TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling.

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

Tctp, a unique Ing5-binding partner, inhibits the chromatin binding of Enok in Drosophila.

The MOZ/MORF histone acetyltransferase complex is highly conserved in eukaryotes and controls transcription, development, and tumorigenesis. However, little is known about how its chromatin localization is regulated. Inhibitor of growth 5 (ING5) tumor suppressor is a subunit of the MOZ/MORF complex. Nevertheless, the in vivo function of ING5 remains unclear. Here, we report an antagonistic interaction between Drosophila Translationally controlled tumor protein (TCTP) (Tctp) and ING5 (Ing5) required for chromatin localization of the MOZ/MORF (Enok) complex and H3K23 acetylation. Yeast two-hybrid screening using Tctp identified Ing5 as a unique binding partner. In vivo, Ing5 controlled differentiation and down-regulated epidermal growth factor receptor signaling, whereas it is required in the Yorkie (Yki) pathway to determine organ size. Ing5 and Enok mutants promoted tumor-like tissue overgrowth when combined with uncontrolled Yki activity. Tctp depletion rescued the abnormal phenotypes of the Ing5 mutation and increased the nuclear translocation of Ing5 and chromatin binding of Enok. Nonfunctional Enok promoted the nuclear translocation of Ing5 by reducing Tctp, indicating a feedback mechanism between Tctp, Ing5, and Enok to regulate histone acetylation. Therefore, Tctp is essential for H3K23 acetylation by controlling the nuclear translocation of Ing5 and chromatin localization of Enok, providing insights into the roles of human TCTP and ING5-MOZ/MORF in tumorigenesis.

Ultrathin Serpentine Insulation Layer Architecture for Ultralow Power Gas Sensor.

Toxic gases have surreptitiously influenced the health and environment of contemporary society with their odorless/colorless characteristics. As a result, a pressing need for reliable and portable gas-sensing devices has continuously increased. However, with their negligence to efficiently microstructure their bulky supportive layer on which the sensing and heating materials are located, previous semiconductor metal-oxide gas sensors have been unable to fully enhance their power efficiency, a critical factor in power-stringent portable devices. Herein, an ultrathin insulation layer with a unique serpentine architecture is proposed for the development of a power-efficient gas sensor, consuming only 2.3 mW with an operating temperature of 300 °C (≈6% of the leading commercial product). Utilizing a mechanically robust serpentine design, this work presents a fully suspended standalone device with a supportive layer thickness of only ≈50 nm. The developed gas sensor shows excellent mechanical durability, operating over 10 000 on/off cycles and ≈2 years of life expectancy under continuous operation. The gas sensor detected carbon monoxide concentrations from 30 to 1 ppm with an average response time of ≈15 s and distinguishable sensitivity to 1 ppm (ΔR/R0 = 5%). The mass-producible fabrication and heating efficiency presented here provide an exemplary platform for diverse power-efficient-related devices.

Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development.

Regulation of microtubule stability is crucial for the maintenance of cell structure and function. While the acetylation of α-tubulin lysine 40 by acetylase has been implicated in the regulation of microtubule stability, the in vivo functions of N-terminal acetyltransferases (NATs) involved in the acetylation of N-terminal amino acids are not well known. Here, we identify an N-terminal acetyltransferase, Mnat9, that regulates cell signaling and microtubule stability in Drosophila Loss of Mnat9 causes severe developmental defects in multiple tissues. In the wing imaginal disc, Mnat9 RNAi leads to the ectopic activation of c-Jun N-terminal kinase (JNK) signaling and apoptotic cell death. These defects are suppressed by reducing the level of JNK signaling. Overexpression of Mnat9 can also inhibit JNK signaling. Mnat9 colocalizes with mitotic spindles, and its loss results in various spindle defects during mitosis in the syncytial embryo. Furthermore, overexpression of Mnat9 enhances microtubule stability. Mnat9 is physically associated with microtubules and shows a catalytic activity in acetylating N-terminal peptides of α- and ß-tubulin in vitro. Cell death and tissue loss in Mnat9-depleted wing discs are restored by reducing the severing protein Spastin, suggesting that Mnat9 protects microtubules from its severing activity. Remarkably, Mnat9 mutated in the acetyl-CoA binding site is as functional as its wild-type form. We also find that human NAT9 can rescue Mnat9 RNAi phenotypes in flies, indicating their functional conservation. Taken together, we propose that Mnat9 is required for microtubule stability and regulation of JNK signaling to promote cell survival in developing Drosophila organs.

Regulation of epithelial integrity and organ growth by Tctp and Coracle in Drosophila.

Regulation of cell junctions is crucial for the integrity of epithelial tissues and organs. Cell junctions also play roles in controlling cell proliferation for organ growth. Translationally controlled tumor protein (TCTP) is a conserved protein involved in growth control, but its role in cell junctions is unknown. Here we show that Drosophila Tctp directly interacts with the septate junction protein Coracle (Cora) to regulate epithelial integrity and organ growth. Tctp localizes together with Cora in the epidermis of the embryo. Loss of Cora reduces the level of Tctp in the epidermis but not vice versa. cora/+ or Tctp/+ single heterozygotes develop normally to adulthood. However, double heterozygotes for cora and Tctp mutations show severe disruption of epithelia causing synthetic lethality in the embryo. Double knockdown of Cora and Tctp in eye imaginal disc synergistically leads to disruption of the eye disc, resulting in a severe reduction or loss of eye and head. Conversely, double knockdown of Cora and Tctp in wing disc causes overgrowth as well as cell death. Inhibition of cell death under this condition causes hyperplastic growth of the wing disc. Tctp also shows direct and functional interaction with Cora-associated factors like Yurt and Na+/K+-ATPase. This study suggests that proper levels of Tctp and Cora are essential for the maintenance of the Cora complex and the integrity of epithelia. Our data also provide evidence that both Cora and Tctp are required to suppress overgrowth in developing wing.

Crumbs, Galla and Xpd are required for Kinesin-5 regulation in mitosis and organ growth in Drosophila.

Xeroderma Pigmentosum D (XPD, also known as ERCC2) is a multi-functional protein involved in transcription, DNA repair and chromosome segregation. In Drosophila, Xpd interacts with Crumbs (Crb) and Galla to regulate mitosis during embryogenesis. It is unknown how these proteins are linked to mitosis. Here, we show that Crb, Galla-2 and Xpd regulate nuclear division in the syncytial embryo by interacting with Klp61F, the Drosophila mitotic Kinesin-5 associated with bipolar spindles. Crb, Galla-2 and Xpd physically interact with Klp61F and colocalize to mitotic spindles. Knockdown of any of these proteins results in similar mitotic defects. These phenotypes are restored by overexpression of Klp61F, suggesting that Klp61F is a major effector. Mitotic defects of galla-2 RNAi are suppressed by Xpd overexpression but not vice versa. Depletion of Crb, Galla-2 or Xpd results in a reduction of Klp61F levels. Reducing proteasome function restores Klp61F levels and suppresses mitotic defects caused by knockdown of Crb, Galla-2 or Xpd. Furthermore, eye growth is regulated by Xpd and Klp61F. Hence, we propose that Crb, Galla-2 and Xpd interact to maintain the level of Klp61F during mitosis and organ growth.

Solution process manufacture of a simple, multifunctional flexible sensor based on capacitance measurement.

Conventional sensors are rigid, involve complex processes and structures, and one sensor can detect only one type of stimulus. The manufacturing costs of such devices are high owing to the use of vacuum processes for the formation of thin films and electrodes and the complicated fabrication processes required to construct multiple layers. In addition, the multiple-layer design increases the risk of peeling due to mechanical movement. In this study, to solve the aforementioned problems, a simple two-layer multi-sensor has been fabricated using a non-vacuum solution process. The sensor consists of a light absorption layer comprising polyvinyl butyral and semiconductor particles and a top layer comprising two spiral-shaped Ag nanowire electrodes. The sensor experiences minimal damage by external adhesives and has a light-sensitive optical response at 420 nm and at 1.2 mW cm-2. Herein, the capacitance of the sensor applied to the two-electrode structure was determined, along with the light sensitivity and change in noise with frequency. We believe that the proposed multi-sensor can be applied in a wide range of fields because it can act as a touch sensor and light sensor.

Realization of Nanolene: A Planar Array of Perfectly Aligned, Air-Suspended Nanowires.

Air suspension and alignment are fundamental requirements to make the best use of nanowires' unique properties; however, satisfying both requirements is very challenging due to the mechanical instability of air-suspended nanowires. Here, a perfectly aligned air-suspended nanowire array called "nanolene" is demonstrated, which has a high mechanical stability owing to a C-channel-shaped cross-section of the nanowires. The excellent mechanical stability is provided through geometrical modeling and finite element method simulations. The C-channel cross-section can be realized by top-down fabrication procedures, resulting in reliable demonstrations of the nanolenes with various materials and geometric parameters. The fabrication process provides large-area uniformity; therefore, nanolene can be considered as a 2D planar platform for 1D nanowire arrays. Thanks to the high mechanical stability of the proposed nanolene, perfectly aligned air-suspended nanowire arrays with an unprecedented length of 1 mm (aspect ratio ≈5100) are demonstrated. Since the nanolene can be used in an energy-efficient nanoheater, two energy-stringent sensors, namely, an air-flow sensor and a carbon monoxide gas sensor, are demonstrated. In particular, the gas sensor achieves sub-10 mW operations, which is a requirement for application in mobile phones. The proposed nanolene will pave the way to accelerate nanowire research and industrialization by providing reliable, high-performance nanowire devices.

Ebi modulates wing growth by ubiquitin-dependent downregulation of Crumbs in Drosophila.

Notch signaling at the dorsoventral (DV) boundary is essential for patterning and growth of wings in Drosophila The WD40 domain protein Ebi has been implicated in the regulation of Notch signaling at the DV boundary. Here we show that Ebi regulates wing growth by antagonizing the function of the transmembrane protein Crumbs (Crb). Ebi physically binds to the extracellular domain of Crb (Crbext), and this interaction is specifically mediated by WD40 repeats 7-8 of Ebi and a laminin G domain of Crbext Wing notching resulting from reduced levels of Ebi is suppressed by decreasing the Crb function. Consistent with this antagonistic genetic relationship, Ebi knockdown in the DV boundary elevates the Crb protein level. Furthermore, we show that Ebi is required for downregulation of Crb by ubiquitylation. Taken together, we propose that the interplay of Crb expression in the DV boundary and ubiquitin-dependent Crb downregulation by Ebi provides a mechanism for the maintenance of Notch signaling during wing development.

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila.

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/ß-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. Here, we show that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind ß-catenin and rescue klp64D RNAi phenotypes. Taken together, we propose that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm.

High-performance hybrid complementary logic inverter through monolithic integration of a MEMS switch and an oxide TFT.

A hybrid complementary logic inverter consisting of a microelectromechanical system switch as a promising alternative for the p-type oxide thin film transistor (TFT) and an n-type oxide TFT is presented for ultralow power integrated circuits. These heterogeneous microdevices are monolithically integrated. The resulting logic device shows a distinctive voltage transfer characteristic curve, very low static leakage, zero-short circuit current, and exceedingly high voltage gain.

Fast-Response and Low-Power Self-Heating Gas Sensor Using Metal/Metal Oxide/Metal (MMOM) Structured Nanowires.

With the escalating global awareness of air quality management, the need for continuous and reliable monitoring of toxic gases by using low-power operating systems has become increasingly important. One of which, semiconductor metal oxide gas sensors have received great attention due to their high/fast response and simple working mechanism. More specifically, self-heating metal oxide gas sensors, wherein direct thermal activation in the sensing material, have been sought for their low power-consuming characteristics. However, previous works have neglected to address the temperature distribution within the sensing material, resulting in inefficient gas response and prolonged response/recovery times, particularly due to the low-temperature regions. Here, we present a unique metal/metal oxide/metal (MMOM) nanowire architecture that conductively confines heat to the sensing material, achieving high uniformity in the temperature distribution. The proposed structure enables uniform thermal activation within the sensing material, allowing the sensor to efficiently react with the toxic gas. As a result, the proposed MMOM gas sensor showed significantly enhanced gas response (from 6.7 to 20.1% at 30 ppm), response time (from 195 to 17 s at 30 ppm), and limit of detection (∼1 ppm) when compared to those of conventional single-material structures upon exposure to carbon monoxide. Furthermore, the proposed work demonstrated low power consumption (2.36 mW) and high thermal durability (1500 on/off cycles), demonstrating its potential for practical applications in reliable and low-power operating gas sensor systems. These results propose a new paradigm for power-efficient and robust self-heating metal oxide gas sensors with potential implications for other fields requiring thermal engineering.

Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase.

Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. Here we show that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. Our studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease.

Ultrafast (∼0.6 s), Robust, and Highly Linear Hydrogen Detection up to 10% Using Fully Suspended Pure Pd Nanowire.

The high explosiveness of hydrogen gas in the air necessitates prompt detection in settings where hydrogen is used. For this reason, hydrogen sensors are required to offer rapid detection and possess superior sensing characteristics in terms of measurement range, linearity, selectivity, lifetime, and environment insensitivity according to the publicized protocol. However, previous approaches have only partially achieved the standardized requirements and have been limited in their capability to develop reliable materials for spatially accessible systems. Here, an electrical hydrogen sensor with an ultrafast response (∼0.6 s) satisfying all demands for hydrogen detection is demonstrated. Tailoring structural engineering based on the reaction kinetics of hydrogen and palladium, an optimized heating architecture that thermally activates fully suspended palladium (Pd) nanowires at a uniform temperature is designed. The developed Pd nanostructure, at a designated temperature distribution, rapidly reacts with hydrogen, enabling a hysteresis-free response from 0.1% to 10% and durable characteristics in mechanical shock and repetitive operation (>10,000 cycles). Moreover, the device selectively detects hydrogen without performance degradation in humid or carbon-based interfering gas circumstances. Finally, to verify spatial accessibility, the wireless hydrogen detection system has been demonstrated, detecting and reporting hydrogen leakage in real-time within just 1 s.

Opposing interactions between homothorax and Lobe define the ventral eye margin of Drosophila eye.

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.

Modulation of Hippo signaling by Mnat9 N-acetyltransferase for normal growth and tumorigenesis in Drosophila.

Hippo signaling is a conserved mechanism for controlling organ growth. Increasing evidence suggests that Hippo signaling is modulated by various cellular factors for normal development and tumorigenesis. Hence, identification of these factors is pivotal for understanding the mechanism for the regulation of Hippo signaling. Drosophila Mnat9 is a putative N-acetyltransferase that is required for cell survival by affecting JNK signaling. Here we show that Mnat9 is involved in the negative regulation of Hippo signaling. RNAi knockdown of Mnat9 in the eye disc suppresses the rough eye phenotype of overexpressing Crumbs (Crb), an upstream factor of the Hippo pathway. Conversely, Mnat9 RNAi enhances the eye phenotype caused by overexpressing Expanded (Ex) or Warts (Wts) that acts downstream to Crb. Similar genetic interactions between Mnat9 and Hippo pathway genes are found in the wing. The reduced wing phenotype of Mnat9 RNAi is suppressed by overexpression of Yorkie (Yki), while it is suppressed by knockdown of Hippo upstream factors like Ex, Merlin, or Kibra. Mnat9 co-immunoprecipitates with Mer, implying their function in a protein complex. Furthermore, Mnat9 overexpression together with Hpo knockdown causes tumorous overgrowth in the abdomen. Our data suggest that Mnat9 is required for organ growth and can induce tumorous growth by negatively regulating the Hippo signaling pathway.

Tctp regulates the level and localization of Foxo for cell growth in Drosophila.

Regulation of cell size is crucial for organ development. Insulin signaling regulates organ size by antagonizing the subgroup O of forkhead box transcription factor (Foxo) through 14-3-3 in Drosophila. However, mechanisms for controlling the level and the nuclear localization of Foxo in developing organs are not well understood. Here, we investigate the role of Drosophila Translationally controlled tumor protein (Tctp) and its interacting partner 14-3-3 in Foxo regulation during organ development. Foxo overexpression in the developing eye disc results in growth inhibition. We show that Tctp overexpression antagonizes the Foxo effect by downregulating the Foxo level in the eye disc. Foxo overexpression or knockdown of Tctp in the larval salivary gland results in reduced gland size, mainly due to reduced cell size by defects in endoreplication. Whereas 14-3-3ζ knockdown has a negligible effect, knockdown of 14-3-3ε mimics the effect of Foxo overexpression or Tctp knockdown, suggesting an isoform-specific role of 14-3-3. Unlike nuclear enrichment of the endogenous Foxo in the salivary gland, overexpressed Foxo protein is largely distributed in the cytoplasm, and this mislocalization is restored by Tctp overexpression. Opposite to the effect of Tctp overexpression, Tctp knockdown increases cytoplasmic Foxo levels while decreasing nuclear Foxo levels. Together, our data suggest that Tctp and 14-3-3ε play critical roles in cell growth by reducing cytoplasmic Foxo levels. Knockdown of human TCTP also elevates the level of cytoplasmic FOXO1 in HeLa cells, suggesting that human TCTP may have a conserved role in downregulating FOXO in human cells.

Wireless and Linear Hydrogen Detection up to 4% with High Sensitivity through Phase-Transition-Inhibited Pd Nanowires.

Palladium (Pd) has been drawing increasing attention as a hydrogen (H2) detecting material due to its highly selective sensitivity to H2. However, at H2 concentrations above 2%, Pd undergoes an inevitable phase transition, causing undesirable electrical and mechanical alterations. In particular, nonlinear gas response (ΔR/R0) that accompanies phase transition has been a great bottleneck for detecting H2 in high concentrations, which is especially important as there is a risk of explosion over 4% H2. Here, we propose a phase-transition-inhibited Pd nanowire H2 sensor that can detect up to 4% H2 with high linearity and high sensitivity. Based on the calculation of the change in free energy, we designed Pd nanowires that are highly adhered to the substrate to withstand the stress that leads to phase transition. We theoretically optimized the Pd nanowire dimensions using a finite element method simulation and then experimentally fabricated the proposed sensor by exploiting a developed nanofabrication method. The proposed sensor exhibits a high sensing linearity (98.9%) with high and stable sensitivity (ΔR/R0/[H2] = 875%·bar-1) over a full range of H2 concentrations (0.1-4%). Using the fabricated Pd sensors, we have successfully demonstrated a wireless sensor module that can detect H2 with high linearity, notifying real-time H2 leakage through remote communication. Overall, our work suggests a nanostructuring strategy for detecting H2 with a phase-transition-inhibited pure Pd H2 sensor with rigorous scientific exploration.

Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.

Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc.

Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple ex-vivo assay for identifying potential microtubule regulators in the wing imaginal disc of Drosophila melanogaster. This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, we designed an assay using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, we can test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in Drosophila. This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms. Graphic abstract: Graphical summary of Ex-vivo microtubule stability assay using Drosophila wing disc.