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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.

Kavar(21g), a dominant female-sterile mutation of Drosophila, identifies the alphaTubulin67C gene that encodes alpha4-tubulin, the maternally provided alpha-tubulin isoform. Although alpha4-tubulin is synthesized during oogenesis, its function is required only in the early cleavage embryos. However, once present in the developing oocyte, much of the alpha4-tubulin and the Kavar(21g)-encoded E426K-alpha4-tubulin molecules become incorporated into the microtubules. We analyzed ooplasmic streaming and lipid-droplet transport, with confocal reflection microscopy, in the developing egg primordia in the presence and absence of alpha4-tubulin and E426K-alpha4-tubulin and learnt that the E426K-alpha4-tubulin molecules eliminate ooplasmic streaming and alter lipid-droplet transport. Apparently, Glu426 is involved in stabilization of the microtubule-kinesin complexes when the kinesins are in the most labile, ADP-bound state. Replacement of Glu426 by Lys results in frequent detachments of the kinesins from the microtubules leading to reduced transport efficiency and death of the embryos derived from the Kavar(21g)-carrying females. Glu426 is a component of the twelfth alpha-helix, which is the landing and binding platform for the mechanoenzymes. Since the twelfth alpha-helix is highly conserved in the alpha-tubulin family, Glu415, which corresponds to Glu426 in the constitutively expressed alpha-tubulins, seems be a key component of microtubule-kinesin interaction and thus the microtubule-based transport.

Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that ∼50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in vivo fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation ΔkinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtbule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

The highly elongated bristles of Drosophila have proven to be a valuable model system for studying cellular morphogenesis. Extending bristles contain a series of large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules. Models to explain the extension of the bristle have principally focused on the assembly of actin filaments at the distal tip of the bristle. We have used time-lapse observations of wild-type and mutant bristles and the related arista laterals and come to the conclusion that growth takes place throughout the growing cellular extension. This distributed growth can explain the behavior of split laterals and the shape changes seen at the tip during bristle and lateral outgrowth. Inhibitor studies suggest that the microtubule cytoskeleton is essential for maintaining the highly biased axial growth of these structures. We have used fluorescence recovery after photo-bleaching to study the dynamics of the cytoskeleton during bristle growth. Our experiments show that actin bundles in growing bristles are quite stable and move in a retrograde fashion. The bristle microtubules are less stable. The retrograde movement of the peripheral actin appears to be counterbalanced by the distally directed movement of cytoplasm in the center of the bristle.

Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction. These results validate the use of Drosophila as a tool to study the molecular mechanism of viral gene products and suggest that Tat-tubulin interaction is responsible for neurodegenerative diseases associated with AIDS.

Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few micrometre in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The movement of the protoplasm is retarded by increases in calcium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain preferentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The complete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the presence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Conditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plasmolysis and laser microperforation. This laser technique should be suitable for the performance of electrophysiological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells.

The vacuole system in growing hyphal tips of Pisolithus tinctorius is a dynamic continuum of vacuoles and extensible tubular elements. The system varies from a tubular reticulum with few vacuoles across a spectrum of intermediate forms to clusters of vacuoles with few tubules. Spherical vacuoles interconnected in clusters are situated at intervals along the hyphal tip and are transiently linked by tubules that extend from a vacuole in one cluster and fuse with that of another. Extension and retraction of the tubules is independent of cytoplasmic streaming, can occur in either direction, and covers distances as great as 60 micrometre. The tubules pulsate and peristalsis-like movements transfer globules of material along them between the vacuoles in different clusters. The tubules also generate vacuoles. The tubular system has the potential for intracellular transport of solutes in the hyphal tips without concomitant transfer of large amounts of membrane. This contrasts with models of intracellular transport via vesicles, where the ratio of membrane transferred to internal content is very much higher. The system has many features in common with tubular endosomal and lysosomal systems in cultured animal cells.

Cytasters were induced in the unfertilized eggs of the sea urchin Lytechinus pictus by two different methods: (1) treatment with hexylene glycol or taxol, which are known to lower the tubulin critical concentration in vitro, but do not activate the nuclear cycle, and (2) by raising the cytoplasmic pH from that of unfertilized cytoplasm to that of fertilized, which activated the nuclear cycle and initiated tubulin polymerization. In the unactivated eggs, with increasing concentrations of the inducing agents, temperature and duration of treatment, microtubule structures that formed showed a progression from loose microtubule networks and spiral arrays to tightly organized cytasters. In eggs incubated in sea water containing 2.5 or 10 mM ammonium acetate titrated with HCl or NaOH in steps from pH 5.0 to pH 9.0, the nuclear cycle and tubulin polymerization were initiated at about pH 7.0. The degree of development attainable after three hours was dependent on the pH, with spirals forming at the threshold level of pH 7.0, monasters at pH 7.5, and at pH 8.5 cells formed cytasters, multipolar spindles and even completed multipolar divisions. In unactivated eggs, tubulin was made available by lowering of the tubulin critical concentration; in activated eggs it was made available from some previously unavailable store. The evidence suggests that the amount of assembly-competent tubulin available, regardless of how it is made available, determines the type of structure that is formed, with microtubule bundles and spirals at the lower concentrations and functional cytasters at the upper. We also describe some details of cytaster formation, including the role of microtubule interactions and the movement of dense granules to the aster centers in hexylene glycol-treated eggs.

Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein Mast/Orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that Mast accumulates at kinetochores early in mitosis, which is followed by a poleward streaming upon microtubule attachment. This leads to a reduction of Mast levels at kinetochores during metaphase and anaphase that depends largely on the microtubule minus end-directed motor cytoplasmic dynein. Surprisingly, we also found that co-depletion of Dynein rescues spindle bipolarity in Mast-depleted cells, while restoring normal microtubule poleward flux. Our results suggest that Mast and Dynein have antagonistic roles in the local regulation of microtubule plus-end dynamics at kinetochores, which are important for the maintenance of spindle bipolarity and normal spindle length.

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal ∼30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 micrograms/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB.(ABSTRACT TRUNCATED AT 250 WORDS)

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained. The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 °C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a positionspecific manner throughout the embryo, the shortest cycles being of 65 min duration. During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator. The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid. The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.

Previous investigators have proposed that cytoplasmic streaming in Chara internodal cells results from the interaction between an endoplasmic factor and fibrils composed of microfilaments in the stationary cortex. Using the internal perfusion technique, we confirmed the observation that organelles which had been attached to the fibrils by decreasing the internal concentration of ATP moved along the fibrils after ATP was introduced. Thin-sectioned specimens revealed that endoplasmic organelles of various shapes were linked to microfilament bundles in the absence of ATP. Linkage was effected by regularly arranged electron-dense materials with a spacing of 100–130 nm at definite regions on each organelle. The organelles in question were studied in negatively stained preparations of endoplasm. The organelles had some common features. (1) They were all membrane-limited.(2) Their sizes and configurations varied largely. (3) One or more protuberances were present on them. (4) The protuberances were usually rod- or horn-like. (5) Small globular bodies 20–30 nm in diameter were found in ordered array with the same spacing as those in thin sections at the surface of the protuberances. (6) Many fine filaments were always attached to the surface of the protuberances. These fine filaments differed from F-actin in diameter (less than 4 nm) and inability to react with heavy meromyosin from rabbit skeletal muscle. The role of such components of the organelles in cytoplasmic streaming is discussed. A paracrystalline array of microfilaments with a transverse periodicity of about 38 nm is presented, together with its optical diffraction pattern.

Algae-free Paramecium bursaria was exposed to cells of Chlorella sp. for 30s (pulse) and then incubated in algae-free medium for periods between 0 and 15 min. During this chase the fate of the vacuoles formed during the exposure to algae was followed in order to reveal the moment of perialgal vacuole (PV) formation. PVs are characterized by always enclosing an individual algal cell and thus differ from digestive vacuoles (DVs). PVs did not appear immediately after the pulse, but were found only in cells chased for at least 3 min. In those ciliates the algae-containing vacuoles were more than 3 min old and located in the middle part of the cell. These results showed that PVs were not formed directly at the cytopharynx, but many algae were at first enclosed together in large DVs. After the release from the cytopharynx DVs undergo a sequence of fusion events during their cyclosis: fusion with acidosomes apparently occurs at the cell's posterior end, not later than 2 min, and fusion with lysosomes in the middle region of the cell at the earliest at about 7 min, after the pinching off of a DV from the cytopharynx. Thus, PVs appeared to develop from condensing DVs after acidosomal but before lysosomal fusion. As the first step, part of the DV enclosing an individual algal cell must detach from the large vacuole. Further steps and the implications of the mechanism of PV formation resulting in the re-establishment of endosymbionts in P. bursaria are discussed.

As a contribution to the understanding of the mechanism of the collective motion of the cell mass in the migrating pseudoplasmodium its motive force has been determined. The measurements in the present paper were made by an original method using centrifugal force. The maximum value of the motive force of a migrating pseudoplasmodium in the dark was estimated as 0.6-1.6 dyn. This value is comparable to the motive force of protoplasmic streaming in Physarum polycephalum. In addition, the motive force in phototactic migration, which has not previously been estimated, was measured. Illumination from the front increased the motive force, whereas illumination from the back produced the opposite results. That is to say, illumination could directly influence the motive force. Furthermore, some interesting results were obtained by measuring the changes in the velocity of the pseudoplasmodia placed under four conditions (combinations of illuminated or non-illuminated, and parallel or antiparallel to the direction of the external force). These results suggest the necessity of taking into consideration the direct effect of light upon the motility of the cells in the migrating pseudoplasmodium.

During the last phase of oogenesis in Drosophila, nurse cell cytoplasm can be seen to be streaming into the growing oocyte when visualized in time-lapse films. This process can be reversibly inhibited by cytochalasins. The distribution of F-actin filaments in the nurse cells has been studied by staining with rhodamine-conjugated phalloidin. At the beginning of cytoplasmic streaming (stage 10B) increasingly thick bundles of microfilaments formed, many of which spanned the nurse cell cytoplasm from the cell membrane to the nuclear membrane. The association of F-actin with the nuclear membrane persisted when nurse cell nuclei were isolated mechanically. The experimental evidence suggests that microfilament contraction in the nurse cells leads to cytoplasmic streaming by pressure flow.

Induction and cessation of the rotational cytoplasmic streaming in Vallisneria mesophyll cells could be controlled by external stimuli. In cells that had been kept in darkness the cytoplasm remained quiescent. However, when the cells were treated in the dark with EGTA solution (10 mM or 20 mM buffered with 10 mM-Tris-maleate at pH 7.0), rotational cytoplasmic streaming was induced. When the cells were transferred again to artificial pond water in the dark, the induced streaming was inhibited; that is, only 50% of the observed cells exhibited active streaming after 2 h. When the cells were irradiated continuously with far-red light (lambda max = 750 nm, 0.4 W/m2) in the same external medium, the induced streaming was inhibited almost completely within 2 h. The relative quantum effectiveness of monochromatic light (450–800 nm) in producing cessation of streaming was also investigated. Irradiation with light of 450, 550 and 600 nm was almost as effective as darkness. Light of 500 and 650 nm was less effective than dark exposure. Only irradiation at 750 nm stopped streaming in almost all cells. But when calcium was excluded from the external medium, the effect of far-red light decreased to almost the dark control level. Light of 800 nm also inhibited the streaming but the effect was much less than that of far-red light. Microfilaments in bundles with the long axis parallel to the streaming direction were localized in the vicinity of the cell membrane. Their configuration, localization and distribution were the same in the present experimental system irrespective of whether the cytoplasm was streaming or quiescent. Intracellular calcium was examined by electron microscopic cytochemistry and X-ray microanalysis. In cells with streaming induced by EGTA, only a small amount of calcium-containing precipitates formed in the cytoplasm in the presence of antimony. A few precipitates were found in the chloroplasts, the middle lamella of the cell wall and at the border between the cytoplasm and the cell wall. On the other hand, in cells treated with EGTA and subsequently irradiated with far-red light in artificial pond water, many precipitates were observe in the cytoplasm, chloroplasts, mitochondria and endoplasmic reticulum. The middle lamella was also heavily stained. On the basis of these observations, it was concluded that rotational cytoplasmic streaming in Vallisneria cells can be induced when the free calcium concentration in the cytoplasm decreases and that the induced streaming is arrested when the free calcium concentration in the cytoplasm increases. Far-red light accelerates the increase of calcium in the cytoplasm.

SUMMARY Reticulomyxa is a large multinucleated freshwater protozoan that provides a new model system in which to study intracellular transport and cytoskeletal dynamics. Within the cell body and reticulopodial network, rapid, visually striking saltatory organelle motility as well as bulk cytoplasmic streaming can be readily observed. In addition, the cytoskeletal elements within these strands undergo dynamic splaying and fusing rearrangements, which can be visualized by video-enhanced light microscopy. A reactivatable lysed cell model has been developed that appears to preserve, and therefore permits examination of, these three forms of motility in a more controlled environment. Individual organelle movements are microtubule-based and have similarities to, but also differences from, the recently described kinesin-based transport. This lysed cell model can be further manipulated to provide native, ordered, completely exposed networks of either microtubules or microfilaments, or a combination of both, and thus may serve as a versatile motility assay system in which to examine the movement of exogenously added isolated organelles or latex beads.

Pollen germination and tube growth of Lilium longiflorum in vitro are affected by 10(−5) M-nifedipine. Germinating ‘tubes’ form broad protuberances along the whole colpus. Short tubes show a high tendency to grow ‘amoeboid-like’ and to branch; or a second tube emerges in another region of the colpus. Longer tubes (greater than or equal to 100 micron) broaden irregularly or swell at their tips. The diameter of the tube can vary drastically within the same tube. With increasing time of treatment many tubes burst. Normal tip growth stops within 10 min, but protoplasmic streaming continues even after 15 h. More or less regularly, wall thickenings are formed along the whole tube or on the flanks of the germinating region after some hours. The internal calcium gradient, visualized by chlorotetracycline (CTC) fluorescence, is also disturbed. Nifedipine treatment results in uniform or irregular CTC fluorescence. Branching tubes temporarily show a new subapical CTC gradient. After 6–8 h of nifedipine treatment many cells are no longer stainable with CTC. The results indicate the presence of nifedipine-sensitive calcium channels in pollen tubes.

The study shows that diacylglycerols (DAGs) as physiological activators of protein kinase C induce characteristic shape changes in human neutrophil granulocytes. In contrast to chemotactic peptides, which can induce front-tail polarity characterized by a contracted tail and an expanding front, DAGs elicit the formation of non-polar cells with surface projections. These cells exhibit a distinct type of motility characterized by vigorous and continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in polarized cells. In neutrophils exposed to DAGs, F-actin is shifted to the cell periphery and mainly into the surface projections of activated cells. DAGs induce the formation of large intracellular vacuoles in neutrophils producing surface projections, and these vacuoles persist after the cells have reacquired a spherical shape. Combined stimulation of human neutrophils with DAG and fNLPNTL results in a suppression of peptide-induced polarity and the formation of non-polar motile cells resembling those stimulated with DAG alone. These results suggest that the diacylglycerol-protein kinase C pathway may be instrumental in transducing or modulating signals to both the locomotor apparatus and the exocytotic and/or pinocytic system of the cell. Neutrophil stimulation with DAGs thus represents a useful model with which to study further the hypothesis that distinct types of neutrophil shapes and movements are preferentially associated with distinct functions and to characterize signalling pathways.

A study of cytokinesis in the heliozoan Actinophrys sol has been made using low-temperature treatments, antimitotic drugs, cytochalasin B, light microscopy and electron microscopy. It reveals that microtubular axopodia remain extended during cell division and play a major role in it. Data indicate that when organisms are attached to the bases of culture dishes the normal locomotive mechanism of the presumptive daughter cells pulls them apart. However, when Actinophrys are floating freely in their culture medium, they are still able to undertake division. In this situation interactions between axopodia from opposite daughter cell bodies appear to facilitate the movement apart of the prospective daughter cells. The present study and other published accounts indicate that a type of cytokinesis exists which is not explicable in terms of the ‘contractile ring’ or ‘fusing vesicle’ theories. To account for these observations a ‘barge pole’ model of division is suggested.

Roots of seedling tomato (Lycopersicon esculenturn) and oilseed rape (Brassica napus) plants were grown on agarose slopes formed on microscope slides. Root hairs were used for microelectrode insertions, for observations of the rate of cytoplasmic streaming and for measurements of fluorescence of the calcium indicator fura-2. The resting potential across the plasma membrane of root hair cells was between −140 and −160mV in both species. Insertion of microelectrodes caused immediate cessation of cytoplasmic streaming; 1–4 min later streaming restarted and quickly recovered its normal velocity of 1–3 μms−1. Once the electrode had been inserted, current pulses had no obvious effect on streaming. Thus, fura-2 injected by iontophoresis was quickly distributed throughout the length of the hair and the epidermal cell body. Injections were made into the cap of cytoplasm at the tip of the hair. Movement of fura-2 from the cytoplasm into the vacuole was observed; this was marked by a great increase in its fluorescence after excitation at 350 nm and occurred after about 15–30 min in tomato, but more rapidly (5–10 min) in oilseed rape hairs. No fluorescence was associated with the cell walls; this was demonstrated by plasmolysing the cells. The distribution and concentration of Ca2+ was estimated from the ratio of fura-2 fluorescence excited at 350 and 385 nm and from digital image analysis. Resting levels of calcium in the cytoplasm near the tip were in the range 30–90 nM, while much higher levels, which eventually saturated the fura-2 response (>5μM), were seen in vacuoles once the indicator had crossed the tonoplast. This distribution was rapidly perturbed by the addition of the calcium channel blocker, verapamil, to the outer solution. There was a return to the original distribution as the verapamil was diluted. Separate experiments showed that verapamil inhibited cytoplasmic streaming over a time-course similar to these perturbations in cytoplasmic Ca2+ and caused a small depolarization (by approx. 30 mV) of the membrane potential. The application of 3x10−5 or 3 × 10−4 M-N-ethyl maleimide to the hairs strongly and irreversibly depolarized the plasma membrane electric potential, caused cytoplasmic streaming to stop or slow down and resulted in a very rapid rise in cytoplasmic Ca2+, which became uniformly distributed with saturating 350:385 nm ratios.

The reversible inhibition of cytoplasmic streaming in cells of Nitella translucens by cytochalasin B was found to be accompanied by an increase in the number of mini-vacuoles in the endoplasm. Fibrils thought to drive the streaming were still present in inhibited cells. In droplets of cytoplasm obtained from cut cells of Chara corralina, the final number of fibrils formed was not sensitive to cytochalasin B, although the motility of these fibrils was highly so. The movement of organelles in the absence of visible fibrils and the rotation of chloroplasts were also inhibited. The evidence for the involvement of the microfilament system in cytoplasmic streaming is discussed. The way in which such microfilaments seem to be operating in characean cells differs from the most likely mechanism for their operation in other cytochalasinsensitive processes.

Cytochalasin B reversibly inhibits cytoplasmic streaming in both Nitella and Avena cells. Colchicine, on the other hand, has no effect on streaming in either plant; nor does colchicine prevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of protein synthesis by cycloheximide has no effect on either streaming itself or on the recovery of streaming after cytochalasin withdrawal. All this suggests that microfilaments may provide one component of the structure that generates the streaming force and that microtubules play little, if any,role in the process. Ultrastructural studies of Nitella demonstrate that microfilaments are localized at the boundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments are organized in discrete bundles, with possible cross-bridges between individual filaments in each bundle. These bundles are closely associated with the extensive endoplasmic reticulum. Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does in some animal-cell filaments. Since the molecular mechanism of cytochalasin's action is unknown, there may be no necessary correlation between functional inhibition by the drug and altered microfilament morphology. A model is advanced which proposes that streaming is generated by an interaction between microfilaments and the endoplasmic reticulum.

Two distinct patterns of cytoplasmic streaming in the leaf of Caulerpa prolifera are described. Broad, longitudinally running, two-way streams are restricted to the endoplasm of one leaf surface. Also present are large numbers of narrow, two-way streams that coil helically through-out the endoplasm surrounding the central vacuole. Numerous unique bundles of aggregated, evenly spaced, oriented microtubules are distributed within the inner cytoplasm some distance from the cell wall. Cortical microtubules, as described for other plant material, have been only very infrequently encountered in Caulerpa and appear to be sparsely distributed. Apart from the prominent bundles of oriented microtubules, no other significant ultrastructural differences were noted between the stationary ectoplasm and streaming endoplasm. The possible cyto-skeletal role of the oriented microtubules in the development and maintenance of asymmetries in organ differentiation is discussed in relation to their direct or indirect influence on the directional migration of cytoplasmic components.

After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under synchronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (approximately 99.5%), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 7S) to 9 min (K401); the involvement of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidin microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes.

The organization of the microfilaments in epidermal cells of Vallisneria leaves was investigated with respect to the induction of cytoplasmic streaming (secondary streaming). In many of the epidermal cells, cytoplasm exhibited rotational streaming along the anticlinal wall of the cell after exposure to intense light. In these cells, many bundles of 6–7 nm microfilaments were observed in the cortex around the anticlinal wall. The bundles were arrayed in parallel to the streaming direction. They were recognized usually as 10–40 closely packed dense dots in cross-section. The spacing between bundles was not even. Bundles tended to form groups of 4 to 5 in which the spacing between bundles was usually 0·3 and 0·5 μm. The microfilaments were identified as F-actin. Together with the fact that rotational streaming in Vallisneria cells was inhibited by cytochalasin B, the motile mechanism of secondary streaming was concluded to be similar in its essential features to the cytoplasmic streaming seen in Characean cells (primary streaming). In epidermal cells that had been kept under low-intensity light the cytoplasm and the chloroplasts were mostly localized along the wall facing the exterior (the periclinal wall). No cytoplasmic streaming occurred in these cells. The bundles of microfilaments remained in the very thin layer of cytoplasm lining the anticlinal wall, although they were fewer and somewhat loosely packed. EGTA at appropriate concentration could induce cytoplasmic streaming in these cells. The mechanism of the induction is discussed on the basis of the effectiveness of EGTA and the requirement of a low concentration of free Ca2+ for cytoplasmic streaming in Characean cells.

Plasmodia of Physarum polycephalum grown on agar or filter paper and fed with rolled oats as food or with a partially defined medium were morphologically analysed in the living state and after fixation. Observation of the living plasmodium growing on agar reveals plasmalemma indentations in the outer regions of protoplasmic strands, which were studied in more detail by phase-contrast microscopy of unstained 1-µm sections. Plasmodia fixed and embedded in situ, i.e. in close contact to their substrate, exhibit an extensive system of plasmalemma invaginations as characteristic constituents throughout all regions. In plasmodial strands measuring between 40 µm and 1.5 mm in diameter and involved in shuttle streaming, the plasmalemma invaginations are found within the outer ectoplasmic wall. Rounded-up parts of this branched extracellular labyrinth limit the endoplasmic core engaged in the mass transport of protoplasm by shuttle streaming. Despite this clearcut borderline, the central endoplasmic core and the ectoplasmic cortex are connected by occasional protoplasmic bridges. The extracellular phase within the ectoplasmic regions of the strands can be interpreted either as a result of plasmalemma invaginations from the outer border of the strand, or as a consequence of pseudopodial-like processes originating from the central core and extending into the surrounding medium. The invagination system provides an extensive enlargement of the surface area within the multinucleate protoplasmic mass, probably important for food absorption, excretion processes and motility phenomena. In thick protoplasmic strands with diameters between 0.2 and 1.5 mm, there is an intimate connexion between the actomyosin fibrils and the invagination system. The fibrils are attached to the plasmalemma invaginations and/or run parallel to the invaginated plasmalemma sheets. The close relations between the invagination system and actomyosin fibrils will be described in detail in a subsequent paper.

Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells without disassembling their actin bundles. Lower cytochalasin concentrations than those needed for streaming inhibition are now shown to disrupt bundle assembly and, over longer periods, assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the production of polygons and straight, discontinuous bundles that rarely connected to bundles outside the wound. The regenerated bundles supported only scattered organelle movements, whereas long, oriented bundles of control cells were connected to those outside the wound and supported bulk endoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 microM-cytochalasin maintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number of bundles per file of chloroplasts fell from 5.2 in controls to 2.0 in growing cells and 3.4 in nongrowing cells. These structural effects seem more likely than the streaming inhibition to reflect cytochalasin's in vitro effect of blocking extension at the barbed but not the pointed end of F-actin. In particular, cytochalasin inhibited the extension into the wound of bundles in which only the barbed ends of filaments would be exposed. However, short lengths of isolated bundles grew within the wound and bundle growth in the intact cell continued, albeit in modified form. It is suggested that these examples of continuing bundle growth involve cytochalasin-resistant mechanisms that are not wholly dependent on barbed-end filament growth.

The microtubular cytopharyngeal basket acts as a jet engine when Nassula ingests filaments of blue-green algae. Prolonged and highly directed cytoplasmic streaming forms the main mechanical basis for propulsion of algal filaments through the basket. Cytoplasm surrounding such filaments streams at the same rate as that at which filaments travel through the basket’s interior. This cytoplasm appears to be very highly gelated. The arm-bearing surfaces of certain rows of microtubules seem to be located in the active shear zone where the forces responsible for cytoplasmic streaming are generated. Algal ingestion represents a rapid and large scale form of endocytosis. An algal filament is contained in a long membranous invagination from the ciliate’s cell surface as it is drawn through the basket. The invagination moves through the basket at the same velocity as the algal filament and surrounding cytoplasm. The arm-bearing microtubules do not appear to bind directly to the invagination which consists of two unit membranes. One of these is probably an invagination of the cell surface membrane. The origin of the other membrane has not been ascertained. There are indications that microtubule sliding and a contractile ring of microfilaments operate antagonistically during feeding to evert and re-invaginate a cell surface depression where the top of the basket is located.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46·7 μm s-1, S.D. ±4·8 at 20 °C) similar to those obtained for the same cells using the light microscope (44·3 μm s-1, S.D. ± 5·3 at 20 °C). A narrow distribution of streaming velocities is indicated. The technique described proUdes a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.

After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50 mum S minus 1, but but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2nlevels, free Ca2nat 10 minus 7 M or less and was inhibited by high levels of Clminus and by pH's on either side of 7.0. The reactivated movements were rapidly and completely inhibited by 25mug ml minus 1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.

The migration of human neutrophilic granulocytes in hydrated collagen lattices was studied by a combination of cinemicroscopy, and scanning and transmission electron microscopy. The basic pattern of cell migration in collagen was similar to that observed previously for these cells on inert material surfaces; i.e., a cycle of cell extension and cytoplasmic flow into the leading extension. In general, however, neutrophils in collagen were less spread than on glass or plastic surfaces. Thin lamellipodia were absent and the leading extension of the cells was often an elaborately folded pseudopodium. In addition, neutrophils migrating in collagen were never observed to have retraction fibres at the tail end of the cells, although a uropod was usually seen. In the region of the uropod, extensive blebbing of the cells often occurred, and when this happened, forward movement of the cells ceased. At the ultrastructural level, both the leading pseudopodium and the blebs at the tail of the cell were found to contain a dense cytoskeletal network from which cell organelles were excluded. Finally, the cells were found to be coated with an extensive glycocalyx, and individual collagen fibres were sometimes observed within the glycocalyx.

The telotrophic ovary of Notonecta glauca glauca consists of 7 ovarioles. Each ovanole comprises, from front to rear, a terminal filament, a trophic region, a prefolhcular region, and a series of 10-15 follicles of progressively increasing size The trophic region is largely syncytial and is made up of polyploid trophic nuclei packed around a central trophic core The cytoplasm of the trophic core is continuous with the cytoplasm of each oocyte through a system of trophic tubes. There is one trophic tube per oocyte. The trophic nuclei have large nucleoh. There are a few small nucleoh in the oocyte nuclei The cytoplasm of the trophic core, the trophic tubes, and the oocytes is rich in RNA. Autoradiographs of sections of ovarioles fixed 2 h after injection of PHjundine into animals show label over the trophic nuclei only. Eight-hour autoradiographs show heavy labelling of the trophic region and label over the front ends of the trophic tubes, but little label over the posterior regions of the tubes or the oocyte cytoplasm. Later autoradiographs indicate that label gradually spreads backwards from the trophic core, along the trophic tubes, and progressively builds up in the oocyte cytoplasm These observations are thought to indicate synthesis of RNA in the trophic region and movement of RNA from the trophic core along the trophic tubes to the oocytes The trophic core and tubes show brilliant positive form birefringence with respect to their lengths. This birefringence can be reduced by keeping animals at 2 °C for 12 h, and eliminated by placing ovanoles in 1 % colchicine for 6 h. Electron micrographs of thin sections of ovanoles show that trophic core and tubes are densely and uniformly packed with nbosomes and microtubules The latter are lined up along the trophic tubes. There are about 30000 microtubules evident m a TS through a trophic tube 15 fim wide. Lengths of microtubules up to 2 /*m have been observed. Ribosomes are packed between the microtubules but are excluded from regions where the spacing between adjacent microtubules is less than 25 ran The contribution of the trophic region to the oocytes and the role of the microtubules in maintaining or facilitating the movement of nbosomes along the trophic tubes is discussed

Preliminary observations on the fine structure of the club-shaped glands on Phaseolus vulgaris leaves are reported. The perinuclear cytoplasm of the apical cells of these glands contains an abundance of microtubules. These occur either as aggregates of 2–8 or more tubules, or they may be organized around a central core of material to form a fibre-like structure. The cells also contain cortical microtubules and are rich in rough endoplasmic reticulum and dictyosomes. The nuclei of these cells also contain a proteinaceous fibre, visible in the light microscope. The possible significance of these structures is discussed in relation to cytoplasmic streaming, maintenance of cellular asymmetry, and reaction to virus infection.

In Difflugia corona, a free-living amoeboid cell, locomotion is hampered by a heavy shell or test made of sand grains and other debris. Locomotion involves pseudopod extension, attachment to the substratum, and forcible pseudopod retraction which pulls the shelled cell body forward. When observed through a polarizing microscope, the extending pseudopodia appear isotropic or very weakly birefringent. Upon attachment to the substratum a positively birefringent fibrillar array develops rapidly at the attachment point and extends from this region back to the cell body within the test. These birefringent fibrils extend through and parallel to the long axis of the pseudopod. As the pseudopod retracts, the birefringent fibrillar array disappears, and hyaline blebs, suggestive of syneresis, appear on the pseudopodial surface. The birefringent fibrils correspond in position and approximate diameter (1 μ) to refractile fibrils visible with the Nomarski differential interference microscope. Individual organisms were fixed for electron microscopy at a time when the pseudopodia were firmly attached to the substratum. Electron-microscopic examination of thin sections of pseudopodia revealed many 1-μ bundles of intimately associated, aligned, 55–75 Å micro-filaments. The orientation and size of the bundles indicate that they probably correspond to the birefringent, refractile fibrils observed in living cells. Microfilaments have also been observed both as randomly oriented and dispersed cytoplasmic components, and as aligned filaments in the ectoplasm adjacent to the plasmalemma. During pseudopod extension with sporadic streaming, birefringent ‘flashes’ have been observed at the front of the pseudopod. These flashes are believed to represent a photo-elastic phenomenon.