Arrest of intravitelline mitoses in Drosophila embryos by u.v. irradiation of the egg surface

Development ◽  
1984 ◽  
Vol 80 (1) ◽  
pp. 43-61
Author(s):  
Shin Togashi ◽  
Masukichi Okada

The intravitelline mitosis in Drosophila was arrested at the anaphase within the span of a single cell cycle after irradiation with 300 nm u.v. Embryos at and before the 8-nucleus stage were influenced by the u.v. only when irradiated anteriorly, while at and after the 16-nucleus stage, embryos are sensitive to either anterior or posterior irradiation. In embryos anteriorly irradiated at or before the 8-nucleus stage all nuclei in the embryo were prevented from performing mitosis. When irradiated at or after the 16-nucleus stage, inhibition of the intravitelline mitosis is limited to the nuclei in approximately anterior-half region of embryos in anterior irradiation, and to those inapproximately posterior-half region in posterior irradiation, resulting in incomplete blastoderm formation. Sites sensitive to 300 nm u.v. are postulated to be present in the peripheral cytoplasmic region of the embryo and not in the nucleus, because the half-attenuation thickness of 300 nm u.v. light for the Drosophila egg cytoplasm is 3 µm and nuclei are at least 50 µm away from the periphery at the stage of irradiation. In addition lateral irradiation of a portion of an egg where there is no nucleus underneath was also effective in arresting division of nuclei in the same egg. It is suggested that the effects of 300nm u.v. may not be conveyed to the nuclei from the periphery by simple diffusion of a substance, and a hypothesis is proposed for the involvement of cytoskeletal elements associated with the u.v. sensitive sites on the surface to the control mechanism of the intravitelline mitosis of the Drosophila embryo.

Author(s):  
William Theurkauf

Cell division in eucaryotes depends on coordinated changes in nuclear and cytoskeletal components. In Drosophila melanogaster embryos, the first 13 nuclear divisions occur without cytokinesis. During the final four divisions, nuclei divide in a uniform monolayer at the surface of the embryo. These surface divisions are accompanied by dramatic changes in cortical actin and microtubule structure (Karr and Alberts, 1986), and inhibitor studies indicate that these changes are essential to orderly mitosis (Zalokar and Erk, 1976). Because the early embryo is syncytial, fluorescent probes introduced by microinjection are incorporated in structures associated with all of the nuclei in the blastoderm. In addition, the nuclei divide synchronously every 10 to 20 min. These characteristics make the syncytial blastoderm embryo an excellent system for the analysis of mitotic reorganization of both nuclear and cytoskeletal elements. However, the Drosophila embryo is a large cell, and resolution of cytoskeletal filaments and nuclear structure is hampered by out-of focus signal.


mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Azuma Taoka ◽  
Ayako Kiyokawa ◽  
Chika Uesugi ◽  
Yousuke Kikuchi ◽  
Zachery Oestreicher ◽  
...  

ABSTRACT Magnetotactic bacteria are a unique group of bacteria that synthesize a magnetic organelle termed the magnetosome, which they use to assist with their magnetic navigation in a specific type of bacterial motility called magneto-aerotaxis. Cytoskeletal filaments consisting of the actin-like protein MamK are associated with the magnetosome chain. Previously, the function of MamK was thought to be in positioning magnetosome organelles; this was proposed based on observations via electron microscopy still images. Here, we conducted live-cell time-lapse fluorescence imaging analyses employing highly inclined and laminated optical sheet microscopy, and these methods enabled us to visualize detailed dynamic movement of magnetosomes in growing cells during the entire cell cycle with high-temporal resolution and a high signal/noise ratio. We found that the MamK cytoskeleton anchors magnetosomes through a mechanism that requires MamK-ATPase activity throughout the cell cycle to prevent simple diffusion of magnetosomes within the cell. We concluded that the static chain-like arrangement of the magnetosomes is required to precisely and consistently segregate the magnetosomes to daughter cells. Thus, the daughter cells inherit a functional magnetic sensor that mediates magneto-reception. IMPORTANCE Half a century ago, bacterial cells were considered a simple “bag of enzymes”; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles. IMPORTANCE Half a century ago, bacterial cells were considered a simple “bag of enzymes”; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles.


Nature ◽  
2021 ◽  
Vol 590 (7847) ◽  
pp. 649-654
Author(s):  
Diana Mahdessian ◽  
Anthony J. Cesnik ◽  
Christian Gnann ◽  
Frida Danielsson ◽  
Lovisa Stenström ◽  
...  
Keyword(s):  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karl Olofsson ◽  
Valentina Carannante ◽  
Madoka Takai ◽  
Björn Önfelt ◽  
Martin Wiklund

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.


Cell ◽  
2019 ◽  
Vol 177 (4) ◽  
pp. 925-941.e17 ◽  
Author(s):  
Victoria E. Deneke ◽  
Alberto Puliafito ◽  
Daniel Krueger ◽  
Avaneesh V. Narla ◽  
Alessandro De Simone ◽  
...  

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1245-1254 ◽  
Author(s):  
W. Sullivan ◽  
P. Fogarty ◽  
W. Theurkauf

Cytoplasmic organization, nuclear migration, and nuclear division in the early syncytial Drosophila embryo are all modulated by the cytoskeleton. In an attempt to identify genes involved in cytoskeletal functions, we have examined a collection of maternal-effect lethal mutations induced by single P-element transposition for those that cause defects in nuclear movement, organization, or morphology during the syncytial embryonic divisions. We describe three mutations, grapes, scrambled, and nuclear-fallout, which define three previously uncharacterized genes. Females homozygous for these mutations produce embryos that exhibit extensive mitotic division errors only after the nuclei migrate to the surface. Analysis of the microfilament and microtubule organization in embryos derived from these newly identified mutations reveal disruptions in the cortical cytoskeleton. Each of the three mutations disrupts the actin-based pseudocleavage furrows and the cellularization furrows in a distinct fashion. In addition to identifying new genes involved in cytoskeletal organization, these mutations provide insights into cytoskeletal function during early Drosophila embryogenesis.


Development ◽  
1996 ◽  
Vol 122 (1) ◽  
pp. 205-214 ◽  
Author(s):  
D.N. Arnosti ◽  
S. Barolo ◽  
M. Levine ◽  
S. Small

Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo. The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo, bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively. Here, we report the findings of extensive cis- and trans- complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos. This study reaches two primary conclusions. First, the strip 2 enhancer is inherently ‘sensitized’ for repression by gt. We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex. Second, we find that the synergistic activity of hb and bcd is ‘promiscuous’. For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer. This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.


Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Riham Abouleisa ◽  
Qinghui Ou ◽  
Xian-liang Tang ◽  
Mitesh Solanki ◽  
Yiru Guo ◽  
...  

Rationale: The regenerative capacity of the heart to repair itself after myocardial infarction (MI)is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 andCdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in vitro and in vivo andimproves cardiac function after MI. However, its clinical application is limited due to the concernsfor tumorigenic potential in other organs. Objectives: To first, identify on a single cell transcriptomic basis the necessary reprogrammingsteps that cardiomyocytes need to undertake to progress through the proliferation processfollowing 4F overexpression, and then, to determine the pre-clinical efficacy of transient andcardiomyocyte specific expression of 4F in improving cardiac function after MI in small and largeanimals. Methods and Results: Temporal bulk and single cell RNAseq of mature hiPS-CMs treated with4F or LacZ control for 24, 48, or 72 h revealed full cell cycle reprogramming in 15% of thecardiomyocyte population which was associated with sarcomere disassembly and metabolicreprogramming. Transient overexpression of 4F specifically in cardiomyocytes was achievedusing non-integrating lentivirus (NIL) driven by TNNT2 (TNNT2-4F-NIL). One week after inductionof ischemia-reperfusion injury in rats or pigs, TNNT2-4F-NIL or control virus was injectedintramyocardially. Compared with controls, rats or pigs treated with TNNT2-4F-NIL showed a 20-30% significant improvement in ejection fraction and scar size four weeks after treatment, asassessed by echocardiography and histological analysis. Quantification of cardiomyocyteproliferation in pigs using a novel cytokinesis reporter showed that ~10% of the cardiomyocyteswithin the injection site were labelled as daughter cells following injection with TNNT2-4F-NILcompared with ~0.5% background labelling in control groups. Conclusions: We provide the first understanding of the process of forced cardiomyocyteproliferation and advanced the clinical applicability of this approach through minimization ofoncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specificviral construct.


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