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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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Export of reducing power from chloroplasts to cytoplasm serves to balance the NADPH/ATP ratio that is optimal for CO 2 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 Q A . 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We introduce a filament-based simulation model for coarse-grained, effective motor-mediated interaction between microtubule pairs to study the time-scales that compose cytoplasmic streaming. We characterise microtubule dynamics in two-dimensional systems by chronologically arranging five distinct processes of varying duration that make up streaming, from microtubule pairs to collective dynamics. The structures found were polarity sorted due to the propulsion of antialigned microtubules. This also gave rise to the formation of large polar-aligned domains, and streaming at the domain boundaries. Correlation functions, mean squared displacements, and velocity distributions reveal a cascade of processes ultimately leading to microtubule streaming and advection, spanning multiple microtubule lengths. The characteristic times for the processes extend over three orders of magnitude from fast single-microtubule processes to slow collective processes. Our approach can be used to directly test the importance of molecular components, such as motors and crosslinking proteins between microtubules, on the collective dynamics at cellular scale., Competing Interests: AR, ÖD, MH, GS, GV, TA, GG No competing interests declared, (© 2019, Ravichandran et al.)

During physiological processes, cells can undergo morphological changes that can result in a significant redistribution of the cytoskeleton causing anisotropic behavior. Evidence of anisotropy in cells under mechanical stimuli exists; however, the role of cytoskeletal restructuring resulting from changes in cell shape in mechanical anisotropy and its effects remain unclear. In the present study, we examine the role of cell morphology in inducing anisotropy in both intracellular mechanics and dynamics. We change the aspect ratio of cells by confining the cell width and measuring the mechanical properties of the cytoplasm using optical tweezers in both the longitudinal and transverse directions to quantify the degree of mechanical anisotropy. These active microrheology measurements are then combined with intracellular movement to calculate the intracellular force spectrum using force spectrum microscopy (FSM), from which the degree of anisotropy in dynamics and force can be quantified. We find that unrestricted cells with aspect ratio (AR) ∼1 are isotropic; however, when cells break symmetry, they exhibit significant anisotropy in cytoplasmic mechanics and dynamics.

Cytoplasmic streaming is essential for intracellular communications but its specific functions are not well known. In Chara corallina internodes, long-distance interactions mediated by cyclosis are clearly evident with microscopy-pulse amplitude modulation (PAM) fluorometer under application of localized light (LL) pulses to a remote cell region. Measurements of LL-induced profiles of chlorophyll fluorescence F' at various distances from the LL source suggest that illuminated chloroplasts release into the streaming cytoplasm excess reducing equivalents that are entrained by the fluid flow and transiently reduce the intersystem electron carriers in chloroplasts of downstream shaded areas. The reducing equivalents propagate to distances up to 4.5 mm from the LL source, with the transport rate nearly equal to the velocity of liquid flow. The F' transients disappeared after the arrest of streaming with cytochalasin D and reappeared upon its recovery in washed cells. The F' responses to a distant LL were used as an indicator for the passage of cytosolic reductants across the analyzed cell area during measurements of cell surface pH (pH o ) in intact and microperforated internodes. In microwounded cell regions, the LL-induced increase in F' occurred synchronously with the increase in pH o , by contrast to a slight decrease in pH o observed prior to perforation. The results show that reducing agents transported with the cytoplasmic flow are involved in rapid pH changes on the surface of microinjured cells. A possibility is considered that cytoplasmic reductants are processed by stress-activated plasmalemmal NADPH oxidase carrying electrons to oxygen with the eventual H + consumption on the outer cell side.

Explanations for protoplasmic streaming began with appeals to contraction in the eighteenth century and ended with appeals to contraction in the twentieth. During the intervening years, biologists proposed a diverse array of mechanisms for streaming motions. This paper focuses on the re-emergence of contraction among the molecular mechanisms proposed for protoplasmic streaming during the twentieth century. The revival of contraction is a result of a broader transition from colloidal chemistry to a macromolecular approach to the chemistry of proteins, the recognition of the phenomena of shuttle streaming and the pulse of protoplasm, and the influential analogy between protoplasmic streaming and muscle contraction., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, we show that RNP particle motility is required for long-term germ plasm retention. We propose that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

There is a paucity of knowledge on how mRNA transcripts in the spatially crowded, but molecularly organized bacterial cytoplasm contact the 30S ribosomal subunits. Does simple diffusion in the cytoplasm account for transcript-ribosome interactions given that a large number of ribosomes (e.g., about 72,000 in Escherichia coli during exponential growth) can be present in the cytoplasm? Or are undiscovered mechanisms present where specific transcripts are directed to specific ribosomes at specific cytoplasmic locations, while others are mobilized in a random manner? Moreover, is it possible that cytoplasmic mobilization occurs in bacteria, driven possibly by thermal infrared (IR) radiation and the generation of exclusion zone (EZ) water? These aspects will be discussed in this article and hypotheses presented., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-gamma mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

An attempt is made to reconcile experimental data dealing with, inter alia, cytoplasmic streaming in Characean algae, contraction in actomyosin systems. Na+- and -K+-simtulated ATPase activity and the ultrastructure of brush border microvilli. It is postulated that myosin molecules transfer energy from ATP to an actin-containing filament and that a high energy conformation is subsequently propagated along the filament. At regularly spaced intervals corresponding to the length of an actin-tropomyosin subunit, the propagation of high energy involves rejection of a pressure pulse in the direction of cytoplasmic streaming. Proteins in solution capable of storing the thermodynamic energy represented by the pressure pulse will either migrate in the opposite direction or conserve the quantized cytoplasmic flow generated by the actin-containing filaments. At sites where actin filaments are attached to the plasma membrane the high energy is propagated in another direction leading to expulsion of sodium ions and neutralization of the vectorial pressure pulse.

Fluorescence microphotolysis was used to measure nucleocytoplasmic flux in single rat hepatocytes for a series of dextrans ranging in molecular mass from 3 to 150 kd. The cytoplasmic translational diffusion coefficient DC and the nucleoplasmic diffusion coefficient DN of a 62-kd dextran were also determined. DC was approximately 2 X 10(-8) and DN approximately 3 X 10(-8) cm2/s, i.e., 1/20-1/15 of the value in free solution. The mobile fraction amounted to 0.7-0.8 in measurements of both intracellular diffusion and nucleo-cytoplasmic flux. The flux of dextrans from cytoplasm to nucleus depended inversely on molecular mass with an exclusion limit between 17 and 41 kd suggesting that the nuclear envelope has functions of a molecular sieve. Employing the Pappenheimer-Renkin equations, a functional pore radius of 50-56 A was derived. By comparison with recent measurements on isolated liver cell nuclei, large quantitative differences between the intracellularly located and the isolated nucleus were revealed.

We report the first direct demonstration that the cytoplasmic transport of organelles and vesicles (collectively called particles) takes place along microtubules. Living keratocytes from the corneal stroma of the frog, Rana pipiens were observed with Allen video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy [Allen et al, 1981]. In sufficiently thin regions of these cells a network of linear elements was visible. When particles were observed in motion, they always moved along these linear elements. The linear elements remained intact and in focus on the microscope when lysed in a cell lysis solution that stabilized microtubules. Preparations were then fixed in formaldehyde, washed with phosphate-buffered saline (PBS), incubated with rabbit antitubulin, washed with PBS, stained with rhodamine-conjugated goat antirabbit, and washed with PBS. The extracted cells continued to remain in place and in focus on the microscope throughout these procedures. The same cells were then observed using epifluorescence optics and a silicon-intensified target (SIT) video camera. A network of fluorescent linear elements was seen to correspond in number, form, and position to the linear elements seen in the live AVEC-DIC image. Taken together, the AVEC-DIC and fluorescence microscopy observations prove that the linear elements along which particles move are microtubules (MTLEs). The observed particle speeds, pause times, and distances moved varied widely, even for the same particle on the same microtubule. Particles were also observed to switch from one microtubule to another as they were transported. The polarity of the microtubules did not seem to affect the particle direction, since particles were observed to move in both directions on the same MTLE. When not in motion these particles behaved as if anchored to the microtubules since they showed negligible Brownian motion. Finally, it was observed that an elongate particle could move onto two intersecting linear elements such that it was deformed into an inverted "Y" shape. This indicates that there may be more than a single site of attachment between the force generator and the particle.

Fully polymerization-competent fluorescein-labeled actin from skeletal muscle was microinjected into both normal moving and experimentally treated Amoeba proteus. Its intracellular distribution was followed by integral image intensification of the fluorescence on a television screen and compared with controls injected with rhodamine-labeled serum albumin. The labeled actin was incorporated into the endogenous actin pool and exhibited a characteristic redistribution depending on the cellular morphology. Increased amounts of labeled actin could be detected within a thin layer separating the hyalo- and granuloplasm or running immediately beneath the plasma membrane when hyaloplasmic regions were absent. The topography of the fluorescent layer demonstrated in living cells is in agreement with the cortical microfilament layer described ultrastructurally recently in corresponding cells. The combined results emphasize the important role of the cortical filament layer in both morphogenetic processes (e.g., hyalo-granuloplasm separation or changes in cell shape) and motive force generation for cytoplasmic streaming and amoeboid movement.

The streaming endoplasm of characean cells has been shown to contain previously unreported endoplasmic filaments along which bending waves are observed under the light microscope using special techniques. The bending waves are similar to those propagated along sperm tails causing propulsion of sperm. In Nitella there is reason to believe that nearly all of the filaments are anchored in the cortex and that their beating propels the endoplasm in which they are suspended. This hypothesis is supported by calculations in which typical and average wave parameters have been inserted into the classical hydrodynamic equations derived for sperm tail bending waves. These calculations come within an order of magnitude of predicting the velocity of streaming and they show that waves of the character described, propagated along an estimated 52 m of endoplasmic filaments per cell, must generate a total motive force per cell within less than an order of magnitude of the forces measured experimentally by others. If we assume that undulating filaments produce the force driving the endoplasm, then the method described for measuring the motive force could lead to a lower than actual value for the motive force, since both centrifugation and vacuolar perfusion would reverse the orientation of some filaments. Observations of the initiation of particle translation in association with the filaments suggest that particle transport and wave propagation, which occur at the same velocity, may both be dependent on the same process. The possibility that some form of contractility provides the motive force for filament flection and particle transport is discussed.

In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 11 a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.

The electric potential difference (1 to 15 mv.) between two loci of the slime mold connected with a strand of protoplasm changes rhythmically with the same period (60 to 180 seconds) as that of back and forth protoplasmic streaming along the strand. When atmospheric pressure at a part of the plasmodium is increased (about 10 cm. H(2)O), the electric potential at this part becomes positive (0 to 20 mv.) to another part with a time constant of 2 to 15 minutes. If the atmospheric pressure at a part of the plasmodium is changed (about 10 cm. H(2)O) periodically, the electric potential rhythm also changes with the same period as that of the applied pressure change, and the amplitude of the former grows to a new level (i.e., forced oscillation). The electric potential rhythm, in this case, is generally delayed about 90 degrees in phase angle from the external pressure change. The period of the electric potential rhythm which coincided with that of the pressure change is maintained for a while after stopping the application of the pressure change, if the period is not much different from the native flow rhythm. Such a pressure effect is brought about by the forced transport of protoplasm and is reversible as a rule. In the statistical analysis made by Kishimoto (1958) and in the rheological treatment made in the report, the rhythmic deformation of the contractile protein networks is supposed to be the cause of the protoplasmic flow along the strand and of the electric potential rhythm. The role of such submicroscopic networks in the protoplasm in various kinds of protoplasmic movement is emphasized.

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q(10) of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.

Plasmodia of the acellular slime mold, Physarum polycephalum, reveal a complex and changing pattern of birefringence when examined with a sensitive polarizing microscope. Positively birefringent fibrils are found throughout the ectoplasmic region of the plasmodium. In the larger strands they may be oriented parallel to the strand axis, or arranged circularly or spirally along the periphery of endoplasmic channels. Some fibrils exist for only a few minutes, others for a longer period. Some, particularly the circular fibrils, undergo changes in birefringence as they undergo cyclic deformations. In the ramifying strand region and the advancing margin there is a tendency for fibrils of various sizes to become organized into mutually orthogonal arrays. In some plasmodia the channel wall material immediately adjacent to the endoplasm has been found to be birefringent. The sign of endoplasmic birefringence is negative, and its magnitude is apparently constant over the streaming cycle. The pattern of plasmodial birefringence and its changes during the shuttle streaming cycle of Physarum are considered in the light of several models designed to explain either cytoplasmic streaming alone or the entire gamut of plasmodial motions. The results of this and other recent physical studies suggest that both streaming and the various other motions of the plasmodium may very likely be explained in terms of coordinated contractions taking place in the fibrils which are rendered visible in polarized light.

The electric potential difference (1 to 15 mv.) between two loci of the slime mold connected with a strand of protoplasm changes rhythmically with the same period (60 to 180 seconds) as that of the back and forth protoplasmic streaming along the strand. Generally some phase difference is observed between them. Periods of the electric potential rhythm show a Gaussian distribution. Amplitudes give a somewhat different distribution curve. Wave forms are not always simple harmonic ones, but are distorted more or less. However, auto-correlation analysis proves that there is a dominant rhythm of a nearly constant period which coincides with the mean period of the Gaussian distribution curve. Calculations made on an assumption that the electric potential rhythm is the result of many elementary rhythms (i.e., same periodicity, arbitrary phase angles) distributed throughout the plasmodium, give a satisfactory coincidence with the observed distribution for the amplitude. The predominance of a rhythm of a nearly constant periodicity suggests the existence of well organized interactions among components of a contractile protein network, the rhythmic deformation of which is supposed to be responsible for the protoplasmic streaming and for the electric potential rhythm.