Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.

Cerebral Small Vessel Disease Burden for Bleeding Risk during Antithrombotic Therapy: Bleeding with Antithrombotic Therapy 2 Study.

OBJECTIVE: This study was undertaken to determine the excess risk of antithrombotic-related bleeding due to cerebral small vessel disease (SVD) burden. METHODS: In this observational, prospective cohort study, patients with cerebrovascular or cardiovascular diseases taking oral antithrombotic agents were enrolled from 52 hospitals across Japan between 2016 and 2019. Baseline multimodal magnetic resonance imaging acquired under prespecified conditions was assessed by a central diagnostic radiology committee to calculate total SVD score. The primary outcome was major bleeding. Secondary outcomes included bleeding at each site and ischemic events. RESULTS: Of the analyzed 5,250 patients (1,736 women; median age = 73 years, 9,933 patient-years of follow-up), antiplatelets and anticoagulants were administered at baseline in 3,948 and 1,565, respectively. Median SVD score was 2 (interquartile range = 1-3). Incidence rate of major bleeding was 0.39 (per 100 patinet-years) in score 0, 0.56 in score 1, 0.91 in score 2, 1.35 in score 3, and 2.24 in score 4 (adjusted hazard ratio [aHR] for score 4 vs 0 = 5.47, 95% confidence interval [CI] = 2.26-13.23), that of intracranial hemorrhage was 0.11, 0.33, 0.58, 0.99, and 1.06, respectively (aHR = 9.29, 95% CI = 1.99-43.35), and that of ischemic event was 1.82, 2.27, 3.04, 3.91, and 4.07, respectively (aHR = 1.76, 95% CI = 1.08-2.86). In addition, extracranial major bleeding (aHR = 3.43, 95% CI = 1.13-10.38) and gastrointestinal bleeding (aHR = 2.54, 95% CI = 1.02-6.35) significantly increased in SVD score 4 compared to score 0. INTERPRETATION: Total SVD score was predictive for intracranial hemorrhage and probably for extracranial bleeding, suggesting the broader clinical relevance of cerebral SVD as a marker for safe implementation of antithrombotic therapy. ANN NEUROL 2024;95:774-787.

BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination.

BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

The ATPase asymmetry: Novel computational insight into coupling diverse FO motors with tripartite F1.

ATP synthase, a crucial enzyme for cellular bioenergetics, operates via the coordinated coupling of an FO motor, which presents variable symmetry, and a tripartite F1 motor. Despite extensive research, the understanding of their coupling dynamics, especially with non-10-fold symmetrical FO motors, remains incomplete. This study investigates the coupling patterns between eightfold and ninefold FO motors and the constant threefold F1 motor using coarse-grained molecular dynamics simulations. We unveil that in the case of a ninefold FO motor, a 3-3-3 motion is most likely to occur, whereas a 3-3-2 motion predominates with an eightfold FO motor. Furthermore, our findings propose a revised model for the coupling method, elucidating that the pathways' energy usage is primarily influenced by F1 rotation and conformational changes hindered by the b-subunits. Our results present a crucial step toward comprehending the energy landscape and mechanisms governing ATP synthase operation.

Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging.

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.

StayGold photostability under different illumination modes.

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.

To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20 µm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

α-Spectrin regulates cell shape changes during disassembly of microtubule-driven protrusions in Drosophila wings.

The dynamics of microtubule-mediated protrusions, termed Interplanar Amida Network (IPAN) in Drosophila pupal wing, involve cell shape changes. The molecular mechanisms underlying these processes are yet to be fully understood. This study delineates the stages of cell shape alterations during the disassembly of microtubule protrusions and underscores the pivotal role of α-Spectrin in driving these changes by regulating both the microtubule and actomyosin networks. Our findings also demonstrate that α-Spectrin is required for the apical relaxation of wing epithelia during protrusion disassembly, indicating its substantial contribution to the robustness of 3D tissue morphogenesis.

Dimeric transport mechanism of human vitamin C transporter SVCT1.

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.

Blood pressure during long-term cilostazol-based dual antiplatelet therapy after stroke: a post hoc analysis of the CSPS.com trial.

We determined the associations of follow-up blood pressure (BP) after stroke as a time-dependent covariate with the risk of subsequent ischemic stroke, as well as those of BP levels with the difference in the impact of long-term clopidogrel or aspirin monotherapy versus additional cilostazol medication on secondary stroke prevention. In a sub-analysis of a randomized controlled trial (CSPS.com), patients between 8 and 180 days after stroke onset were randomly assigned to receive aspirin or clopidogrel alone, or a combination of cilostazol with aspirin or clopidogrel. The percent changes, differences, and raw values of follow-up BP were examined. The primary efficacy outcome was the first recurrence of ischemic stroke. In a total of 1657 patients (69.5 ± 9.3 years, female 29.1%) with median 1.5-year follow-up, ischemic stroke recurred in 74 patients. The adjusted hazard ratio for ischemic stroke of a 10% systolic BP (SBP) increase from baseline was 1.19 (95% CI 1.03-1.36), that of a 10 mmHg SBP increase was 1.14 (1.03-1.28), and that of SBP as the raw value with the baseline SBP as a fixed (time-independent) covariate was 1.14 (1.00-1.31). Such significant associations were not observed in diastolic BP-derived variables. The estimated adjusted hazard ratio curves for the outcome showed the benefit of dual therapy over a wide SBP range between ≈120 and ≈165 mmHg uniformly. Lower long-term SBP levels after ischemic stroke were associated with a lower risk of subsequent ischemic events. The efficacy of dual antiplatelet therapy including cilostazol for secondary stroke prevention was evident over a wide SBP range.

Transient sensorimotor projections in the developmental song learning period.

Memory recall and guidance are essential for motor skill acquisition. Like humans learning to speak, male zebra finches learn to sing by first memorizing and then matching their vocalization to the tutor's song (TS) during specific developmental periods. Yet, the neuroanatomical substrate supporting auditory-memory-guided sensorimotor learning has remained elusive. Here, using a whole-brain connectome analysis with activity-dependent viral expression, we identified a transient projection into the motor region, HVC, from neuronal ensembles responding to TS in the auditory forebrain, the caudomedial nidopallium (NCM), in juveniles. Virally induced cell death of the juvenile, but not adult, TS-responsive NCM neurons impaired song learning. Moreover, isolation, which delays closure of the sensory, but not the motor, learning period, did not affect the decrease of projections into the HVC from the NCM TS-responsive neurons after the song learning period. Taken together, our results suggest that dynamic axonal pruning may regulate timely auditory-memory-guided vocal learning during development.