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GFP-Forked, a genetic reporter for studying <i>Drosophila</i> oocyte polarity
摘要： The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). In this study, we used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. We generated a null allele of the forked gene using the CRISPR-Cas9 system and found that forked is not required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, we demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal microtubule-organizing centers. Our results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.
关键词： ncMTOC,Oocyte,Drosophila,Polarity,Forked,Microtubules,CRISPR
更新于2025-09-23 15:22:29
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Guided by light: optogenetic control of microtubule gliding assays
摘要： Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.
关键词： motor proteins,optogenetics,optical control,Microtubules
更新于2025-09-23 15:21:01
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Reply to Comment on a??Photo-Controlled Reversible Microtubule Assembly Mediated by Paclitaxel-Modified Cyclodextrina??
摘要： In a Correspondence on our previous study “Photo-Controlled Reversible Microtubule Assembly Mediated by Paclitaxel-Modified Cyclodextrin” published in this journal in 2018,[1a] Thorn-Seshold comments on our results.[1b] First of all, we would like to appreciate his comments and interest in our work. The aggregation behavior of microtubules (MTs) in our work has been demonstrated from the viewpoint of macrocycle-based host–guest complexation at the supramolecular level and subsequently, the MT stabilizers based on azobenzene-modified paclitaxel (PTX) derivatives as photoswitchable small molecules have been investigated by Thorn-Seshold and co-workers in 2019.[2] In our case, the microscopy results showed that the MT morphology was dramatically affected by the photoisomeric complexation between cyclodextrin (CD) and arylazopyrazole (AAP). No fibrous assembly as free MT could be observed in the presence of free PTX-CD, PTX-AAP, or their inclusion complex in the cis/trans states. Therefore, the introduction of CD and AAP definitely influenced the self-assembling behavior between PTX and MT. Moreover, fluorescent-dye-staining assays demonstrated that the PTX-derived host and guest compounds still possessed MT-targeting ability to some extent, because MT could be co-labeled by FITC-tagged antibodies and adamantane-containing RhB. Thus, the microtubular aggregation was proposed as one of the possible assembling modes in Scheme 1 (cartoon presentation). The binding mode of MT with CD and AAP was directly deduced from our microscopy images and cellular confocal experiments. The biological effect in our work may be jointly attributed to both the PTX-dependent pathway (PTX-induced microtubular stabilization) and the PTX-independent pathway (complexation-induced multivalent supramolecular cross-linkage) at the nanometer scale.[3] Under these circumstances, one reasonable explanation is that the latter (independent) effect may become comparable to the former (dependent) one when the MT affinity is reduced by chemical modification at the 2’-OH position of PTX. Moreover, as a widely studied macrocyclic receptor in supramolecular chemistry, CD can form a diversity of supramolecular assemblies.[4] To determine the precise binding mode, in addition to the viewpoint of structural biology for evaluating the original PTX–MT interaction at the single-molecule level, many other factors and multiple supramolecular noncovalent interactions (e.g., self-inclusion, self-exclusion, amphiphilicity, extensive hydrogen bonding, and supramolecular multivalency/cooperativity) between PTX-CD and PTX-AAP should also be taken into account. For example, the multivalent inclusion complexation between multiple CD and PTX molecules may confer high stability to the nano-assembly.[5] Therefore, in our opinion, no binding mode can be exclusively confirmed at the present time until the hyperfine structures of such multicomponent CD–protein assemblies have been obtained both in solution and in the solid state (e.g., in a single crystal). Moreover, azobenzene/CD is one of the most frequently used host–guest pairs in adjusting the assembling/disassembling behavior of proteins and other biomacromolecules.[6] Meanwhile, AAP is a new type of azo compound, which possesses quite distinct photophysical behavior compared to conventional azobenzenes, such as enhanced photostability and photoconversion efficiency. The biological effect of pristine AAP on pure MT may deserve further attention, but this aspect was outside the scope of our previous study. Overall, based on NMR, TEM, UV/Vis transmittance, and confocal microscopy experiments, we clearly demonstrated in our previous study that 1) the MT self-assembling morphology can be strongly affected by the host–guest complexation between CD and AAP, and that 2) complexation-induced MT aggregation can be realized in a cellular environment. Thus, our work provides an alternative supramolecular chemistry method to modulate a biomacromolecular assembling process. Finally, we would like to thank Dr. Thorn-Seshold for his constructive suggestions and express our hope that we can improve the chemical simulation and gain further insight into the biological mechanism in further work.
关键词： molecular recognition,photo-responsiveness,cyclodextrin,supramolecular assembly,microtubules
更新于2025-09-23 15:19:57
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Reply to Comment on a??Photo-Controlled Reversible Microtubule Assembly Mediated by Paclitaxel-Modified Cyclodextrina??
摘要： In a Correspondence on our previous study ?Photo-Controlled Reversible Microtubule Assembly Mediated by Paclitaxel-Modified Cyclodextrin“ published in this journal in 2018,[1a] Thorn-Seshold comments on our results.[1b] First of all, we would like to appreciate his comments and interest in our work. The aggregation behavior of microtubules (MTs) in our work has been demonstrated from the viewpoint of macrocycle-based host–guest complexation at the supramolecular level and subsequently, the MT stabilizers based on azobenzene-modified paclitaxel (PTX) derivatives as photoswitchable small molecules have been investigated by Thorn-Seshold and co-workers in 2019.[2] In our case, the microscopy results showed that the MT morphology was dramatically affected by the photoisomeric complexation between cyclodextrin (CD) and arylazopyrazole (AAP). No fibrous assembly as free MT could be observed in the presence of free PTX-CD, PTX-AAP, or their inclusion complex in the cis/trans states. Therefore, the introduction of CD and AAP definitely influenced the self-assembling behavior between PTX and MT. Moreover, fluorescent-dye-staining assays demonstrated that the PTX-derived host and guest compounds still possessed MT-targeting ability to some extent, because MT could be co-labeled by FITC-tagged antibodies and adamantane-containing RhB. Thus, the microtubular aggregation was proposed as one of the possible assembling modes in Scheme 1 (cartoon presentation). The binding mode of MT with CD and AAP was directly deduced from our microscopy images and cellular confocal experiments. The biological effect in our work may be jointly attributed to both the PTX-dependent pathway (PTX-induced microtubular stabilization) and the PTX-independent pathway (complexation-induced multivalent supramolecular cross-linkage) at the nanometer scale.[3] Under these circumstances, one reasonable explanation is that the latter (independent) effect may become comparable to the former (dependent) one when the MT affinity is reduced by chemical modification at the 2’-OH position of PTX.
关键词： molecular recognition,photo-responsiveness,cyclodextrin,supramolecular assembly,microtubules
更新于2025-09-23 15:19:57
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Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy
摘要： Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.
关键词： super-resolution microscopy,live-cell imaging,multi-angle interference microscopy,mitochondria,microtubules
更新于2025-09-10 09:29:36
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[IEEE 2018 25th IEEE International Conference on Image Processing (ICIP) - Athens, Greece (2018.10.7-2018.10.10)] 2018 25th IEEE International Conference on Image Processing (ICIP) - Detection and Tracking of Astral Microtubules in Fluorescence Microscopy Images
摘要： In this paper we explore detection and tracking of astral micro-tubules, a sub-population of microtubules which only exists during and immediately before mitosis and aids in the spindle orientation by connecting it to the cell cortex. Its analysis can be useful to deter-mine the presence of certain diseases, such as brain pathologies and cancer. The proposed algorithm focuses on overcoming the prob-lems regarding fluorescence microscopy images and microtubule behaviour by using various image processing techniques and is then compared with three existing algorithms, tested on consistent sets of images.
关键词： image segmentation,Medical diagnostic imaging,kalman filter,fluorescence microscopy,microtubules
更新于2025-09-04 15:30:14