Unraveling the fastest myosin: Discovery history and structure-function relationships of algae Chara myosin XI.

Plant myosins have higher velocities than animal myosins. Among them, myosins in freshwater algae of the genus Chara have extremely high velocities. We have biochemically studied myosins that perform high-speed movements in the alga Chara. Our studies have elucidated the structural and enzymatic basis for the fast movement of Chara myosins. This review outlines the history leading to the discovery of the fastest myosin, algae Chara myosin XI, and the structure-function correlation of the fastest myosin. This review article is an extended version of the Japanese article, "Structure-function Relationship of the Fastest Myosin" by Ito et al., published in SEIBUTSU BUTSURI Vol. 63, p. 91-96 (2023).

Emergence and stability of endoplasmic reticulum network streaming in plant cells.

The endoplasmic reticulum (ER) network is highly complex and highly dynamic in its geometry, and undergoes extensive remodeling and bulk flow. It is known that the ER dynamics are driven by actin-myosin dependent processes. ER motion through the cytoplasm will cause forces on the cytoplasm that will induce flow. However, ER will also clearly be passively transported by the bulk cytoplasmic streaming. We take the complex ER network structure into account and propose a positive-feedback mechanism among myosin-like motors, actin alignment, ER network dynamics for the emergence of ER flow. Using this model, we demonstrate that ER streaming may be an emergent feature of this three-way interaction and that the persistent-point density may be a key driver of the emergence of ER streaming.

Cytoplasmic stirring by active carpets.

Large cells often rely on cytoplasmic flows for intracellular transport, maintaining homeostasis, and positioning cellular components. Understanding the mechanisms of these flows is essential for gaining insights into cell function, developmental processes, and evolutionary adaptability. Here, we focus on a class of self-organized cytoplasmic stirring mechanisms that result from fluid-structure interactions between cytoskeletal elements at the cell cortex. Drawing inspiration from streaming flows in late-stage fruit fly oocytes, we propose an analytically tractable active carpet theory. This model deciphers the origins and three-dimensional spatiotemporal organization of such flows. Through a combination of simulations and weakly nonlinear theory, we establish the pathway of the streaming flow to its global attractor: a cell-spanning vortical twister. Our study reveals the inherent symmetries of this emergent flow, its low-dimensional structure, and illustrates how complex fluid-structure interaction aligns with classical solutions in Stokes flow. This framework can be easily adapted to elucidate a broad spectrum of self-organized, cortex-driven intracellular flows.

Mechanosensing and anesthesia of single internodal cells of Chara.

The giant (2-3 × 10-2 m long) internodal cells of the aquatic plant, Chara, exhibit a rapid (>100 × 10-6 m s-1) cyclic cytoplasmic streaming which stops in response to mechanical stimuli. Since the streaming - and the stopping of streaming upon stimulation - is easily visible with a stereomicroscope, these single cells are ideal tools to investigate mechanosensing in plant cells, as well as the potential for these cells to be anesthetized. We found that dropping a steel ball (0.88 × 10-3 kg, 6 × 10-3 m in diameter) through a 4.6 cm long tube (delivering ca. 4 × 10-4 J) reliably induced mechanically-stimulated cessation of cytoplasmic streaming. To determine whether mechanically-induced cessation of cytoplasmic streaming in Chara was sensitive to anesthesia, we treated Chara internodal cells to volatilized chloroform in a 9.8 × 10-3 m3 chamber for 2 minutes. We found that low doses (15,000-25,000 ppm) of chloroform did not always anesthetize cells, whereas large doses (46,000 and higher) proved lethal. However, 31,000 ppm chloroform completely, and reversibly, anesthetized these cells in that they did not stop cytoplasmic streaming upon mechanostimulation, but after 24 hours the cells recovered their sensitivity to mechanostimulation. We believe this single-cell model will prove useful for elucidating the still obscure mode of action of volatile anesthetics.

Principles of organelle positioning in motile and non-motile cells.

Cells are equipped with asymmetrically localised and functionally specialised components, including cytoskeletal structures and organelles. Positioning these components to specific intracellular locations in an asymmetric manner is critical for their functionality and affects processes like immune responses, tissue maintenance, muscle functionality, and neurobiology. Here, we provide an overview of strategies to actively move, position, and anchor organelles to specific locations. By conceptualizing the cytoskeletal forces and the organelle-to-cytoskeleton connectivity, we present a framework of active positioning of both membrane-enclosed and membrane-less organelles. Using this framework, we discuss how different principles of force generation and organelle anchorage are utilised by different cells, such as mesenchymal and amoeboid cells, and how the microenvironment influences the plasticity of organelle positioning. Given that motile cells face the challenge of coordinating the positioning of their content with cellular motion, we particularly focus on principles of organelle positioning during migration. In this context, we discuss novel findings on organelle positioning by anchorage-independent mechanisms and their advantages and disadvantages in motile as well as stationary cells.

Oscillations of chlorophyll fluorescence after plasma membrane excitation in Chara originate from nonuniform composition of signaling metabolites in the streaming cytoplasm.

Excitable cells of higher plants and characean algae respond to stressful stimuli by generating action potentials (AP) whose regulatory influence on chlorophyll (Chl) fluorescence and photosynthesis extends over tens of minutes. Unlike plant leaves where the efficiency of photosystem II reaction (YII) undergoes a separate reversible depression after an individual AP, characean algae exhibit long-lasting oscillations of YII after firing AP, provided that Chl fluorescence is measured on microscopic cell regions. Internodal cells of charophytes feature an extremely fast cytoplasmic streaming that stops immediately during the spike and recovers within ~10 min after AP. In this study a possibility was examined that multiple oscillations of YII and Chl fluorescence parameters (F', Fm') result from the combined influence of metabolic rearrangements in chloroplasts and the cyclosis cessation-recovery cycle induced by the Ca2+ influx during AP. It is shown that the AP-induced Fm' and YII oscillations disappear when the fluidic communications between the analyzed area (AOI) and surrounding cell regions are restricted or eliminated. The microfluidic signaling was manipulated in two ways: by narrowing the illuminated cell area and by arresting the cytoplasmic streaming with cytochalasin D (CD). The inhibition of Fm' and YII oscillations was not caused by the loss of cell excitability, since CD-treated cells retained the capacity of AP generation. The mechanism of AP-induced oscillations of YII and Chl fluorescence seems to involve the lateral microfluidic transport of signaling substances in combination with the distribution pattern of these substances that was enhanced during the period of streaming cessation.

Cortex-driven cytoplasmic flows in elongated cells: fluid mechanics and application to nuclear transport in Drosophila embryos.

The Drosophila melanogaster embryo, an elongated multi-nucleated cell, is a classical model system for eukaryotic development and morphogenesis. Recent work has shown that bulk cytoplasmic flows, driven by cortical contractions along the walls of the embryo, enable the uniform spreading of nuclei along the anterior-posterior axis necessary for proper embryonic development. Here, we propose two mathematical models to characterize cytoplasmic flows driven by tangential cortical contractions in elongated cells. Assuming Newtonian fluid flow at low Reynolds number in a spheroidal cell, we first compute the flow field exactly, thereby bypassing the need for numerical computations. We then apply our results to recent experiments on nuclear transport in cell cycles 4-6 of Drosophila embryo development. By fitting the cortical contractions in our model to measurements, we reveal that experimental cortical flows enable near-optimal axial spreading of nuclei. A second mathematical approach, applicable to general elongated cell geometries, exploits a long-wavelength approximation to produce an even simpler solution, with errors below [Formula: see text] compared with the full model. An application of this long-wavelength result to transport leads to fully analytical solutions for the nuclear concentration that capture the essential physics of the system, including optimal axial spreading of nuclei.

Go with the flow - bulk transport by molecular motors.

Cells are the smallest building blocks of all living eukaryotic organisms, usually ranging from a couple of micrometers (for example, platelets) to hundreds of micrometers (for example, neurons and oocytes) in size. In eukaryotic cells that are more than 100 µm in diameter, very often a self-organized large-scale movement of cytoplasmic contents, known as cytoplasmic streaming, occurs to compensate for the physical constraints of large cells. In this Review, we discuss cytoplasmic streaming in multiple cell types and the mechanisms driving this event. We particularly focus on the molecular motors responsible for cytoplasmic movements and the biological roles of cytoplasmic streaming in cells. Finally, we describe bulk intercellular flow that transports cytoplasmic materials to the oocyte from its sister germline cells to drive rapid oocyte growth.

Cytoplasmic streaming induced by intracytoplasmic spindle translocation contributes to developmental competence through mitochondrial distribution in mouse oocytes.

OBJECTIVE: To evaluate the developmental competency of mouse metaphase II oocytes and the pattern of mitochondrial positioning through cytoplasmic streaming in mouse metaphase II oocytes. DESIGN: We observed cytoplasmic streaming as movement indicated by fluorescently stained mitochondria using a newly developed method in which the spindle is translocated to the opposite site of the oocyte. This method is termed as intracytoplasmic spindle translocation (ICST). SETTING: University research laboratory. ANIMALS: Female B6D2F1 mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fresh oocytes, postovulatory-aged oocytes, and oocytes treated with cytochalasin B were classified based on the presence of cytoplasmic streaming induced by ICST. The pattern of redistributed mitochondria and developmental competence caused by parthenogenetic activation were evaluated in oocytes with or without cytoplasmic streaming. RESULT(S): Induced cytoplasmic streaming occurred in 84% of the fresh oocytes but not in the postovulatory-aged oocytes and the oocytes treated with cytochalasin B. Abnormal mitochondrial aggregation was observed in oocytes in which cytoplasmic streaming was not induced. Furthermore, the developmental competence was significantly lower in oocytes without cytoplasmic streaming. CONCLUSION(S): Cytoplasmic streaming induced by ICST contributes to developmental competence through the redistribution of mitochondria and may be a valuable criterion for predicting early developmental competence in mouse oocytes.

Microfluidic interactions involved in chloroplast responses to plasma membrane excitation in Chara.

Adaptation of plants to environmental changes involves the mechanisms of long-distance signaling. In characean algae, these mechanisms comprise the propagation of action potential (AP) and the rotational cytoplasmic streaming acting in cooperation with light-dependent exchange of ions and metabolites across the chloroplast envelope. Both excitability and cyclosis exert conspicuous effects on photosynthetic activity of chloroplasts but possible influence of cyclosis arrest on the coupling of AP stimulus to photosynthetic performance remained unexplored. In this study, fluidic interactions between anchored chloroplasts were allowed or restricted by illuminating the whole internode or a confined cell area (2 mm in diameter), respectively. Measurements of chlorophyll fluorescence parameters (F' and Fm') in cell regions located close to calcium crystal depositions revealed that the AP generation induced long-lasting Fm' oscillations that persisted in illuminated cells. The AP generation often induced the F' oscillations, whose number diminished upon the transfer of internodal cells from total to local background light. The results indicate that the AP-induced changes in photosynthetic parameters, F' in particular, have a complex origin and comprise the internal processes caused by the elevation of stromal Ca2+ concentration in the analyzed chloroplasts and the stages related to ion and metabolite exchange mediated by cytoplasmic streaming. It is supposed that the composition of flowing cytoplasm is heterogeneous due to the spatial alteration of calcified and noncalcified cell sites, but this heterogeneity is enhanced and can be visualized after the transient cessation and restoration of cytoplasmic streaming.

Discovery of ultrafast myosin, its amino acid sequence, and structural features.

Cytoplasmic streaming with extremely high velocity (∼70 µm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 µm s-1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 µm s-1, one-third of the cytoplasmic streaming velocity in Chara Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 µm s-1 These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

Control of the Actin Cytoskeleton Within Apical and Subapical Regions of Pollen Tubes.

In flowering plants, sexual reproduction involves a double fertilization event, which is facilitated by the delivery of two non-motile sperm cells to the ovule by the pollen tube. Pollen tube growth occurs exclusively at the tip and is extremely rapid. It strictly depends on an intact actin cytoskeleton, and is therefore an excellent model for uncovering the molecular mechanisms underlying dynamic actin cytoskeleton remodeling. There has been a long-term debate about the organization and dynamics of actin filaments within the apical and subapical regions of pollen tube tips. By combining state-of-the-art live-cell imaging with the usage of mutants which lack different actin-binding proteins, our understanding of the origin, spatial organization, dynamics and regulation of actin filaments within the pollen tube tip has greatly improved. In this review article, we will summarize the progress made in this area.

Organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana depend on class XI myosins.

F-actin and myosin XI play important roles in plant organelle movement. A few myosin XI genes in the genome of Arabidopsis are mainly expressed in mature pollen, which suggests that they may play a crucial role in pollen germination and pollen tube tip growth. In this study, a genetic complementation assay was conducted in a myosin xi-c (myo11c1) myosin xi-e (myo11c2) double mutant, and fluorescence labeling combined with microscopic observation was applied. We found that myosin XI-E (Myo11C2)-green fluorescent protein (GFP) restored the slow pollen tube growth and seed deficiency phenotypes of the myo11c1 myo11c2 double mutant and Myo11C2-GFP partially colocalized with mitochondria, peroxisomes and Golgi stacks. Furthermore, decreased mitochondrial movement and subapical accumulation were detected in myo11c1 myo11c2 double mutant pollen tubes. Fluorescence recovery after photobleaching experiments showed that the fluorescence recoveries of GFP-RabA4d and AtPRK1-GFP at the pollen tube tip of the myo11c1 myo11c2 double mutant were lower than those of the wild type were after photobleaching. These results suggest that Myo11C2 may be associated with mitochondria, peroxisomes and Golgi stacks, and play a crucial role in organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana.

Tracking organelle movements in plant cells.

Cytoplasmic streaming is characterized by the rapid movement of organelles and other cellular components throughout the cell. In plants, the process depends on actin filaments and myosin motor proteins and plays an important role in cell growth. Detailed quantification of organelle movements can yield important insights into the dynamics of intracellular organization and its functional aspects. This quantification is hindered by a lack of knowledge of the different types of movements and generic tracking algorithms that yield erroneous output. This protocol provides a step-by-step guide to the detection and measurement of organelle motility as well as a description of additional analysis steps distinguishing undirected, diffusion-like movements from directional movements along the actin cytoskeleton.

Going with the flow: insights from Caenorhabditis elegans zygote polarization.

Cell polarity is the asymmetric distribution of cellular components along a defined axis. Polarity relies on complex signalling networks between conserved patterning proteins, including the PAR (partitioning defective) proteins, which become segregated in response to upstream symmetry breaking cues. Although the mechanisms that drive the asymmetric localization of these proteins are dependent upon cell type and context, in many cases the regulation of actomyosin cytoskeleton dynamics is central to the transport, recruitment and/or stabilization of these polarity effectors into defined subcellular domains. The transport or advection of PAR proteins by an actomyosin flow was first observed in the Caenorhabditis elegans zygote more than a decade ago. Since then a multifaceted approach, using molecular methods, high-throughput screens, and biophysical and computational models, has revealed further aspects of this flow and how polarity regulators respond to and modulate it. Here, we review recent findings on the interplay between actomyosin flow and the PAR patterning networks in the polarization of the C. elegans zygote. We also discuss how these discoveries and developed methods are shaping our understanding of other flow-dependent polarizing systems. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Characterization of ancestral myosin XI from Marchantia polymorpha by heterologous expression in Arabidopsis thaliana.

Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 µm s-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.

Transient depletion of transported metabolites in the streaming cytoplasm of Chara upon shading the long-distance transmission pathway.

Export of reducing power from chloroplasts to cytoplasm serves to balance the NADPH/ATP ratio that is optimal for CO2 assimilation. Rapid cytoplasmic streaming in characean algae conveys the exported metabolites downstream towards the shaded plastids where envelope transporters may operate for the import of reducing power in accordance with the direction of concentration gradients. Import of reducing equivalents by chloroplasts in the analyzed area transiently enhances the pulse-modulated chlorophyll fluorescence F' controlled by the redox state of photosystem II acceptor QA. When the microfluidic pathway was transferred to darkness while the analyzed cell area remained in dim background light, the amplitude of cyclosis-mediated F' changes dropped sharply and then recovered within 5-10 min. The suppression of long-distance signaling indicates temporal depletion of transmitted metabolites in the streaming cytoplasm. The return to overall background illumination induced an exceptionally large F' response to the first local light pulse admitted to a remote cell region. This indicates the appearance of excess reductants in the streaming cytoplasm at a certain stage of photosynthetic induction. The results suggest highly dynamic exchange of metabolites between stationary chloroplasts lining the microfluidic pathway and the streaming cytoplasm upon light-dark and dark-light transitions. Evidence is obtained that slow stages of chlorophyll fluorescence induction in algae with rapid cytoplasmic streaming directly depend on cyclosis-mediated long-distance delivery of metabolites produced far beyond the analyzed cell area.

Role of Proton Motive Force in Photoinduction of Cytoplasmic Streaming in Vallisneria Mesophyll Cells.

In mesophyll cells of the aquatic monocot Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.

The diffusive injection micropipette (DIMP).

The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.

Profiles of Physarum Microplasmodial Phosphatase Activity Crucial to Cytoplasmic Streaming and Spherule Formation.

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.