Immotile cilia mechanically sense the direction of fluid flow for left-right determination.

Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.

Life cycle and functional genomics of the unicellular red alga Galdieria for elucidating algal and plant evolution and industrial use.

Sexual reproduction is widespread in eukaryotes; however, only asexual reproduction has been observed in unicellular red algae, including Galdieria, which branched early in Archaeplastida. Galdieria possesses a small genome; it is polyextremophile, grows either photoautotrophically, mixotrophically, or heterotrophically, and is being developed as an industrial source of vitamins and pigments because of its high biomass productivity. Here, we show that Galdieria exhibits a sexual life cycle, alternating between cell-walled diploid and cell wall-less haploid, and that both phases can proliferate asexually. The haploid can move over surfaces and undergo self-diploidization or generate heterozygous diploids through mating. Further, we prepared the whole genome and a comparative transcriptome dataset between the diploid and haploid and developed genetic tools for the stable gene expression, gene disruption, and selectable marker recycling system using the cell wall-less haploid. The BELL/KNOX and MADS-box transcription factors, which function in haploid-to-diploid transition and development in plants, are specifically expressed in the haploid and diploid, respectively, and are involved in the haploid-to-diploid transition in Galdieria, providing information on the missing link of the sexual life cycle evolution in Archaeplastida. Four actin genes are differently involved in motility of the haploid and cytokinesis in the diploid, both of which are myosin independent and likely reflect ancestral roles of actin. We have also generated photosynthesis-deficient mutants, such as blue-colored cells, which were depleted in chlorophyll and carotenoids, for industrial pigment production. These features of Galdieria facilitate the understanding of the evolution of algae and plants and the industrial use of microalgae.

Long-term live cell cycle imaging of single Cyanidioschyzon merolae cells.

Live cell imaging by fluorescence microscopy is a useful tool for elucidating the localization and function of proteins and organelles in single cells. Especially, time-lapse analysis observing the same field sequentially can be used to observe cells of many organisms and analyze the dynamics of intracellular molecules. By single-cell analysis, it is possible to elucidate the characteristics and fluctuations of individual cells, which cannot be elucidated from the data obtained by averaging the characteristics of an ensemble of cells. The primitive red alga Cyanidioschyzon merolae has a very simple structure and is considered a useful model organism for studying the mechanism of organelle division, since the division is performed synchronously with the cell cycle. However, C. merolae does not have a rigid cell wall, and environmental changes such as low temperature or high pH cause morphological change and disruption easily. Therefore, morphological studies of C. merolae typically use fixed cells. In this study, we constructed a long-term time-lapse observation system to analyze the dynamics of proteins in living C. merolae cells. From the results, we elucidate the cell division process of single living cells, including the function of intracellular components.

The Physical Role of Mesenchymal Cells Driven by the Actin Cytoskeleton Is Essential for the Orientation of Collagen Fibrils in Zebrafish Fins.

Fibrous collagen imparts physical strength and flexibility to tissues by forming huge complexes. The density and orientation of collagen fibers must be correctly specified for the optimal physical property of the collagen complex. However, little is known about its underlying cellular mechanisms. Actinotrichia are collagen fibers aligned at the fin-tip of bony fish and are easily visible under the microscope due to their thick, linear structure. We used the actinotrichia as a model system to investigate how cells manipulate collagen fibers. The 3D image obtained by focused ion beam scanning electron microscopy (FIB-SEM) showed that the pseudopodia of mesenchymal cells encircle the multiple actinotrichia. We then co-incubated the mesenchymal cells and actinotrichia in vitro, and time-lapse analysis revealed how cells use pseudopods to align collagen fiber orientation. This in vitro behavior is dependent on actin polymerization in mesenchymal cells. Inhibition of actin polymerization in mesenchymal cells results in mis-orientation of actinotrichia in the fin. These results reveal how mesenchymal cells are involved in fin formation and have important implications for the physical interaction between cells and collagen fibers.

Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry.

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome.

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Multiplexed 129Xe HyperCEST MRI Detection of Genetically Reconstituted Bacterial Protein Nanoparticles in Human Cancer Cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4 and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive, and genetically encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

Prediction of Sequential Organelles Localization under Imbalance using A Balanced Deep U-Net.

Assessing the structure and function of organelles in living organisms of the primitive unicellular red algae Cyanidioschyzon merolae on three-dimensional sequential images demands a reliable automated technique in the class imbalance among various cellular structures during mitosis. Existing classification networks with commonly used loss functions were focused on larger numbers of cellular structures that lead to the unreliability of the system. Hence, we proposed a balanced deep regularized weighted compound dice loss (RWCDL) network for better localization of cell organelles. Specifically, we introduced two new loss functions, namely compound dice (CD) and RWCD by implementing multi-class variant dice and weighting mechanism, respectively for maximizing weights of peroxisome and nucleus among five classes as the main contribution of this study. We extended the Unet-like convolution neural network (CNN) architecture for evaluating the ability of our proposed loss functions for improved segmentation. The feasibility of the proposed approach is confirmed with three different large scale mitotic cycle data set with different number of occurrences of cell organelles. In addition, we compared the training behavior of our designed architectures with the ground truth segmentation using various performance measures. The proposed balanced RWCDL network generated the highest area under the curve (AUC) value in elevating the small and obscure peroxisome and nucleus, which is 30% higher than the network with commonly used mean square error (MSE) and dice loss (DL) functions. The experimental results indicated that the proposed approach can efficiently identify the cellular structures, even when the contour between the cells is obscure and thus convinced that the balanced deep RWCDL approach is reliable and can be helpful for biologist to accurately identify the relationship between the cell behavior and structures of cell organelles during mitosis.

Cyanidioschyzon merolae aurora kinase phosphorylates evolutionarily conserved sites on its target to regulate mitochondrial division.

The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis.

Roles of basal keratinocytes in actinotrichia formation.

The embryonic fins and the tip of adult fins of teleost fish are supported by rows of straight, unmineralized fibrils called actinotrichia. The proximal ends of the actinotrichia are bundled and the mineralized bones called lepidotrichia are made along them. Since malformation in actinotrichia causes wavy fin bones, the correct configuration of actinotrichia is essential for the correct construction of the fin shape. Past studies suggested that two types of cells, basal keratinocytes, and mesenchymal cells involve in the formation of actinotrichia. However, the mechanism how these cells contribute is unknown. In this study, we elucidated the role of basal keratinocytes in actinotrichia formation. First, we developed the imaging tool that specifically visualizes the basal keratinocytes and actinotrichia. Then, we established the in vitro culture method of the basal keratinocytes and found that the keratinocytes developed fine needle-like structures in it. The TEM image of them showed the specific shadow pattern of actinotrichia, indicating that the fine needle-like structures are the newly made actinotrichia. Finally, we cultured the basal keratinocytes with mature actinotrichia and observed that the basal keratinocytes actively holded actinotrichia with their membrane, and often generated a linear array of cells holding a single actinotrichium. This behavior suggested a mechanism with which long actinotrichia are made by relatively small cells. Our results clarified the role of basal keratinocyte and provided a novel insight into understanding the mechanism of actinotrichia formation.

The role of structural dynamics of actin in class-specific myosin motility.

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

Local heat activation of single myosins based on optical trapping of gold nanoparticles.

Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.

Spontaneous structural changes in actin regulate G-F transformation.

Transformations between G- (monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Förster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.

Switching of myosin-V motion between the lever-arm swing and brownian search-and-catch.

Motor proteins are force-generating nanomachines that are highly adaptable to their ever-changing biological environments and have a high energy conversion efficiency. Here we constructed an imaging system that uses optical tweezers and a DNA handle to visualize elementary mechanical processes of a nanomachine under load. We apply our system to myosin-V, a well-known motor protein that takes 72 nm 'hand-over-hand' steps composed of a 'lever-arm swing' and a 'brownian search-and-catch'. We find that the lever-arm swing generates a large proportion of the force at low load (<0.5 pN), resulting in 3 k(B)T of work. At high load (1.9 pN), however, the contribution of the brownian search-and-catch increases to dominate, reaching 13 k(B)T of work. We believe the ability to switch between these two force-generation modes facilitates myosin-V function at high efficiency while operating in a dynamic intracellular environment.

Simultaneous observation of the lever arm and head explains myosin VI dual function.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V.

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.

Mechanical characterization of one-headed myosin-V using optical tweezers.

Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (approximately 36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 k(B)T from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.

Direct visualization of secondary structures of F-actin by electron cryomicroscopy.

F-actin is a helical assembly of actin, which is a component of muscle fibres essential for contraction and has a crucial role in numerous cellular processes, such as the formation of lamellipodia and filopodia, as the most abundant component and regulator of cytoskeletons by dynamic assembly and disassembly (from G-actin to F-actin and vice versa). Actin is a ubiquitous protein and is involved in important biological functions, but the definitive high-resolution structure of F-actin remains unknown. Although a recent atomic model well reproduced X-ray fibre diffraction intensity data from a highly oriented liquid-crystalline sol specimen, its refinement without experimental phase information has certain limitations. Direct visualization of the structure by electron cryomicroscopy, however, has been difficult because it is relatively thin and flexible. Here we report the F-actin structure at 6.6 Å resolution, made obtainable by recent advances in electron cryomicroscopy. The density map clearly resolves all the secondary structures of G-actin, such as α-helices, ß-structures and loops, and makes unambiguous modelling and refinement possible. Complex domain motions that open the nucleotide-binding pocket on F-actin formation, specific D-loop and terminal conformations, and relatively tight axial but markedly loose interprotofilament interactions hydrophilic in nature are revealed in the F-actin model, and all seem to be important for dynamic functions of actin.

Switch between large hand-over-hand and small inchworm-like steps in myosin VI.

Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

Single molecular observation of self-regulated kinesin motility.

Kinesin-1 is an ATP-driven molecular motor that transports various cargoes in cells, a process that can be regulated by the kinesin tail domain. Here, kinesin ATPase activity and motility were inhibited in vitro by interacting the kinesin heavy chain C-terminal tail domain with the kinesin N-terminal motor domain. Though the tail domain can directly interact with microtubules, we found 70% of tail domains failed to bind in the presence of >100 mM (high) KCl, which also modulated the ATPase inhibition manner. These observations suggest that self-inhibition of kinesin depends on electrostatic interactions between the motor domain, the tail domain, and a microtubule. Furthermore, we observed self-regulated behavior of kinesin at the single molecule level. The tail domain did not affect motility velocity, but it did lower the binding affinity of the motor domain to the microtubule. The decrement in binding was coupled to ATPase inhibition. Meanwhile, the tail domain transfected into living cells not only failed to bind to microtubules but also inhibited the motor domain and microtubule interaction, in agreement with our in vitro results. Furthermore, at high potassium concentrations, the self-regulation of kinesin observed in cells was like that in vitro. The results favor a way tail inhibition mechanism where the tail domain masks the microtubule binding site of the motor domain in high potassium concentration.