Photoinduction of circular F-actin on chloroplast in a fern protonemal cell

Summary Circular F-actin on a photooriented chloroplast was observed by rhodamine-phalloidin staining in the fernAdiantum protonemal cells in which phytochrome- or blue light receptor-mediated intracellular photoorientation of chloroplasts was induced. The circular structure located along the edge of chloroplast on the side facing the plasma membrane but not on the opposite side. Most of the chloroplasts in protonemal cell have dumbbell-shape and the circular ring-like structure was found on each half of the dumbbell. The structure was not observed in the cells which were kept in the dark, indicating the change of F-actin organization by the light condition. Possible role of the structure on the anchorage of chloroplast in its intracellular photoorientation was discussed.     

Photoorientation of chloroplasts in protonemal cells of the fernAdiantum as analyzed by use of a video-tracking system

Photoorientation of chloroplasts mediated by phytochrome and blue light-absorbing pigment in protonemal cells of the fernAdiantum was studied by use of inhibitors of the cytoskeleton and was analyzed with a video-tracking system. The photoorientation responses were inhibited by cytochalasin B and by N-ethylmaleimide (NEM) but not by colchicine, suggesting that the photomovement depends on the actomyosin system. In the dark, chloroplasts moved randomly, being independent of one another. After induction of photoorientation by polarized red light, most chloroplasts that had been located at the margin of cells moved almost perpendicularly to the cell axis toward the site of photoorientation. This type of movement was hardly ever observed in the dark. Under polarized blue light, such specific movements were less evident but were still observed in the case of a few chloroplasts. After photoorientation was complete, chloroplasts still moved in random directions but their mobility was lower than that in the dark, indicating the presence of some anchoring mechanism. When EGTA was applied, photoorientation was inhibited but this inhibition was overcome by the addition of CaCl₂. Video-tracking of chloroplasts in the dark revealed that the mobility of chloroplasts was higher in medium with EGTA than in medium with EGTA plus CaCl₂ and that many of the chloroplasts moved jerkily in the medium with EGTA. This change in the nature of movements was also seen under polarized light, resulting in the disturbance of photoorientation. These results indicate that the inhibition of photoorientation at low concentrations of Ca²⁺ ions may be due to change in the nature of chloroplast movement.     

Cytoskeletal changes during resumption of tip growth in nongrowing protonemal cells of the fernAdiantum capillus-veneris L.

Summary Nongrowing, two-celled protonemata of the fernAdiantum capillus-veneris L. resume tip growth within the apical cell upon irradiation with red light. In this study, the phenomenon of growth resumption was analyzed with reference to changes in cytoskeletal organization. Continuous observations of apical cells with time lapse video-microscopy revealed that the nucleus migrated toward the tip ca. 1.9 h after the onset of red light, much earlier than the initiation of tip growth, which took place ca. 8.5 h after irradiation. Cytoskeletal organization was observed at various time points during growth resumption by fluorescent staining of microfilaments (MFs) and microtubules (MTs) with rhodamine-phalloidin and anti-tubulin antibodies. At 2 h after red-light irradiation, endoplasmic MF and MT strands appeared at the apical end of nucleus. These strands extended into the apical endoplasm, where filaments were rare prior to irradiation. Many fine filaments branched from the strands to the cell periphery, including the cortex of the apical-dome region. At this time, cortical circular arrays of MTs and MFs, normally found in the growing apex of protonemal cells, were absent. Both MT and MF circular arrays appeared during the resumption of tip growth concomitantly. The half-maximum appearance of MT and MF circular arrays within a population occurred at 5.4 h and 5.8 h after red-light irradiation, respectively. Thus, the process of red-light-induced resumption of tip growth in fern protonemal cell is composed of a series of events. These events include: (1) the appearance of strands extending from the nucleus toward the apical cortex and the migration of nucleus toward the apex; (2) the formation of circular MT and MF arrays at the sub-apical cortex; and (3) the initiation of cell growth at the apex. These results reflect the significant roles of MF and MT cytoskeleton in the resumption of tip growth.Abbreviations MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule     

Reorganization of microfilaments in protonemal tip cells of the mossCeratodon purpureus during the phototropic response

Summary The F-actin distribution in caulonemal tip cells of the mossCeratodon purpureus was examined by rhodamine-phalloidin staining. Gravitropically-growing caulonemal tip cells of the moss possess a distinct alignment of microfilaments (MFs) in their apices. Axially oriented actin bundles run from subapical regions to the apex where they converge towards a central area of the tip, although bundles are absent from the central area itself thus forming a collar-like structure. During a unilateral red light irradiation the actin strands of the apical dome become reoriented towards the irradiated apical flank and still surround an area free of MFs, the point of prospective outgrowth. This process is closely correlated with the morphological effect of bulging and precedes the light-directed outgrowth. The collar structure is essential for the tubular growth form. In darkness, under the influence of antimicrotubule agents the structure is decomposed, the actin strands drift along the cell flanks and finally accumulate in randomly distributed areas where further growth takes place. The microtubules (MTs) are not involved in the phytochromemediated reorientation of the microfilaments. Unilateral red light suppresses the distorting effect of antimicrotubule drugs and restores the collar structure with a pronounced light-directed orientation. Instead, the MTs seem to be responsible for restricting the reorientation to the cell tip. This notion is based on the observation that the small area in the apical dome, which is normally the exclusive location of the light-regulated MF rearrangement, extends towards the cell base when MT inhibitors are applied before the unilateral red light irradiation. This in turn leads to a non-tubular expansion of the light-directed cell flank.Abbreviations DIG differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(beta-aminoethylether) N,N,N,N-tetraacetic acid - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - MBS 3-maleimidobenzoic     

Cytoskeletal aspects of nuclear migration during tip-growth in the fernAdiantum protonemal cell

Summary In the tip-growing protonemal cell, the nucleus migrates with the tip as it grows, keeping a constant distance between them. Cytoskeletal control of this nuclear migration was analyzed inAdiantum capillus-veneris. Using rhodamine-phalloidin (Rh-Phal), tubulin antibodies and confocal laser scanning microscopy, we found the presence of microtubule (MT) and microfilament (MF) strands connecting the cell nucleus to the cortex of the growing apex. The strands come from the apical end of the spindle-shaped nucleus and run through the endoplasm, arriving at the apical cortex, where a circular arrangement of MTs and MFs is present. Strands of MFs and MTs were also found to emanate from the proximal end of the nucleus and extend towards the cortex of the basal part of the cell. Double staining of MTs and MFs revealed a co-localization of these cytoskeletal elements. When MF strands were disrupted by cytochalasin B (CB), tip-growth ceased and nuclear movement stopped. After the application of colchicine, MT structures disappeared, tip-growth was largely inhibited, and the nucleus moved towards the basal part of the cell. When both CB and colchicine were applied to the cell, no basipetal migration of cell nucleus was observed. These results suggest that the MT strands between the apex and the nucleus may have a role in the anchorage of the cell nucleus to the tip during tip-growth, and that the MF strands may be important for basipetal movement of the nucleus. When the nucleus was dislocated basipetally by centrifugation, cytoskeletal strands between the cell apex and the nucleus were still observed, and by acropetal movement the nucleus resumed its previous position. The acropetal movement of the nucleus was inhibited by the application of both CB and colchicine but not by CB alone nor by colchicine alone, indicating that both cytoskeletal elements are involved in the forward movement of cell nucleus.Abbreviations CB cytochalasin B - DAPI4 6-diamino-2-phenylin-dole - DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N,N,N-tetraacetic acid - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule - PMSF phenylmethylsulfonyl fluoride - PSM polyoxyethylene sorbitan monolaurate - Rh-Phal rhodamine-labeled phalloidin     

Reorganization of the cortical cytoskeleton in tip-growing fern protonemal cells during phytochrome-mediated phototropism and blue light-induced apical swelling

Summary Circular arrays of cortical microtubules (MTs) and microfilaments (MFs) are found in the subapical region of tip-growing protonemal cells of the fernAdiantum capillus-veneris. Reorganization of the two cytoskeletal structures during phytochrome-mediated phototropism and blue light-induced apical swelling was investigated by double-staining of MTs and MFs with rhodaminephalloidin and an indirect immunofluorescence method with tubulinspecific antibody. Before any growth responses were detectable, the MF and MT structures were reorganized according to similar patterns in both photoresponses, that is, oblique orientation and transient disappearance of the structures occurred during the phototropic response, and the disappearance of the structures occurred during apical swelling. The reorganization of MF structures clearly preceded that of the MT structures in the phototropic response. In the case of apical swelling, both types of circular array disappeared with an almost identical time course.These results provide evidence for the significant role of the circular organization of MFs as well as of MTs, in the light-induced growth responses of tip-growing fern protonemal cells. Possible roles of the circular array of MFs in the regulation of tip growth are discussed.Abbreviations DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - MF microfilament - MT microtubule - Rh-Phal rhodaminelabeled phalloidin     

Dynamic changes in the organization of microfilaments associated with the photocontrolled motility of chloroplasts in epidermal cells ofVattisneria

Summary Using time-lapse video microscopy, we performed a semiquantitative investigation of the movement of chloroplasts on the cytoplasmic layer that faces the outer periclinal wall (P side) of epidermal cells of leaves of the aquatic angiospermVallisneria gigantea Graebner. Under continuous irradiation with red light (650 nm, 0.41 W/m²), the movement of chloroplasts on the P side was transiently accelerated within 5 min. The increased movement began to decrease at around 20 min and fell below the original level after 40 to 60 min of irradiation with red light. The acceleration and deceleration of movement of chloroplasts on the P side seemed to lead directly to the increase and the subsequent decrease in the rate of migration of chloroplasts from the P side to the anticlinal layers of cytoplasm, which are responsible for the accumulation of chloroplasts on the P side, as we demonstrated previously. In the presence of inhibitors of photosynthesis, the accelerated movement of chloroplasts was maintained for as long as the chloroplasts were irradiated with red light. The rapid acceleration and deceleration of the movement of chloroplasts could be observed repeatedly with sequential irradiation with red and then far-red light (746 nm, 0.14 W/m²). Concomitantly with the loss of motility of chloroplasts on the P side, a dynamic change in the configuration of microfilaments, from a network to a honeycomb, occurred on the P side.Abbreviations APW artificial pond water - A side cytoplasmic layer that faces the anticlinal wall - ATP adenosine triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F-actin fibrous actin - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.     

Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fernAdiantum

Orientational movement of chloroplasts was induced by a brief irradiation with red light (R) or blue light (B) in dark-adapted prothallial cells ofAdiantum, whose chloroplasts had gathered along the cell dividing wall (i.e., the anticlinal wall). When the whole dark-adapted prothallia were irradiated from a horizontal direction (i.e., from their lobes) with horizontally vibrating polarized R (H pol. R) for 10 or 3 min, the chloroplast left the anticlinal walls and spread over the prothallial surface (i.e., the periclinal walls) within 1–2 hr after the onset of irradiation, returning to the anticlinal wall (dark-position) within 10 hr. However, vertically vibrating polarized R (V pol. R) for 10 min did not induce the movement towards periclinal walls. The R effect was cancelled by non-polarized far-red light (FR) irradiation just after the R irradiation. Irradiation with H pol. B for 10 or 3 min but not with V pol. B could also induce a similar movement of chloroplasts, although the chloroplasts returned within 4 hr. The effect of H pol. B, however, was not cancelled by the subsequent FR irradiation. When a part of the dark-adapted cell at the prothallial surface was irradiated from above with a microbeam of R or B for 1 min, chloroplasts of the cell in the dark-position moved towards the irradiated locus in subsequent darkness. However, in the neighboring cells, orientational movement was not induced by either R or B microbeams. These results show that in dark-adapted prothallial cells, both brief irradiation with R and B can induce chloroplast photo-orientation and that the photoreceptors are phytochrome and blue light-absorbing pigment, respectively. It is also clear that effects of both R and B irradiation do not transfer to neighboring cells.     

Regulation of the orientation movement of chloroplasts in epidermal cells of Vallisneria: cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light

In epidermal cells of the leaves of the aquatic angiosperm Vallisneria gigantea Graebner, the chloroplasts accumulate in the outer periclinal layer of cytoplasm (P side) under light at low fluence rates. The nature of such intracellular orientation of chloroplasts was investigated in a semiquantitative manner. Time-lapse video microscopy revealed that, while irradiation with red light (650 nm, 0.41 W · m^–2) rapidly accelerated the migration of chloroplasts, not only from the anticlinal layers of cytoplasm (A sides) to the P side but also from the P side to the A sides, the increased rate of migration in both directions returned to the control rate upon subsequent irradiation with far-red light (746nm, 0.14W · m^–2). These effects of red and far-red light could be observed repeatedly, both in the presence and in the absence of inhibitors of photosynthesis, suggesting the involvement of phytochrome as the photoreceptor. After saturating irradiation with red light, the increased rate of migration of chloroplasts from the P side to the A sides declined more rapidly than the increased rate of migration in the opposite direction. This imbalance in the migration of chloroplasts between the two opposing directions resulted in the accumulation of chloroplasts on the P side. The more rapid decline in the rate of migration of chloroplasts from the P side to the A sides than in the opposite direction was not observed in the presence of an inhibitor of photosynthesis. It appears, therefore, that phytochrome and photosynthetic pigment cooperatively regulate the accumulation of chloroplasts on the P side through modulation of the nature of the movement of the chloroplasts.Abbreviations A side cytoplasmic layer that faces the anticlinal wall - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture to S.T. and R.N. The authors are indebted to the Osaka branch of Kashimura Inc. for their kind cooperation in preparing the GREEN software.     

Detection of intranuclear forces by the use of laser optics during the recovery process of elongated interphase nuclei in centrifuged protenemal cells ofAdiantum capillus-veneris

For the direct investigation of intranuclear dynamics in living cells, extremely deformed nuclei of basipetally centrifuged protonemal cells of the fernAdiantum capillus-veneris were manipulated by the laser trap and the laser scalpel. Whereas the nucleolus was tightly fixed at the central position inside the non-centrifuged nucleus and proved to be immovable by the optical trap, it could easily be trapped and moved towards three directions inside the bubble-like terminal widening of the basal thread-like extension of centrifuged nuclei. Due to the connection of the nucleolus to the chromatin inside the nuclear thread (NT), moving was not possible against the direction of the nuclear apical main body. Nucleoli in recovered nuclei were again immovable, thus indicating the presence of a dynamic nucleolar anchoring system inside the nucleus. When the nucleolus in the bubble was arrested during the thread shortening process by the optical trap, the acropetal movement of the bubble continued. Probably due to dragging forces, some nucleoli became stretched, and a thick strand of a still unknown composition stretched between the nucleolus and the insertion site of the shortening NT. To assess whether the shrinking of the nuclear envelope (NE) and the shortening of the chromatin inside the NT were independent processes, the chromatin above the bubble was cut inside the NT by the laser scalpel. After severance, a gap between the nucleolus and the end of the chromatin strand in the NT indicated the shortening of the chromatin inside the NT. From these findings it was concluded that a shortening force was existing in the chromatin of the NT and that probably no physical link existed between the chromatin and the NE.     

The role of microfilaments in the organization and orientation of microtubules during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells

Summary During cell cycle transition from M to G₁ phase, micro-tubules (MTs), organized on the perinuclear region, reached the cell cortex. Microfilaments (MFs) were not involved in this process, however, MFs accumulated to form a ring-like structure in the division plane and from there they elongated toward the distal end in the cell cortex. Subsequently, when MTs elongated along the long axis of the cells, towards the distal end, the MTs ran into and then associated with the predeveloped MFs in the cell cortex, suggesting the involvement of MFs in organizing the parallel oriented MTs in the cell cortex. When cortical MTs were formed in the direction transverse to the long axis of cells, the two structures were again closely associated. Therefore, with regards to the determination of the direction of organizing MTs, predeveloped MFs may have guided the orientation of MTs at the initial stage. Disorganization of MFs in this period, by cytochalasins, prevented the organization of cortical MTs, and resulted in the appearance of abnormal MT configurations. We thus demonstrate the involvement of MFs in determining the orientation and organization of cortical MTs, and discuss the possible role of MFs during this process.Abbreviations CB cytochalasin B - CD cytochalasin D - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - EF-1 elongation factor 1 - MF microfilament - MT microtubule     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

Differences in phytochrome action on the formation of carotenoids and quinones during chloroplast development in seedlings of barley (Hordeum vulgare)

The influence of phytochrome on the light induced formation of carotenoids and quinones was investigated using etiolated seedlings of barley ( Hordeum vulgare L. cv. Villa). The biosynthesis of both the quinones and the carotenoids was enhanced by active phytochrome, but the formation of the individual carotenoids and quinones was influenced by quite different thresholds. In both younger and older plants the biosynthesis of β-carotene, lutein and violaxanthin was promoted by a low P_fr threshold. The formation of plastoquinone-9, plastohydroquinone-9, α-tocoquinone and phylloquinone was also influenced by a low P_fr threshold. The biosynthesis of zeaxanthin and neoxanthin required a much higher amount of P_fr. Only antheraxanthin-likeα-tocopherol, desmethylphylloquinone and, in older leaves, α-tocoquinone exhibited a complete reversibility of the phytochrome action in their biosynthesis. The effect of phytochrome on the biosynthesis of carotenoids and quinones was different in seedlings of different age.     

The transition of early translocation intermediates in chloroplasts is accompanied by the movement of the targeting signal on the precursor protein

During protein import into chloroplasts, precursor proteins are docked to these organelles under stringent energy conditions to form early translocation intermediates. Depending on the temperature and the requirement for ATP, different types of early-intermediates are present, for which the extent of precursor protein translocation differs [H. Inoue, M. Akita, J. Biol. Chem. 283 (2008) 7491–7502]. However, it has not been determined whether the environment surrounding the precursor differs for each intermediate. We therefore employed a site-specific photo-crosslinking strategy in our current study to capture any components in close proximity to the targeting signal of the precursors within the early-intermediates. Various crosslinked products, one of which contains Toc75, were identified. The appearance of these products was found to be dependent on the position of the precursor upon modification by the crosslinker and also the intermediate state. This indicated that the transition of early translocation intermediates is accompanied with the movement of the targeting signal within the early-intermediates.     

Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of<Emphasis Type="Italic"> Vallisneria gigantea</Emphasis>

In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts.Preliminary results of part of the local irradiation study have already been reported in abstract form [N. Sakurai et al. (2002) J Photosci 9:326–328].     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.