation show spinal cord regeneration and spinal neurogenesis defects. Live imaging shows abnormal asymmetric neurogenic divisions and spindle orientation defects in mutant neural progenitor cells. In line with this, the expression of rassf7a is enriched in neural progenitor cells. Subcellular analysis shows that Rassf7a localizes to the centrosome and is essential for cell cycle progression. Our data indicate a role for Rassf7a in modulating spindle orientation and the proliferation of neural progenitor cells after spinal cord injury.

Abstract P4-08-01: Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy

Abstract Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Surprisingly, PLK2 is frequently deleted in human breast cancer, preferentially in triple negative breast cancer (TNBC). Pilot studies using an RNAi screen and colony formation assays suggested that PLK2 might act as a tumor suppressor in breast cancer. This paradox stimulated us to investigate the functions of PLK1 and PLK2 in preclinical models and to understand their clinical implications. In preliminary experiments, we found that knockdown of PLK1 mitigated PLK2-deletion induced phenotypes including inhibiting colony formation, and rescued Plk2 null mammary epithelial cell phenotypes in vivo, including gland hyperbranching, epithelial cell hyperproliferation, and abnormal spindle orientation. Furthermore, we demonstrated that PLK2 interacted with PLK1 using a bimolecular fluorescence complementation assay and by co-immunoprecipitation, and using a proximity ligation assay this interaction was shown to occur in prometaphase. Due to the lack of targeted therapy, chemotherapy, such as carboplatin usually in combination with Taxol, is still one of the only systematic treatments for TNBC. Although PLK2 is frequently deleted in TNBC, there is currently no viable strategy to target this loss of function. The relationship between PLK2 and PLK1 suggests targeting PLK1 might provide a potential target to treat TNBC patients with loss of PLK2. In order to test this hypothesis, we used p53 null mammary epithelial cells as a sensitized background to generate a cohort of p53/Plk2 double null TNBC tumors. RNA-seq analysis showed that these tumors can be classified into three distinct molecular TNBC sublines: luminal-like, basal-like and claudin-low. Combinational treatment with the PLK1 inhibitor Volasertib and carboplatin showed a strong synergistic effect on tumor growth and led to regression of the Plk2/p53 null claudin-low tumors, but not control Plk2+/p53 null claudin-low tumors. The effect of combinational treatment of PLK1 inhibitor and carboplatin on the other two sublines as well as PLK2-low TNBC patient-derived xenograft (PDX) models is currently under investigation. We hope that these preclinical experiments will provide a rationale for the application PLK1 inhibitors in combination with chemotherapy in a subset of TNBC patients with deletion of PLK2. [Supported by Susan G. Komen Foundation grant SAC110031] Citation Format: Yang Gao, Elena Kabotyanski, Deanna Acosta, Elizabeth Villegas, Thomas F Westbrook, Charles M Perou, Jeffrey M Rosen. Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-01.

Junctional tumor suppressors interact with 14-3-3 proteins to control planar spindle alignment.

Proper orientation of the mitotic spindle is essential for cell fate determination, tissue morphogenesis, and homeostasis. During epithelial proliferation, planar spindle alignment ensures the maintenance of polarized tissue architecture, and aberrant spindle orientation can disrupt epithelial integrity. Nevertheless, in vivo mechanisms that restrict the mitotic spindle to the plane of the epithelium remain poorly understood. Here we show that the junction-localized tumor suppressors Scribbled (Scrib) and Discs large (Dlg) control planar spindle orientation via Mud and 14-3-3 proteins in the Drosophila wing disc epithelium. During mitosis, Scrib is required for the junctional localization of Dlg, and both affect mitotic spindle movements. Using coimmunoprecipitation and mass spectrometry, we identify 14-3-3 proteins as Dlg-interacting partners and further report that loss of 14-3-3s causes both abnormal spindle orientation and disruption of epithelial architecture as a consequence of basal cell delamination and apoptosis. Combined, these biochemical and genetic analyses indicate that 14-3-3s function together with Scrib, Dlg, and Mud during planar cell division.

Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Abstract A23: The dual roles of microRNA-200: From inclusion cyst formation to cell migration.

Abstract Background: Historically, metaplasia of ovarian surface epithelial (OSE) cells in the cortical inclusion cysts is associated with ovarian cancer development. Recent studies have also suggested fallopian tube epithelium as another origin of ovarian cancer cells. MicroRNA-200 (miR200) family is related to epithelial-mesenchymal transition (EMT) and overexpressed in ovarian cancer. However, the potential involvement of miR200 in ovarian pathogenesis is largely unknown. Objective: To study any morphological and signaling differences between immortalized normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells with changes in miR200 expression and control cells growing in three-dimensional (3D) cultures. Methods: OSE cells with stable ectopic expression of miR200 and ovarian cancer cells with stable miR200 knockdown were established using lentiviral agents. The resulting cells and control cells were grown as 3D cultures first in Matrigel to form the cyst-like spheroids and then transferred to Collagen I matrix for cell migration study. Immunofluorescence and confocal microscopy was applied to study the morphologies of the 3D structures, and in conjunction with Western blot analysis, to study changes in signaling pathways in response to changes of miR200 expression and in the presence of a variety of pharmacologic inhibitors. Clinical samples were examined for miR200 expression to evaluate the results from 3D in vitro cultures. Results: In comparison with the wild-type control counterparts, OSE cells with ectopic expression of miR200 had elevated E-cadherin expression, showed more compact and prolonged cyst stabilization in both Matrigel and Collagen I 3D cultures; whereas the ovarian cancer cells with miR200 knockdown showed increased TGFβ expression and multiple lumens with enhanced cell death in Matrigel culture, and change from collective migration mode to single cells in Collagen I culture. Both acetylated tubulin and Histone H3 staining of mitotic cells in Matrigel culture demonstrated ovarian cancer cells with miR200 knockdown had abnormal spindle orientation and defective cell cycle division. Application of TGFβ inhibitors reversed the single cell migration phenotype of miR200 knockdown cancer cells to collective movement. However, the downstream pathways responsible for the switching of migration phenotype were not the same between the serous and mucinous cell lines. For the mucinous cell line, miR200 controlled the switching through TGFβ-ROCK pathway, whereas the downstream pathway for the switching in the serous cell line remained unknown. qRT-PCR analysis of micro-dissected cells from clinical samples verified the elevated expression of miR200 in endosalpingiosis cysts and tumor tissues when compared with normal ovarian surface epithelia and fallopian tube epithelia. Conclusion: The qRT-PCR validation study verifies the findings of our 3D in vitro culture study and demonstrates that miR200 is important for inclusion cyst formation and collective movement of ovarian cancer cells during tumor invasion. Perturbation of this pathway in cancer cells will accelerate cell death through abnormal cell division. Hence, miR200 may be an interesting target in ovarian cancer therapy. Citation Format: Pui Wah Choi, Junzheng Yang, Wing Ping Fong, Ross S. Berkowitz, William R. Welch, Gregory J. Goodall, Shu Wing Ng. The dual roles of microRNA-200: From inclusion cyst formation to cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A23.

Cytological, molecular mechanisms and temperature stress regulating production of diploid male gametes in Dianthus caryophyllus L.

In plant evolution, because of its key role in sexual polyploidization or whole genome duplication events, diploid gamete formation is considered as an important component in diversification and speciation. Environmental stress often triggers unreduced gamete production. However, the molecular, cellular mechanisms and adverse temperature regulating diplogamete production in carnation remain poorly understood. Here, we investigate the cytological basis for 2n male gamete formation and describe the isolation and characterization of the first gene, DcPS1 (Dianthus Caryophyllus Parallel Spindle 1). In addition, we analyze influence of temperature stress on diploid gamete formation and transcript levels of DcPS1. Cytological evidence indicated that 2n male gamete formation is attributable to abnormal spindle orientation at male meiosis II. DcPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. DcPS1 expression analysis show DcPS1 gene probably have a role in 2n pollen formation. Unreduced pollen formation in various cultivation was sensitive to high or low temperature which was probably regulated by the level of DcPS1 transcripts. In a broader perspective, these findings can have potential applications in fundamental polyploidization research and plant breeding programs.

Pollen viability and meiotic behaviour in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group x subsp. sargentiana

AbstractPollen viability and male meiosis in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group (2n = 2x = 36) and subsp. sargentiana (2n = 2x = 34) were investigated. Although it had been assumed that they were sterile, pollen viability was observed; it varied from 2.5% to 12.1%. The production of gametes with different chromosome numbers was implied by the analysis of the dispersion of the diameter distribution of pollen grains. Analysis of male meiosis made it possible to identify the origins with two major categories of meiotic aberrations: abnormal chromosome distribution (early chromosome migration at metaphase, lagging chromosomes at anaphase, micronuclei at telophase), leading to the formation of unbalanced tetrads and/or ones with supernumerary microspores; and abnormal spindle orientation in metaphase II (tripolar, fused and parallel spindles), leading to the formation of dyads or triads. The mode of 2n pollen formation is of the First Division Restitution type. The high level of parental heterozygosity that is normally associated with them should facilitate the transfer of a polygenic trait in breeding programme.

Occurrence and cytological mechanism of numerically unreduced pollen in diploid Populus euphratica

AbstractUnreduced gametes are the driving force for the polyploidizaiton of plants in nature, and are also an important tool for breeding of triploid individuals. The final heterozygosity of a 2n pollen grain depends on the cytological mechanism behind 2n pollen formation. In this study, meiotic abnormalities were analysed using fluorescent chromosome staining and indirect immunofluorescence during the microsporogenesis of 18 genotypes of diploid P. euphratica Oliv. (2n = 2x = 38). Among the 18 genotypes, 16 genotypes produce 2n pollen and two genotypes produce only normal n pollen. In all 2n pollen producers, we found that the first meiotic division was normal but that the second division was characterized by frequent abnormal spindle orientation (parallel, tripolar, and fused spindles) and premature cytokinesis. The parallel, fused spindles and premature cytokinesis were considered to be leading dyad formation, and tripolar spindles seemed to be causing triad formation at the tetrad stage. There was a higher frequency of parallel spindles than other spindle forms, but no significant correlations between parallel spindles and dyads were observed. However, a significant association (r = 0.68, P < 0.05) between the tripolar spindles and dyads was found. In some Microspore mother cells (MMCs), an indirect immunofluorescence examination of meiosis II revealed that the parallel spindles led to the gathering of one or two non-sister groups of chromosomes, causing an incorporation of RMSs from two daughter nuclei. Therefore, the incorporated RMSs established two nuclear cytoplasmic domains for the control of division plane, resulting in either triad or dyad formation.

Syntaxin 16 is required for epithelial morphogenesis and single lumen formation

Formation and maintenance of cell‐cell junctions during developmental, physiological, and pathological conditions requires targeting and trafficking of junctional proteins. Syntaxin (Stx) families of proteins are localized at different subcellular membranes and are involved in intracellular trafficking of junctional proteins by regulating vesicle fusion events. Using MDCK II cell line we show that among various endosome‐Golgi localized Stxs, intracellular levels of Stx16 increases progressively as sparsely placed cells were allowed to attain confluency suggesting Stx16 may play a role in the maintenance of cell‐cell contact. Stx16 depleted stable MDCK II cell line when allowed to form confluent monolayer fail to maintain E‐cadherin at the cell‐cell junction. Stx16 depleted confluent monolayer consistently showed lower transepithelial resistance (TER) relative to control monolayer. As expected when these epithelial cells were cultured in matrigel they formed cyst with a hollow single lumen, cysts formed with stx16‐depleted cells show abnormal spindle orientation that lead to multiple lumen formation. Knockdown of zebrafish syntaxin 16 severely affects kidney morphogenesis and lumen formation in morphants. Taken together our in vitro and in vivo studies established role for syntaxin 16 in the maintenance of cell junction integrity and epithelial lumen morphogenesis.Grant Funding Source: N/A

Cytological mechanism of 2n pollen formation in Chinese jujube (Ziziphus jujuba Mill. ‘Linglingzao’)

In order to elucidate the cytological mechanism of 2n pollen formation in Chinese jujube, a cultivar named ‘Linglingzao’ (2n = 2x = 24) which produces relative more 2n pollens naturally was employed for microsporogenesis analysis. Chromosomes paired in 12 bivalents at diakinesis and the first meiotic division was normal, whereas, the second division was characterized by frequent abnormal spindle orientation (parallel and tripolar spindles). Perpendicular, tripolar, and parallel spindles at metaphase II accounted for 72.93, 17.22, and 9.85% respectively. Perpendicular and tripolar spindles led to tetrads and triads formation respectively. Two types of parallel spindles were observed and a significant association correlation (r = 0.84, P < 0.05) between parallel spindle II (fused spindles) and dyads was found which meant only fused spindles can form dyads, while parallel spindle I with a long distance between two sets of chromosomes seemed to lead tetrad formation. At tetrad stage, the observed frequencies of tetrads, triads and dyads were 80.99, 16.40, and 2.61% respectively. By the rule that each tetrad can form four n pollens, each triad can form one 2n pollen and two n pollens, each dyad can form two 2n pollens, the frequency of 2n pollen estimated was 5.71%. Based on the pollen diameter difference between n and 2n pollens, frequency of 2n pollen observed was 6.15%. No significant difference was observed between frequency of 2n pollen estimated and that of observed. The results indicated that, tripolar spindles and fused spindles at metaphase II followed by more triads and few dyads formation at tetrad stage were responsible for the production of 2n pollen in Chinese jujube. 2n gametes observed correspond to first division restitution. Research on the cytological mechanism of unreduced pollen will provide a platform for unreduced pollen induction and polyploidy breeding in Chinese jujube.

Meiotic behavior as a selection tool in silage corn breeding

In breeding programs, commercial hybrids are frequently used as a source of inbred lines to obtain new hybrids. Considering that maize production is dependent on viable gametes, the selection of populations to obtain inbred lines with high meiotic stability could contribute to the formation of new silage corn hybrids adapted to specific region. We evaluated the meiotic stability of five commercial hybrids of silage corn used in southern Brazil with conventional squashing methods. All of them showed meiotic abnormalities. Some abnormalities, such as abnormal chromosome segregation and absence of cytokinesis, occurred in all the genotypes, while others, including cytomixis and abnormal spindle orientation, were found only in some genotypes. The hybrid SG6010 had the lowest mean frequency of abnormal cells (21.27%); the highest frequency was found in the hybrid P30K64 (44.43%). However, the frequency of abnormal meiotic products was much lower in most genotypes, ranging from 7.63% in the hybrid CD304 to 43.86% in Garra. Taking into account the percentage of abnormal meiotic products and, hence, meiotic stability, only the hybrids CD304, P30K64, SG6010, and P30F53 are recommended to be retained in the breeding program to obtain inbred lines to create new hybrids.

Cytological characteristics of numerically unreduced pollen production in Populus tomentosa Carr.

Natural triploids of Populus tomentosa (2n = 3x = 57) are presumably the result of sexual polyploidization through the union of normal n female gametes and numerically unreduced (2n) male gametes. In our microscopic study of microspore mother cells (MMCs) of diploid P. tomentosa (2n = 2x = 38), we observed that the first meiotic division was normal but that the second division was characterized by frequent abnormal spindle orientation (parallel, tripolar, and fused spindles) and premature cytokinesis. The parallel, fused spindles and premature cytokinesis were considered to be leading dyad formation, and tripolar spindles seemed to be leading triad formation at the tetrad stage. There was a higher frequency of parallel spindles than other spindle forms, but there were no significant correlations between parallel spindles and dyads. An indirect immunofluorescence examination of meiosis II revealed that four tetragonally arranged nuclei were formed in MMCs with parallel spindles and that there were radial microtubules systems (RMSs) among these four nuclei, leading to the tetragonal tetrad. In some MMCs, however, the parallel spindles led to the gathering of one or two non-sister groups of chromosomes, causing an incorporation of RMSs from two daughter nuclei. Thus, the incorporated RMSs established three or two nuclear cytoplasmic domains for the control of division plane, resulting in either triad or dyad formation. These results provide new insights on the mechanism of parallel spindles leading to numerically unreduced pollen formation and on the selection and utilization of this type of pollen in polyploid breeding of P. tomentosa.

Spindle misorientation in tumors from APCmin/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the majority of colon cancers, and its mutation may initiate cancer by multiple mechanisms. Recently, abnormal mitotic spindle orientation was shown in normal-appearing tissues from mice heterozygous for APC mutation. To determine the effect of APC mutation on spindle orientation in tumors, and to better understand its mechanism, we measured mitotic spindle orientation in intestinal tumors from APC mutant mice, with three-dimensionally reconstructed confocal stacks of microtubule immunofluorescence images. We found spindle angles were increased in crypts heterozygous for the APC(min) mutation, and further increased in tumors. Astral microtubules of these spindles were clearly evident, suggesting astral microtubule loss is not a major mechanism of spindle misorientation in intestinal cells lacking wild-type APC. beta-Catenin staining was markedly abnormal in crypts and tumors from the mutant mice, suggesting a possible role in spindle orientation. Spindle angles in colon tumors with wild-type APC were equivalent to those in wild-type mice, showing that spindle misorientation is specific to APC mutation and not a general feature of tumors. We suggest spindle misorientation may contribute to the loss of normal tissue organization during tumor formation and could offer new insights into early carcinogenic events.

Mutations in AtPS1 (Arabidopsis thaliana parallel spindle 1) lead to the production of diploid pollen grains.

Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most—if not all—angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes) in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1), implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parent's heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.

Abnormal spindle orientation during microsporogenesis in an interspecific Brachiaria (Gramineae) hybrid

This paper reports a case of abnormal spindle orientation during microsporogenesis in an interspecific hybrid of the tropical grass Brachiaria. In the affected plant, prophase I was normal. In metaphase I, bivalents were regularly co-oriented but distantly positioned and spread over the equatorial plate. In anaphase I, chromosomes failed to converge into focused poles due to parallel spindle fibers. As a consequence, in telophase I, an elongated nucleus or several micronuclei were observed in each pole. In the second division, the behavior was the same, leading to polyads with several micronuclei. A total of 40% of meiotic products were affected. The use of this hybrid in production systems needing good-quality seeds is discussed.

Mitotic impairment by doublecortin is diminished by doublecortin mutations found in patients.

Mutations in doublecortin ( DCX) affect the migration of neuronal precursor cells and cause subcortical band heterotopia and lissencephaly. DCX is known to bind and bundle microtubules; however, the impact of mutation on DCX function and its relation to the manifestation of DCX-associated disorders is still unclear. We analyzed the impact of DCX mutants on COS7 cell microtubule networks. We found that both mutant and wild type DCX are able to bind and bundle microtubules; however, mutants possess a decreased ability to perturb the mitotic machinery, to cause abnormal spindle orientation, and to impair mitotic progression. The magnitude of this decrease is proportional to the severity of the mutation-associated clinical symptoms, thereby providing a cell-based assay for the prognosis of DCX-associated neuronal migration disorders.

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division.

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical–basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ13F to the membrane, indicating an essential role of cortical Gγ1–Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins–Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka–atypical PKC–Par6–Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable–Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.

Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.

The establishment and maintenance of polarity is of fundamental importance for the function of epithelial and neuronal cells. In Drosophila, the multi-PDZ domain protein Bazooka (Baz) is required for establishment of apico-basal polarity in epithelia and in neuroblasts, the stem cells of the central nervous system. In the latter, Baz anchors Inscuteable in the apical cytocortex, which is essential for asymmetric localization of cell fate determinants and for proper orientation of the mitotic spindle. Here we show that Baz directly binds to the Drosophila atypical isoform of protein kinase C and that both proteins are mutually dependent on each other for correct apical localization. Loss-of-function mutants of the Drosophila atypical isoform of PKC show loss of apico-basal polarity, multilayering of epithelia, mislocalization of Inscuteable and abnormal spindle orientation in neuroblasts. Together, these data provide strong evidence for the existence of an evolutionary conserved mechanism that controls apico-basal polarity in epithelia and neuronal stem cells. This study is the first functional analysis of an atypical protein kinase C isoform using a loss-of-function allele in a genetically tractable organism.

Meiotic mutants of Medicago sativa show altered levels of α- and β-tubulin

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).