Cyclin A and B functions in the early Drosophila embryo

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.

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Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos.

The Journal of Cell Biology ◽

10.1083/jcb.116.4.967 ◽

1992 ◽

Vol 116 (4) ◽

pp. 967-976 ◽

Cited By ~ 33

Author(s):

G Maldonado-Codina ◽

D M Glover

Keyword(s):

Metaphase Plate ◽

Cyclin A ◽

Cyclin B ◽

Drosophila Embryo ◽

Polar Regions ◽

Surface Layers ◽

Nuclear Envelope Breakdown ◽

Apical Side ◽

Punctate Staining ◽

Drosophila Embryos

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.

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Quantitative Analysis of Gene Function in the Drosophila Embryo

Genetics ◽

10.1093/genetics/154.1.273 ◽

2000 ◽

Vol 154 (1) ◽

pp. 273-284

Author(s):

William D Tracey ◽

Xiangqun Ning ◽

Martin Klingler ◽

Sunita G Kramer ◽

J Peter Gergen

Keyword(s):

Gene Function ◽

Model Systems ◽

Drosophila Embryo ◽

Direct Role ◽

Developmental Context ◽

Maternal Mrna ◽

Early Drosophila Embryo ◽

Segment Polarity ◽

Pair Rule

Abstract The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

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Regulated nuclear import of the Drosophila rel protein dorsal: structure-function analysis.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.1103 ◽

1996 ◽

Vol 16 (3) ◽

pp. 1103-1114 ◽

Cited By ~ 22

Author(s):

S Govind ◽

E Drier ◽

L H Huang ◽

R Steward

Keyword(s):

Amino Acids ◽

Structure Function ◽

Nuclear Localization ◽

Nuclear Import ◽

Function Analysis ◽

Drosophila Embryo ◽

Nuclear Targeting ◽

Homology Region ◽

Early Drosophila Embryo

The formation of a gradient of nuclear Dorsal protein in the early Drosophila embryo is the last step in a maternally encoded dorsal-ventral signal transduction pathway. This gradient is formed in response to a ventral signal, which leads to the dissociation of cytoplasmic Dorsal from the I kappa B homolog Cactus. Free Dorsal is then targeted to the nucleus. Dorsal is a Rel-family transcription factor. Signal-dependent nuclear localization characterizes the regulation of Rel proteins. In order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus, we have performed an in vivo structure-function analysis. Our results show that all these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolished the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region.

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Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2977 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2977-2991 ◽

Cited By ~ 80

Author(s):

D R Kellogg ◽

C M Field ◽

B M Alberts

Keyword(s):

Affinity Chromatography ◽

Mitotic Spindle ◽

Polyclonal Antibodies ◽

Drosophila Embryo ◽

Microtubule Cytoskeleton ◽

Microtubule Associated Proteins ◽

Early Drosophila Embryo ◽

Individual Mouse ◽

Associated Proteins

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

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Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

10.1101/2022.01.07.475359 ◽

2022 ◽

Author(s):

Jaclyn M Camuglia ◽

Soline Chanet ◽

Adam C Martin

Keyword(s):

Planar Cell Polarity ◽

Adherens Junction ◽

Drosophila Embryo ◽

Spindle Orientation ◽

Planar Polarity ◽

Ventral Furrow ◽

Adherens Junction Protein ◽

Early Drosophila Embryo ◽

Junction Protein

Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

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Behaviour of microtubules and actin filaments in living Drosophila embryos

Development ◽

10.1242/dev.103.4.675 ◽

1988 ◽

Vol 103 (4) ◽

pp. 675-686 ◽

Cited By ~ 18

Author(s):

D.R. Kellogg ◽

T.J. Mitchison ◽

B.M. Alberts

Keyword(s):

Actin Filaments ◽

Drosophila Embryo ◽

Fixation Method ◽

Rabbit Muscle ◽

Specific Staining ◽

Fixation Techniques ◽

Filament Networks ◽

Drosophila Embryos

We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins appear to function normally in vitro and in vivo, and they allow continuous observation of the cytoskeleton in living embryos without perturbing development. By coinjecting labelled actin and tubulin into the early syncytial embryo, the spatial relationships between the distinct filament networks that they form can be followed second by second. The dynamic rearrangements of actin filaments and microtubules observed confirms and extends results obtained from previous studies, in which fixation techniques and specific staining were used to visualize the cytoskeleton in the Drosophila embryo. However, no tested fixation method produces an exact representation of the in vivo microtubule distribution.

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Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm

Development ◽

10.1242/dev.199997 ◽

2022 ◽

Author(s):

Jorge de-Carvalho ◽

Sham Tlili ◽

Lars Hufnagel ◽

Timothy E. Saunders ◽

Ivo A. Telley

Keyword(s):

Drosophila Embryo ◽

Cell Cortex ◽

Similar Distribution ◽

Axis Orientation ◽

Early Drosophila Embryo ◽

Repulsive Interactions ◽

In The Wild ◽

Division Axis ◽

Morphological Transformations

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei to the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, utilising quantification of nuclei position and division orientation together with embryo explants we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

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Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen

Development ◽

10.1242/dev.109.4.811 ◽

1990 ◽

Vol 109 (4) ◽

pp. 811-820 ◽

Cited By ~ 16

Author(s):

W. Driever ◽

V. Siegel ◽

C. Nusslein-Volhard

Keyword(s):

Drosophila Embryo ◽

Beta Globin ◽

P Transformation ◽

Early Drosophila Embryo ◽

Maternal Effect Genes ◽

Translational Block ◽

Maternal Gene ◽

Anterior Posterior

A small number of maternal effect genes determine anterior-posterior pattern in the Drosophila embryo. Embryos from females mutant for the maternal gene bicoid lack head and thorax. bcd mRNA becomes localized to the anterior tip of the egg during oogenesis and is the source for the morphogen gradient of bcd protein. Here we show that in vitro transcribed bicoid mRNA that has its own leader sequences substituted by the Xenopus beta-globin 5′ untranslated sequences is translated more efficiently than bicoid mRNA with the natural 5′ mRNA leader when tested in vitro and in Drosophila Schneider cells. When injected into bicoid mutant embryos, only the bcd mRNA with the beta-globin leader sequence, substituted for the natural leader, is able to induce anterior development. We used P-transformation to show that sequences in the 5′ leader are neither necessary for localization of the transcript nor for the translational block of the bcd mRNA during oogenesis. For our injection experiments, we used only one of the identified splicing forms of bcd mRNA. The bcd protein species derived from this mRNA is able to induce anterior development at any position along the anterior-posterior axis. Thus bicoid protein can induce development of head and thorax independent of any other specifically localized morphogenetic factor. Our findings further support the notion that the concentration gradient of bcd protein, and not the existence of different forms of bcd protein, is responsible for specifying subregions of the embryo.

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Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

Molecular Biology of the Cell ◽

10.1091/mbc.4.1.79 ◽

1993 ◽

Vol 4 (1) ◽

pp. 79-92 ◽

Cited By ~ 93

Author(s):

L Connell-Crowley ◽

M J Solomon ◽

N Wei ◽

J W Harper

Keyword(s):

Mammalian Cells ◽

Specific Activity ◽

Fold Increase ◽

Cyclin A ◽

Cyclin B ◽

Activation Process ◽

Cyclin Dependent Kinase ◽

Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

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