Time-lapse Live Imaging Research Articles

O-GlcNAcylation regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum.

To explore the potential mechanisms which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteogenesis, a publicly RNA-seq dataset was re-analyzed with literature-mining and showed the primary targets of O-GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O-GlcNAcylation-regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt-KO) in MC3T3-E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria-endoplasmic reticulum (Mito-ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt-KO cells. Through artificial intelligence (AI)-predicted cellular structures, the time-lapse live cells imaging with reactive-oxygen-species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt-KO cells were correlated with the Mito-ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O-GlcNAcylation-regulated Mito-ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O-GlcNAcylation-regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.

Effects of EGFR-TKI on epidermal melanin unit integrity: Therapeutic implications for hypopigmented skin disorders.

Epidermal melanin unit integrity is crucial for skin homeostasis and pigmentation. Epidermal growth factor (EGF) receptor (EGFR) is a pivotal player in cell growth, wound healing, and maintaining skin homeostasis. However, its influence on skin pigmentation is relatively unexplored. This study investigates the impact and underlying mechanisms of EGFR inhibitors on skin pigmentation. We evaluated EGF and EGFR expression in various skin cells using quantitative real-time PCR, Western blot, and immunofluorescence. EGF and EGFR were predominantly expressed in epidermal keratinocytes, and treatment with the EGFR tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and PD153035 significantly increased stem cell factor (SCF) and endothelin-1 (ET-1) expression in cultured keratinocytes. Enhanced melanocyte migration and proliferation were observed in co-culture, as evidenced by time-lapse live imaging and single-cell tracking assays. Furthermore, topical application of gefitinib to guinea pig dorsal skin induced increased pigmentation and demonstrated efficacy in mitigating rhododendrol-induced leukoderma. Suppression of EGF signaling indirectly enhanced skin pigmentation by upregulating SCF and ET-1 in epidermal keratinocytes. This novel mechanism highlights the pivotal role of EGF signaling in regulating skin pigmentation, and topical EGFR-TKI therapy at an appropriate dose may be a promising approach for depigmentation disorder management.

Activin is a neural inducer of a male-specific muscle in Drosophila

Drosophila melanogaster has a pair of male-specific muscles called the muscle of Lawrence (MOL) in abdominal segment 5 (A5) of adult flies. The MOL is produced only when its innervating motoneuron expresses FruitlessM (FruM) neural masculinizing proteins. We show that MOL induction is hampered by: (1) silencing electrical activities in the motoneuron, (2) blocking vesicular release from the motoneuron, and (3) knocking down Activin ß (Actß) in the motoneuron or knocking down Actß signaling pathway components in the myoblasts. Our timelapse live imaging of the developing neuromuscular system reveals that, upon contact with the presumptive MOL, the motoneuronal axon retracts concomitant with the progression of MOL degeneration resulting from neural silencing. We conclude that MOL formation depends on the bidirectional trophic interactions between pre- and postsynaptic cells, with motoneuron-derived Actß playing an inducing role in MOL formation.

Ex Vivo Live Imaging in Brain Slices for Analysis of Neurite Formation in Combination with In Utero Electroporation.

The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE).

Directionality of developing skeletal muscles is set by mechanical forces

Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.

Activated EGFR-αVβ3 interactions directly drive integrin-mediated cell mechanics.

Cells sense and respond to physical forces, and aberrations in this exchange are implicated in disease and dysfunction. Integrins and EGFR have robust synergy, with integrins establishing the mechanical link between cytoskeleton and microenvironment and EGFR regulating growth and survival. Previously, we established ligand-activated EGFR tunes integrin mechanics and facilitates cell spreading and focal adhesion (FA) maturation. Sialylation enhanced this mechanical crosstalk by increasing retention of activated EGFR on the plasma membrane. However, the underlying mechanisms orchestrating EGFR's influence on cell mechanics remain unknown. To address if there was a direct interaction between EGFR and integrin, we used a proximity ligation assay. In Cos-7 cells, EGF stimulation increased EGFR-αVβ3 interactions at 1 hour post-plating over untreated controls. The number of interactions were reduced at 24 hours, indicating that they are transient and support initial cell attachment and FA formation. Sites of EGFR-αVβ3 interactions colocalize with paxillin and pFAK with higher efficiency at 1 hour than 24 hours, supporting their role in FA formation but not maintenance. EGFR-αVβ3integrin interactions were confirmed by co-immunoprecipitation with immunoblotting analysis. Applying high-resolution TIRF microscopy, we associate these interactions to lipid raft microdomains which indicate formation of EGFR-integrin based signaling hubs at the membrane to support downstream mechanical outcomes like FA formation. Finally, employing time-lapse live cell TIRF imaging of Cos-7 cells overexpressing tagged EGFR and paxillin we characterize the dynamic nature of EGFR-αVβ3 interactions and associate them with FAs. Activated EGFR accumulates at sites for FA formation before paxillin starts clustering and persists during FA maturation. Our results implicate direct EGFR-integrin interaction as the mechanism driving cellular outcomes including FA formation, maturation and cell spreading during initial cell adhesion. Overall, EGFR-integrin interplay performs a vital synergistic function as a joint-signaling rheostat orchestrating cell mechanics.

Cell Cycle Dynamics during Stomatal Development: Window of MUTE Action and Ramification of Its Loss-of-Function on an Uncommitted Precursor.

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

Septin 9 controls CCNB1 stabilization via APC/CCDC20 during meiotic metaphase I/anaphase I transition in mouse oocytes.

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.

Comparison between phase-field model and coarse-grained model for characterizing cell-resolved morphological and mechanical properties in a multicellular system

Embryonic development is a precise and complex process involving the cell morphology and mechanics interacting in space and time. The difficulty in quantitatively acquiring cellular morphological and mechanical information in vivo makes mathematical modeling a challenging problem and impedes model validation. Recently, the three-dimensional time-lapse live imaging and delineated developmental programs in the roundworm Caenorhabditis elegans provide an excellent platform for establishing quantitative models. In this paper, we study two popular computational models for multicellular systems, i.e., the phase-field model and the coarse-grained model, and compare their performance in characterizing the cell morphologies, cell adhesion, and cell stiffness in a real C. elegans embryo. We show that both models can capture cell–cell contact areas and heterogeneous cell adhesion, but only the phase-field model succeeds in inferring the heterogeneous cell stiffness by fitting cell shapes or cell–cell interface curvatures. Moreover, we demonstrate that the phase-field model converges to the coarse-grained model when increasing cell surface tension to dominance, obtaining a distance-dependent isotropic intercellular force.

Live Imaging of Mitochondria in the Intact Fly Wing.

Detailed mechanisms governing the transport of mitochondria in neurons have recently emerged, although it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. Here, we provide a protocol to study the intracellular dynamics of mitochondria in the wing neurons of adult Drosophila in situ. The mounting and imaging procedures that we describe are suitable for use on most microscopes, and they can be easily implemented in any laboratory. Our noninvasive mounting procedures, combined with the translucency of the wing cuticle in adult animals, makes the wing nervous system accessible to advanced microscopy studies in a physiological environment. Combining the powerful genetics of Drosophila with time-lapse live imaging, users of this protocol will be able to analyze mitochondrial dynamics over time in a subset of sensory neurons in the wing. These cells extend long axons with a stereotypical plus-end-out microtubule orientation that represents a unique model to understand the logic of neuronal cargo transport, including the mitochondria. Finally, the neurons in this tissue respond to mechanical and chemical stimulation of the sensory organs of the wing, opening up the possibility of coupling the study of mitochondrial dynamics with the modulation of neuronal activity in aging Drosophila We anticipate that the unique characteristics of this in vivo system will contribute to the discovery of novel mechanisms that regulate mitochondrial dynamics within an organismal context with relevant implications for the pathogenesis of age-dependent neurological disorders.

Neuroprotective effects of ex vivo-expanded regulatory T cells on trimethyltin-induced neurodegeneration in mice

BackgroundTrimethyltin (TMT) is a potent neurotoxicant that leads to hippocampal neurodegeneration. Regulatory T cells (Tregs) play an important role in maintaining the immune balance in the central nervous system (CNS), but their activities are impaired in neurodegenerative diseases. In this study, we aimed to determine whether adoptive transfer of Tregs, as a living drug, ameliorates hippocampal neurodegeneration in TMT-intoxicated mice.MethodsCD4+CD25+ Tregs were expanded in vitro and adoptively transferred to TMT-treated mice. First, we explored the effects of Tregs on behavioral deficits using the Morris water maze and elevated plus maze tests. Biomarkers related to memory formation, such as cAMP response element-binding protein (CREB), protein kinase C (PKC), neuronal nuclear protein (NeuN), nerve growth factor (NGF), and ionized calcium binding adaptor molecule 1 (Iba1) in the hippocampus were examined by immunohistochemistry after killing the mouse. To investigate the neuroinflammatory responses, the polarization status of microglia was examined in vivo and in vitro using real-time reverse transcription polymerase chain reaction (rtPCR) and Enzyme-linked immunosorbent assay (ELISA). Additionally, the inhibitory effects of Tregs on TMT-induced microglial activation were examined using time-lapse live imaging in vitro with an activation-specific fluorescence probe, CDr20.ResultsAdoptive transfer of Tregs improved spatial learning and memory functions and reduced anxiety in TMT-intoxicated mice. Additionally, adoptive transfer of Tregs reduced neuronal loss and recovered the expression of neurogenesis enhancing molecules in the hippocampi of TMT-intoxicated mice. In particular, Tregs inhibited microglial activation and pro-inflammatory cytokine release in the hippocampi of TMT-intoxicated mice. The inhibitory effects of TMT were also confirmed via in vitro live time-lapse imaging in a Treg/microglia co-culture system.ConclusionsThese data suggest that adoptive transfer of Tregs ameliorates disease progression in TMT-induced neurodegeneration by promoting neurogenesis and modulating microglial activation and polarization.

Vascular endothelial growth factor c regulates hematopoietic stem cell fate in the dorsal aorta.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) during development in a process called endothelial-to-hematopoietic transition. The factors and signals that control HSPC fate decisions from the hemogenic endothelium are not fully understood. We found that Vegfc has a role in HSPC emergence from the zebrafish DA. Using time-lapse live imaging, we show that some HSPCs in the DA of vegfc loss-of-function embryos display altered cellular behavior. Instead of typical budding from the DA, emergent HSPCs exhibit crawling behavior similar to myeloid cells. This was confirmed by increased myeloid cell marker expression in the ventral wall of the DA and the caudal hematopoietic tissue. This increase in myeloid cells corresponded with a decrease in HSPCs that persisted into larval stages. Together, our data suggest that Vegfc regulates HSPC emergence in the hemogenic endothelium, in part by suppressing a myeloid cell fate. Our study provides a potential signal for modulation of HSPC fate in stem cell differentiation protocols.

Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer

BackgroundLiver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients.MethodsIntegrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects.ResultsThrough integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe.ConclusionsOur data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.

Septin 4 controls CCNB1 stabilization via APC/CCDC20 during meiotic G2/M transition in mouse oocytes.

In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.

Timing of meristem initiation and maintenance determines the morphology of fern gametophytes.

The alternation of generations in land plants occurs between the sporophyte phase and the gametophyte phase. The sporophytes of seed plants develop self-maintained, multicellular meristems, and these meristems determine plant architecture. The gametophytes of seed plants lack meristems and are heterotrophic. In contrast, the gametophytes of seed-free vascular plants, including ferns, are autotrophic and free-living, developing meristems to sustain their independent growth and proliferation. Compared with meristems in the sporophytes of seed plants, the cellular mechanisms underlying meristem development in fern gametophytes remain largely unknown. Here, using confocal time-lapse live imaging and computational segmentation and quantification, we determined different patterns of cell divisions associated with the initiation and proliferation of two distinct types of meristems in gametophytes of two closely related Pteridaceae ferns, Pteris vittata and Ceratopteris richardii. Our results reveal how the simple timing of a switch between two meristems has considerable consequences for the divergent gametophyte morphologies of the two ferns. They further provide evolutionary insight into the function and regulation of gametophyte meristems in seed-free vascular plants.

Anti-IL1RAP/CD3 Bispecific Antibody (BsAb) Is a Promising Novel and Effective Therapy for Acute Myeloid Leukemia (AML)

Despite increasing insight of the mechanisms of leukemogenesis, implementation of risk-stratification treatment strategies and use of molecular targeting therapeutics, only a minority of AML patients are cured with chemotherapy-based regimens. Most patients are either primary refractory to induction therapy or subsequently relapse following a brief remission likely due to persistence of chemo-resistant leukemia stem cell (LSC)-enriched subpopulations. Therefore there are urgent unmet therapeutic needs. The principle of CD3 based BsAb therapy is to recruit and activate T cell effector function upon engaging target cells expressing a tumor associated antigen (TAA). Several TAA (e.g.CD33, CD123) have been found to be differentially overexpressed on the surface of AML blasts. Recently, the expression of IL-1 receptor accessory protein (IL1RAP) was reported to be higher on LSCs and more differentiated leukemic progenitor cells (LPCs) than on normal hematopoietic stem cells (HSC), making it an attractive TAA target for immunotherapy. Thus, we hypothesize that an anti-IL1RAP/CD3 BsAb could preferentially target LSCs and LPCs while sparing normal HSCs, thereby offering a window of therapeutic opportunity for AML patients.To test this hypothesis we first determined the expression pattern of IL-1RAP. IL1RAP was differentially expressed on AML cell lines: HL60 and MV4-11 (high), THP-1 (intermediate), and KG-1a (low). Its expression levels were variable on primary blasts from AML patients (n=10). CD34+ peripheral blood mononuclear cells (PBMC) from healthy donors showed little or no IL1RAP expression. Next, we screened 12 anti-IL1RAP monoclonal antibodies from our human phage display antibody library. Based on the results of the BIAcore and antibody dependent cellular cytotoxity (ADCC) assays performed on MV4-11 cells, we selected one lead candidate with binding affinity of ~20nM and EC50 4.9nM for further development. Using knobs-into-holes strategy, we paired this IL1RAP antibody with an anti-CD3 antibody. The dual binding ability of this BsAb was confirmed by flow cytometric analysis. We subsequently proved T cell dependent cytotoxicity (TDCC) of this BsAb using both AML cell lines and AML primary blasts as targets. The anti-IL1RAP/CD3 BsAb induced over 95% of specific killing on all tested AML samples at E: T (Effector : Target) ratio of 1:1 compared with no cytotoxicity effect on the IL-1RAP-negative Raji cells in the 48-hour long term killing (LTK) assay. In line with the cytotoxic data, there was also substantial release of cytokines including IFN-γ, TNF-α and Granzyme B from the AML cells but not Raji cells. TDCC reported over 90% of IL1-RAP specific killing in all but one (only 60%) patient samples (n=10), which had the lowest IL1RAP expression level. The killing of leukemia cells was also observed by time-lapse live cell microscope imaging using luciferase-expressing MV4-11luci+ cells with addition of 100ng/ml BsAb and T cells at E:T ratio of 5:1, which documented effective T cell recruitment and cytolytic activities against MV4-11luci+ cells. We also tested the anti-leukemic activity of this BsAb in vivo using a NSG xenograft mouse model. NSG mice transplanted with 1x106 MV4-11luci+ cells were treated with 200µg anti-IL1RAP/CD3 BsAb plus 3x106 unstimulated T cells, vehicle, T cells alone or anti-CD19/CD3 BsAb (control Ab) plus T cells twice a week for 30 days. In vivo imaging demonstrated effective elimination of the leukemia signal (P<0.001) in MV4-11luci+-engrafted NSG mice treated with T cells and anti-IL1RAP/CD3 BsAb (photon flux (PF) =5.5x105) compared with intense leukemia infiltration in controls treated with either vehicle (PF=3.5x109), T cells alone (PF=4x109), or control anti-CD19/CD3 BsAb plus T cells (PF=2x109). In a separate experiment, treatment with anti-IL1RAP/CD3 BsAb plus T cells of NSG mice engrafted with primary AML blasts led to significant decrease in disease burden than treatment with isotype antibody plusT cells (p=0.004). Further characterization and determination of disease response in these animals is ongoing.In conclusion, we have successfully engineered and characterized an anti-IL1RAP/CD3 BsAb derived from our human naïve phage display library. Our data demonstrated that this BsAb can effectively eradicate AML cells in vitro and in vivo, supporting the clinical translation of this novel immunotherapeutic agent for treatment of AML patients. DisclosuresKwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy. Stein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.

Tunneling nanotubes (TNTs) are actin‐rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down‐regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F‐actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV‐Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.

Glutathione S-transferase Pi (Gstp) proteins regulate neuritogenesis in the developing cerebral cortex.

GSTP proteins are metabolic enzymes involved in the removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, whereas there is only one GSTP in humans. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown (KD) caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation (IUE) to knock down Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared with the control. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.

Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast

SummaryEukaryotic cells position the nucleus within the proper intracellular space, thereby safeguarding a variety of cellular processes. In fission yeast, the interphase nucleus is placed in the cell middle in a microtubule-dependent manner. By contrast, how the mitotic nucleus is positioned remains elusive. Here we show that several cell-cycle mutants that arrest in mitosis all displace the nucleus toward one end of the cell. Intriguingly, the actin cytoskeleton is responsible for nuclear movement. Time-lapse live imaging indicates that mitosis-specific F-actin cables possibly push the nucleus through direct interaction with the nuclear envelope, and subsequently actomyosin ring constriction further shifts the nucleus away from the center. This nuclear movement is beneficial, because if the nuclei were retained in the center, unseparated chromosomes would be intersected by the contractile actin ring and the septum, imposing the lethal cut phenotype. Thus, fission yeast escapes from mitotic catastrophe by means of actin-dependent nuclear movement.

Multifunctional compounds lithium chloride and methylene Blue attenuate the negative effects of diisopropylfluorophosphate on axonal transport in rat cortical neurons

Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need).