NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells.

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.

A case of drug-induced organizing pneumonia caused by amikacin liposome inhalation suspension.

Inhaled liposomal antimicrobials are known to cause hypersensitivity pneumonitis. Amikacin liposome inhalation suspension (ALIS) is a promising novel antimicrobial agent against refractory Mycobacterium avium complex infections. The frequency of drug-induced lung injury caused by ALIS is relatively high. To date, no reports of ALIS-induced organizing pneumonia diagnosed by bronchoscopy are available. We report a case of a 74-year-old female patient presenting with non-tuberculous mycobacterial pulmonary disease (NTM-PD). She was treated with ALIS for refractory NTM-PD. Fifty-nine days after starting ALIS, the patient developed a cough, and her chest radiographs indicated deterioration. She was diagnosed with organizing pneumonia based on pathological findings of the lung tissues obtained by bronchoscopy. After switching from ALIS to amikacin infusion, her organizing pneumonia improved. It is difficult to distinguish between organizing pneumonia and an exacerbation of NTM-PD based on chest radiography alone. Therefore, it is essential to perform an active bronchoscopy for diagnosis.

Determining Response to Treatment for Drug-Induced Bronchocentric Granulomatosis by the Forced Oscillation Technique.

Anti-tumor necrosis factor alpha (TNFα) therapy is widely used to treat various inflammatory conditions. Paradoxically, there are several case reports describing the development of bronchocentric granulomatosis treated with TNFα inhibitors, and it is difficult to determine the effect of treatment using conventional spirometry because the lesions are located in small airways. However, it has been reported that the forced oscillation technique (FOT) is useful in the evaluation of small airway disease in bronchial asthma or chronic obstructive pulmonary disease. We performed the FOT to determine the effect of treatment on bronchocentric granulomatosis and found it to be useful. We report the case of a 55-year-old female with ulcerative colitis who was treated with golimumab and who developed bronchocentric granulomatosis as a sarcoid-like reaction to golimumab. She was successfully treated with prednisone, and the treatment efficacy was confirmed by the FOT. The FOT may be useful in the evaluation of small airway disease in bronchocentric granulomatosis. This case may help inform clinicians of the usefulness of the FOT to assess small airway disease in various diseases.

Simulation of intra-ciliary diffusion suggests a novel role of primary cilia as a cell-signaling enhancer.

Besides the role to generate a fluid flow in the surrounding medium, eukaryotic cilia have a crucial function in sensing external signals such as chemical or mechanical stimuli. A large body of work has shown that cilia are frequently found in various types of sensory cells and are closely related to many regulatory mechanisms in differentiation and development. However, we do not yet have a definitive answer to the fundamental question, "why cilia?" It has been a long-standing mystery why cells use cilia for sensing external signals. To shed light on this, we sought to describe the kinetics of signaling with theoretical approaches. Based on the results, here we propose a new role of cilia as a cell-signaling enhancer. The enhancing effect comes from restricted volume for the free intra-ciliary diffusion of molecules due to the cylindrical shape of cilia, which can facilitate quick accumulation of intracellular signaling molecules. Our simulations demonstrate that both the rate and amplitude of response in signal transduction depend on where the membrane receptors or channels are located along the ciliary shaft. In addition, the calculated transfer function of cilia regarded as a transmitter of external signals also suggests the properties of cilia as a signal enhancer. Since such unique composition of receptors and channels in cilia is found in various types of eukaryotic cells, signal enhancing is presumably one of the most essential and conserved roles of cilia.

Gated entry into the ciliary compartment.

Cilia and flagella play important roles in cell motility and cell signaling. These functions require that the cilium establishes and maintains a unique lipid and protein composition. Recent work indicates that a specialized region at the base of the cilium, the transition zone, serves as both a barrier to entry and a gate for passage of select components. For at least some cytosolic proteins, the barrier and gate functions are provided by a ciliary pore complex (CPC) that shares molecular and mechanistic properties with nuclear gating. Specifically, nucleoporins of the CPC limit the diffusional entry of cytosolic proteins in a size-dependent manner and enable the active transport of large molecules and complexes via targeting signals, importins, and the small G protein Ran. For membrane proteins, the septin protein SEPT2 is part of the barrier to entry whereas the gating function is carried out and/or regulated by proteins associated with ciliary diseases (ciliopathies) such as nephronophthisis, Meckel­Gruber syndrome and Joubert syndrome. Here, we discuss the evidence behind these models of ciliary gating as well as the similarities to and differences from nuclear gating.

Asymmetric distribution of dynamic calcium signals in the node of mouse embryo during left-right axis formation.

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.

A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation.

The visual classification of cell images according to differences in the spatial patterns of subcellular structure is an important methodology in cell and developmental biology. Experimental perturbation of cell function can induce changes in the spatial distribution of organelles and their associated markers or labels. Here, we demonstrate how to achieve accurate, unbiased, high-throughput image classification using an artificial intelligence (AI) algorithm. We show that a convolutional neural network (CNN) algorithm can classify distinct patterns of Golgi images after drug or siRNA treatments, and we review our methods from cell preparation to image acquisition and CNN analysis.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides. This article has an associated interview with the co-first authors of the paper.

Pulmonary Langerhans Cell Histiocytosis That Progressed from a Single-system to a Multisystem Form despite Smoking Cessation.

A 36-year-old Japanese man presented with cavities and nodular shadows in the lower lobes of his lungs and osteolytic lesions in the thoracic spine. He was diagnosed with multisystem Langerhans cell histiocytosis (LCH). Three years earlier, he had been noted to have small cavities and granular lesions noted in the upper lobes of his lungs, which later improved with smoking cessation. It was likely that his single-system pulmonary LCH (PLCH) progressed to multisystem LCH despite smoking cessation. Relapse or progression may occur in cases where PLCH lesions improve after smoking cessation. Thus, close follow-up is vital.

X-ray diffraction recording from single axonemes of eukaryotic flagella.

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 µm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-µm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 µm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly.

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

Geometry-specific heterogeneity of the apparent diffusion rate of materials inside sperm cells.

In sea urchin spermatozoa, the energy source powering flagellar motion is provided as ATP produced by mitochondria located at the proximal ends of flagella. However, the bottleneck structure between the sperm head and the flagellar tail seems to restrict the free entry of ATP from mitochondria into the tail region. To test this possibility, we investigated the diffusion properties in sperm cells using fluorescence recovery after photobleaching. We found that the rate of fluorescence recovery in the head region was approximately 10% of that observed in the flagellar tail regions. We also found that, even within the tail region, rates varied depending on location, i.e., rates were slower at the more distal regions. Using computational analysis, the rate heterogeneity was shown to be caused mainly by the geometry of the sperm structure, even if little or no difference in diffusion rates through the neck region was assumed. Therefore, we concluded that materials such as ATP would generally diffuse freely between the heads and the flagella of sperm cells. We believe these findings regarding the diffusion properties inside spermatozoa provide further insights into material transportation and chemical signaling inside eukaryotic cilia and flagella.

Single-cell electroporation of fluorescent probes into sea urchin sperm cells and subsequent FRAP analysis.

In sea urchin spermatozoa, the energy required for flagellar motility depends only on the diffusional supply from proximal mitochondria, and thus the diffusion rate inside flagella is one of the most crucial factors limiting the practical size and design of the motile machinery. To determine the diffusion rates of materials inside sperm cells, FRAP (fluorescence recovery after photobleaching) analysis of incorporated fluorescent probes is one of the most powerful approaches. However, the only practically possible method until now was to use the ester forms of fluorescence, and our choice was limited to those of relatively small molecular masses, such as fluorescein derivatives. In this report, we show that a modified single-cell electroporation technique can be applied as a new microinjection method for sperm cells of the sea urchin. The method was applied to FRAP analysis to determine the rate of intraflagellar diffusion.

Robust classification of cell cycle phase and biological feature extraction by image-based deep learning.

Across the cell cycle, the subcellular organization undergoes major spatiotemporal changes that could in principle contain biological features that could potentially represent cell cycle phase. We applied convolutional neural network-based classifiers to extract such putative features from the fluorescence microscope images of cells stained for the nucleus, the Golgi apparatus, and the microtubule cytoskeleton. We demonstrate that cell images can be robustly classified according to G1/S and G2 cell cycle phases without the need for specific cell cycle markers. Grad-CAM analysis of the classification models enabled us to extract several pairs of quantitative parameters of specific subcellular features as good classifiers for the cell cycle phase. These results collectively demonstrate that machine learning-based image processing is useful to extract biological features underlying cellular phenomena of interest in an unbiased and data-driven manner.

Structure Optimization of Gatastatin for the Development of γ-Tubulin-Specific Inhibitor.

Gatastatin (O 7-benzyl glaziovianin A) is a γ-tubulin-specific inhibitor that is used to investigate γ-tubulin function in cells. We have previously reported that the unsubstituted phenyl ring of the O 7-benzyl group in gatastatin is important for γ-tubulin inhibition. To obtain further structural information regarding γ-tubulin inhibition, we synthesized several gatastatin derivatives containing a fixed O 7-benzyl moiety. Modifications of the B-ring resulted in drastic decrease in cytotoxicity, abnormal spindle formation activity, and inhibition of microtubule (MT) nucleation. In contrast, various O 6-alkylated gatastatin derivatives showed potent cytotoxicity, induced abnormal spindle formation, and inhibited MT nucleation. We had previously reported that O 6-benzyl glaziovianin A is a potent α/ß-tubulin inhibitor; thus, these new results suggest that the O 6-position restricts affinity for α/ß- and γ-tubulin. Considering that an O 7-benzyl group increases specificity for γ-tubulin, more potent and specific γ-tubulin inhibitors can be generated through O 6-modifications of gatastatin.

Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (approximately 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

A theory of centriole duplication based on self-organized spatial pattern formation.

In each cell cycle, centrioles are duplicated to produce a single copy of each preexisting centriole. At the onset of centriole duplication, the master regulator Polo-like kinase 4 (Plk4) undergoes a dynamic change in its spatial pattern around the preexisting centriole, forming a single duplication site. However, the significance and mechanisms of this pattern transition remain unknown. Using super-resolution imaging, we found that centriolar Plk4 exhibits periodic discrete patterns resembling pearl necklaces, frequently with single prominent foci. Mathematical modeling and simulations incorporating the self-organization properties of Plk4 successfully generated the experimentally observed patterns. We therefore propose that the self-patterning of Plk4 is crucial for the regulation of centriole duplication. These results, defining the mechanisms of self-organized regulation, provide a fundamental principle for understanding centriole duplication.

Feedback loops in the Plk4-STIL-HsSAS6 network coordinate site selection for procentriole formation.

Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in centriole duplication remains poorly understood. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between critical duplication factors coordinate the centriole-duplication process.This article has an associated First Person interview with the first author of the paper.

NPHP proteins are binding partners of nucleoporins at the base of the primary cilium.

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle.

The Cep57-pericentrin module organizes PCM expansion and centriole engagement.

Centriole duplication occurs once per cell cycle to ensure robust formation of bipolar spindles and chromosome segregation. Each newly-formed daughter centriole remains connected to its mother centriole until late mitosis. The disengagement of the centriole pair is required for centriole duplication. However, the mechanisms underlying centriole engagement remain poorly understood. Here, we show that Cep57 is required for pericentriolar material (PCM) organization that regulates centriole engagement. Depletion of Cep57 causes PCM disorganization and precocious centriole disengagement during mitosis. The disengaged daughter centrioles acquire ectopic microtubule-organizing-center activity, which results in chromosome mis-segregation. Similar defects are observed in mosaic variegated aneuploidy syndrome patient cells with cep57 mutations. We also find that Cep57 binds to the well-conserved PACT domain of pericentrin. Microcephaly osteodysplastic primordial dwarfism disease pericentrin mutations impair the Cep57-pericentrin interaction and lead to PCM disorganization. Together, our work demonstrates that Cep57 provides a critical interface between the centriole core and PCM.