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PubMed2004

Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells.

The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.

Actin Cytoskeleton↗
PubMed1980

Contraction rhythm in the plasmodium of Physarum polycephalum: dependence of the period on the amplitude, temperature and chemical environment.

The plasmodium of Physarum polycephalum exhibits a characteristic protoplasmic shuttle streaming and generates periodical tension. We determined the factors which govern the period of the contraction rhythm by measuring isometric tension of the plasmodial strand and the motive force of the protoplasmic streaming under a variety of conditions by changing the size of plasmodia, chemical composition of environment and surrounding temperatures. The results are: (1) The period of contraction rhythm, tau, increased linearly with the amplitude of oscillating tension, F, and was expressed by the following empirical equation when F was lower than a certain critical value, Fc, i.e., tau = aF+tau O. Above Fc, tau stayed at a constant level of tau S. There, a tau O and tau S are numerical constants which are independent of F. A similar relationship is valid for the amplitude of the motive force of the protoplasmic streaming, delta P. (2) Values of tau O were 1.0 min in air and 1.6 min in an aqueous medium and they were independent of temperature. (3) The Arrhenius plots of the parameter "a" exhibited different straight lines in air and in aqueous medium, from which the values of Q10 were determined to be 4.0 and 10, respectively. (4) Lowering of temperature decreased Fc, and eventually diminished it at Tc (= 15 degrees C). (5) The presence of glucose, CaCl2, MgCl2 and NaCl gave the identical tau-F relationship. Applications od D2O and ethanol slowed down the contraction rhythm, while that of KCl accelerated the rhythm. However, all these chemicals did not affect the tau O value. All results described above indicate that the contraction rhythm in the plasmodium is influenced by three components: a limit cycle which is independent of tension generation, a component which is strongly linked to the amplitude of the tension, F, and the part which is independent of the value of F. External factors appear to influence separately these three components of the contraction rhythm.

Cytoplasmic Streaming↗
PubMed1958

Rhythmicity in the protoplasmic streaming of a slime mold, Physarum polycephalum. II. Theoretical treatment of the electric potential rhythm.

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.

Cytoplasm↗
PubMed1998

Interactive computer-assisted position acquisition procedure designed for the analysis of organelle movement in pollen tubes.

An interactive computer-assisted video microscopy method has been developed for the acquisition of extensive data on the sequential positions of pollen tube organelles, which cannot be automatically tracked using geometric or motion patterns. The method consists of video microscopy, analog and digital contrast enhancement, digital time-lapsing of the images, and interactive selection of positions in a coordinate system corresponding to the cell shape and real size. Data on 15,000 positions acquired with this method have been used to make quantitative analyses of the movement patterns of the organelles. From these analyses and the reconstruction of 900 trajectories, it appears that movements are random in the tip of the pollen tube but become more directed in distal regions of the cell, indicating an increase in axial arrangement of the actin filaments. (All custom-made software is available from the authors on request.)

Cell Movement↗
PubMed2001

Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.

Animals↗
PubMed1983

Cytoplasmic transport in keratocytes: direct visualization of particle translocation along microtubules.

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.

Animals↗
PubMed1988

"DMS," a computer-assisted system for quantitating motility, the dynamics of cytoplasmic flow, and pseudopod formation: its application to Dictyostelium chemotaxis.

A computer-assisted Dynamic Morphology System (DMS) is described that allows the rapid quantitation of more than 30 parameters of motility and dynamic morphology for up to 40 amebae in parallel. This system also generates "difference pictures" for characterizing the dynamics of pseudopod formation. A 3-D DMS is described, and application of DMS to problems of motility and chemotaxis in normal and mutant cells of Dictyostelium discoideum is reviewed.

Chemotaxis↗
PubMed1984

Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.

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.

Animals↗
PubMed1976

Furrowing in altered cell surfaces.

Understanding the process which established the cell division mechanism requires analysis of the role of the responding surface as well as that of stimulatory subsurface structures. Cell surface was altered by the expansion which occurs during exovate formation. Exovates appear on the surface of fertilized Arbacia lixula, Paracentrotus lividus and Echinarachnius parma eggs in response to extreme flattening. They result from cytoplasmic outflow initiated in a very restricted portion of the egg surface. Observations of the formation process in pigmented A. lixula eggs revealed that the original surface may be expanded about 100 fold as the exovate swells. When exovates formed 15-30 minutes after fertilization contain the mitotic apparatus, they divide synchronously with flattened controls. If nucleated exovates are established after the beginning of first cleavage, furrows appear in ten minutes. Exovates established after the beginning of second cleavage develop furrows four minutes after the entrance of the the mitsotic apparatus. Cytoplasm beneath damaged exovate surfaces sometimes develops partial constrictions independently of the surface in the plane the furrow would have occupied. These results suggest that normal surface structure is unnecessary for furrow establishment and function.

Animals↗
PubMed2000

The molecular structure of the fastest myosin from green algae, Chara.

Chara myosin in green algae, Chara corallina, is the fastest myosin of all those observed so far. To shed light on the molecular mechanism of this fast sliding, we determined the primary structure of Chara myosin heavy chain (hc). It has a motor domain, six IQ motifs for calmodulin binding, a coiled-coil structure to dimerize, and a globular tail. Chara myosin hc is very similar to some plant myosins and has been predicted to belong to the class XI. Short loop 1 and loop 2 may account for the characteristics of mechanochemical properties of Chara myosin.

Amino Acid Sequence↗
PubMed1994

Elongation factor 1 alpha is a component of the subcortical actin bundles of characean algae.

Antibodies to elongation factor 1 alpha (EF1 alpha), a known 50 kDa actin-bundling protein in Dictyostelium, identified a protein in a whole cell extract of the characean alga Nitella pseudoflabellata that had an apparent molecular weight of 51 kDa. Indirect immunofluorescence microscopy revealed labelling by the EF1 alpha antibodies of the subcortical actin bundles, even after the motile organelles of the endoplasm were removed by perfusion with ATP-containing solutions.

Actins↗