Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted de... more Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. We find that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. We demonstrate that, like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1-dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, we show that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. We conclude that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment.
Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approache... more Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.
In the early embryo of many species, comparatively small spindles are positioned near the cell ce... more In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.
Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechan... more Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fl... more Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL mol(-1) cm(-1) at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.
Although alpha4-tubulin comprises only about one-fifth of the alpha-tubulin pool in every Drosoph... more Although alpha4-tubulin comprises only about one-fifth of the alpha-tubulin pool in every Drosophila egg, in the absence of alpha4-tubulin - in eggs of the kavar(0)/- hemizygous females - only a tassel of short microtubules forms with two barely separated daughter centrosomes. We report that alpha4-tubulin is enriched in the long microtubules that embrace the nuclear envelope and suggest that they push apart daughter centrosomes along the nuclear perimeter during the initial cleavage divisions. In vitro tubulin polymerization showed that alpha4-tubulin is required for rapid tubulin polymerization. Since tubulin polymerization is slow inside eggs of the kavar(0)/- females, only short microtubules can form within the 4 to 5 minutes allowed for the process. A tassel of short microtubules with two barely separated centrosomes forms in every egg of the Kavar(18c)/+ females, in which the cytoplasm contains both wild-type and Kavar(18c)-encoded alpha4-tubulin with an E82K amino acid substitution (E82K-alpha4-tubulin). E82K-alpha4-tubulin is incorporated into the microtubules and renders them unstable. When injected into wild-type early cleavage embryos E82K-alpha4-tubulin slows down the formation of long microtubules and the separation of the daughter centrosomes. Surprisingly, when injected into late cleavage embryos E82K-alpha4-tubulin is non-toxic. Similarly, in the neuroblasts, ectopically expressed E82K-alpha4-tubulin becomes incorporated into the microtubules that grow sufficiently long and function normally.
Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg pri... more Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg primordia. Although similar phenomena have been reported for a number of cultured cell types and some neurons in the zebrafish embryo, the mechanism and importance of the process have remained unexplained. Wriggling involves successive sudden and random minor turns of the nuclei, approximately three twists per minute with roughly 12° per twist, one of which lasts typically for 14 seconds. Wriggling is generated by the growing microtubules seeded throughout the cell cortex, which, while poking the nuclei, buckle and exert 5-40 piconewtons over ∼16 seconds. While wriggling, the nuclei drift ∼5 µm in a day in the immensely growing follicle cells along the apical-basal axis from the apical to the basal cell region. A >2-fold excess of the microtubules nucleated in the apical cell region, as compared with those seeded in the basal cell cortex, makes the nuclei drift along the apical-basal axis. Nuclear wriggling and positioning appear to be tightly related processes: they cease simultaneously when the nuclei become anchored by the actin cytoskeleton; moreover, colchicine or taxol treatment eliminates both nuclear wriggling and positioning. We propose that the wriggling nuclei reveal a thus far undescribed nuclear positioning mechanism.
ABSTRACT Bei der Analyse der RNA-Dynamik in lebenden Zellen werden üblicherweise transgene Method... more ABSTRACT Bei der Analyse der RNA-Dynamik in lebenden Zellen werden üblicherweise transgene Methoden eingesetzt. Diese erfordern modifizierte RNAs und Zellen. Hingegen ermöglichen Hybridisierungs-Fluoreszenzsonden Untersuchungen an Wildtyp-Zellen. Wir haben Nuklease-resistente FIT(“DNA-forced intercalation”)-Sonden entwickelt, in denen hohe Anstiege der Fluoreszenz durch Hybridisierung mit einer großen Helligkeit einhergehen, sodass einzelne Ribonukleotidpartikel (RNP) verfolgt werden können. Hierbei dient ein einzelner Thiazolorange(TO)-Interkalationsfarbstoff als Nukleobasensurrogat während eine benachbarte LNA(“locked nucleic acid”)-Modifikation die Umgebung konformativ fixiert. Dies schließt Fluoreszenzabklingkanäle und erhöht so die Helligkeit von TO in Sonden-Ziel-Komplexen. Zwei FIT-Sonden reichen aus, um oskar-RNPs in lebenden Wildtyp-Oozyten von Drosophila melanogaster zu verfolgen.
Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted de... more Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. We find that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. We demonstrate that, like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1-dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, we show that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. We conclude that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment.
Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approache... more Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.
In the early embryo of many species, comparatively small spindles are positioned near the cell ce... more In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.
Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechan... more Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fl... more Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL mol(-1) cm(-1) at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.
Although alpha4-tubulin comprises only about one-fifth of the alpha-tubulin pool in every Drosoph... more Although alpha4-tubulin comprises only about one-fifth of the alpha-tubulin pool in every Drosophila egg, in the absence of alpha4-tubulin - in eggs of the kavar(0)/- hemizygous females - only a tassel of short microtubules forms with two barely separated daughter centrosomes. We report that alpha4-tubulin is enriched in the long microtubules that embrace the nuclear envelope and suggest that they push apart daughter centrosomes along the nuclear perimeter during the initial cleavage divisions. In vitro tubulin polymerization showed that alpha4-tubulin is required for rapid tubulin polymerization. Since tubulin polymerization is slow inside eggs of the kavar(0)/- females, only short microtubules can form within the 4 to 5 minutes allowed for the process. A tassel of short microtubules with two barely separated centrosomes forms in every egg of the Kavar(18c)/+ females, in which the cytoplasm contains both wild-type and Kavar(18c)-encoded alpha4-tubulin with an E82K amino acid substitution (E82K-alpha4-tubulin). E82K-alpha4-tubulin is incorporated into the microtubules and renders them unstable. When injected into wild-type early cleavage embryos E82K-alpha4-tubulin slows down the formation of long microtubules and the separation of the daughter centrosomes. Surprisingly, when injected into late cleavage embryos E82K-alpha4-tubulin is non-toxic. Similarly, in the neuroblasts, ectopically expressed E82K-alpha4-tubulin becomes incorporated into the microtubules that grow sufficiently long and function normally.
Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg pri... more Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg primordia. Although similar phenomena have been reported for a number of cultured cell types and some neurons in the zebrafish embryo, the mechanism and importance of the process have remained unexplained. Wriggling involves successive sudden and random minor turns of the nuclei, approximately three twists per minute with roughly 12° per twist, one of which lasts typically for 14 seconds. Wriggling is generated by the growing microtubules seeded throughout the cell cortex, which, while poking the nuclei, buckle and exert 5-40 piconewtons over ∼16 seconds. While wriggling, the nuclei drift ∼5 µm in a day in the immensely growing follicle cells along the apical-basal axis from the apical to the basal cell region. A >2-fold excess of the microtubules nucleated in the apical cell region, as compared with those seeded in the basal cell cortex, makes the nuclei drift along the apical-basal axis. Nuclear wriggling and positioning appear to be tightly related processes: they cease simultaneously when the nuclei become anchored by the actin cytoskeleton; moreover, colchicine or taxol treatment eliminates both nuclear wriggling and positioning. We propose that the wriggling nuclei reveal a thus far undescribed nuclear positioning mechanism.
ABSTRACT Bei der Analyse der RNA-Dynamik in lebenden Zellen werden üblicherweise transgene Method... more ABSTRACT Bei der Analyse der RNA-Dynamik in lebenden Zellen werden üblicherweise transgene Methoden eingesetzt. Diese erfordern modifizierte RNAs und Zellen. Hingegen ermöglichen Hybridisierungs-Fluoreszenzsonden Untersuchungen an Wildtyp-Zellen. Wir haben Nuklease-resistente FIT(“DNA-forced intercalation”)-Sonden entwickelt, in denen hohe Anstiege der Fluoreszenz durch Hybridisierung mit einer großen Helligkeit einhergehen, sodass einzelne Ribonukleotidpartikel (RNP) verfolgt werden können. Hierbei dient ein einzelner Thiazolorange(TO)-Interkalationsfarbstoff als Nukleobasensurrogat während eine benachbarte LNA(“locked nucleic acid”)-Modifikation die Umgebung konformativ fixiert. Dies schließt Fluoreszenzabklingkanäle und erhöht so die Helligkeit von TO in Sonden-Ziel-Komplexen. Zwei FIT-Sonden reichen aus, um oskar-RNPs in lebenden Wildtyp-Oozyten von Drosophila melanogaster zu verfolgen.
Uploads
Papers by Imre Gaspar