Regulation of cell shape in Euglena gracilis. III. Involvement of stable microtubules

1985 ◽  
Vol 74 (1) ◽  
pp. 219-237
Author(s):  
C.L. Lachney ◽  
T.A. Lonergan
Keyword(s):  
Euglena Gracilis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Microtubule Depolymerization ◽  
Cytoplasmic Microtubule ◽  
Intact Cells ◽  
Calmodulin Antagonist ◽  
Cell Shapes ◽  
In Cells

The role of cytoplasmic microtubules in a recently reported biological clock-controlled rhythm in cell shape of the alga Euglena gracilis (strain Z) was examined using indirect immunofluorescence microscopy. The resulting fluorescent patterns indicated that, unlike many other cell systems, Euglena cells apparently change from round to long to round cell shape without associated cytoplasmic microtubule assembly and disassembly. Instead, the different cell shapes were correlated with microtubule patterns, which suggested that movement of stable microtubules to accomplish cell shape changes. In live intact cells, these microtubules were demonstrated by immunofluorescence to be stable to lowered temperature and elevated intracellular Ca2+ levels, treatments that are commonly used to depolymerize microtubules. In cells extracted in detergent at low temperature or in the presence of elevated Ca2+ levels, the fluorescent image of the microtubules was disrupted. Transmission electron microscopy confirmed the loss of one subset of pellicle microtubules. The difference in microtubule stability to these agents between live intact cells and cells extracted in detergent suggested the presence of a microtubule-stabilizing factor in live cells, which is released from the cell by extraction with detergent, thereby permitting microtubule depolymerization by Ca2+ or lowered temperature. The calmodulin antagonist trifluoperazine prevented the Ca2+-induced disruption of the fluorescent microtubule pattern in cells extracted in detergent. These results implied the involvement of calmodulin in the sensitivity to Ca2+ of the microtubules of cells extracted in detergent.

scholarly journals Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum

1995 ◽  
Vol 108 (4) ◽  
pp. 1805-1815 ◽  
Author(s):  
J. Hidalgo ◽  
M. Muniz ◽  
A. Velasco
Keyword(s):  
Endoplasmic Reticulum ◽  
G Proteins ◽  
Retrograde Transport ◽  
Microtubule Depolymerization ◽  
High Concentration ◽  
Intact Cells ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Integral Proteins ◽  
In Cells

Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immunofluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTP-gamma S, AIF(3–5), transducin beta gamma subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15 degrees C or 20 degrees C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both alpha s and alpha i trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.

scholarly journals Regulation of cell shape in Euglena gracilis. IV. Localization of actin, myosin and calmodulin

1985 ◽  
Vol 77 (1) ◽  
pp. 197-208
Author(s):  
T.A. Lonergan
Keyword(s):  
Plasma Membrane ◽  
Euglena Gracilis ◽  
Cell Shape ◽  
Biological Clock ◽  
Free Medium ◽  
Calmodulin Antagonist ◽  
Cell Rounding ◽  
Contractile System ◽  
High Degree ◽  
Shape Changes

The immunofluorescence patterns for actin, myosin, calmodulin and tubulin were observed in Euglena gracilis Klebs strain Z during the biological clock-controlled shape changes observed with division-synchronized cells, and during two shock responses that induce cell rounding. The fluorescence patterns for actin, myosin, calmodulin and tubulin show a high degree of coincidence and are visualized as lines running parallel to, and having the same spacing as, the pellicle strips beneath the plasma membrane. The fluorescence patterns remain intact during the daily shape changes, implying that the shape changes do not result from cycles of polymerization and depolymerization of the microtubules and microfilaments. Resuspension of cells in Ca2+-free medium induces cell rounding of many of the cells. The actin and calmodulin patterns are partially disrupted by the Ca2+-free resuspension, while the myosin pattern is almost totally disrupted. Microtubules are unaffected by this treatment. Prior exposure of cells to the calmodulin antagonist trifluoperazine or to the microfilament-stabilizing peptide phalloidin stabilize the actin, myosin and calmodulin patterns against disruption by the Ca2+-free resuspension and other shock responses. The possibility of an actomyosin contractile system controlled by calmodulin is discussed.

Regulation of cell shape in Euglena gracilis. II. The effects of altered extra- and intracellular Ca2+ concentrations and the effect of calmodulin antagonists

1984 ◽  
Vol 71 (1) ◽  
pp. 37-50
Author(s):  
T.A. Lonergan
Keyword(s):  
Euglena Gracilis ◽  
Cell Shape ◽  
Ionophore A23187 ◽  
Free Medium ◽  
Calmodulin Antagonists ◽  
Daily Cycle ◽  
Cell Shapes ◽  
Euglena Gracilis Z ◽  
Shape Changes ◽  
Synchronized Cultures

When cultures of Euglena gracilis Z., normally grown in medium containing 180 microM-Ca2+, are resuspended in Ca2+-free medium cells assume round shapes within 10 min, from which they recover slowly when Ca2+ is returned to the cultures. Cultures grown in 10 microM-Ca2+ do not display the typical circadian rhythm in cell shape even though the photosynthesis and cell division circadian rhythms are unaffected. Elevating intracellular Ca2+ levels by the addition of the Ca2+ ionophore A23187 prevents cells from undergoing the two daily shape changes characteristic of growth-synchronized cultures, but does not alter the ability to maintain the cell shapes found at the time of ionophore addition. When the calmodulin inhibitors trifluoperazine or chlorpromazine are added to cultures, the cells always respond by rounding. Cells are not able to maintain any cell shape other than spherical in the presence of these inhibitors and therefore cannot change shape throughout the daily cycle as is found in the control populations.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

1980 ◽  
Vol 38 ◽  
pp. 552-553
Author(s):  
K.I. Pagh ◽  
M.R. Adelman
Keyword(s):  
Cell Shape ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
Live Cells ◽  
Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

scholarly journals Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

1979 ◽  
Vol 83 (3) ◽  
pp. 623-632 ◽  
Author(s):  
M Schliwa ◽  
U Euteneuer ◽  
W Herzog ◽  
K Weber
Keyword(s):  
Complete Removal ◽  
Cell System ◽  
The State ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Functional Changes ◽  
Organizing Center ◽  
Pigment Distribution ◽  
And Function ◽  
In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

1976 ◽  
Vol 69 (1) ◽  
pp. 106-125 ◽  
Author(s):  
D L Brown ◽  
A Massalski ◽  
R Patenaude
Keyword(s):  
Anterior Part ◽  
Flagellar Apparatus ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Body Region ◽  
Basal Bodies ◽  
Cytoplasmic Microtubule ◽  
Large Numbers ◽  
Attachment Sites ◽  
Cytoplasmic Microtubules

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

scholarly journals Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

1984 ◽  
Vol 98 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
W C Thompson ◽  
D J Asai ◽  
D H Carney
Keyword(s):  
Differential Staining ◽  
Hypotonic Medium ◽  
Cytoplasmic Microtubule ◽  
Alpha Tubulin ◽  
Apparent Correlation ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
Fibroblastic Cells ◽  
In Cells

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

scholarly journals Storage of ram semen in gelatin supplemented extender

2012 ◽  
pp. 7-10
Author(s):  
Csilla Budai ◽  
István Egerszegi ◽  
József Rátky ◽  
András Kovács
Keyword(s):  
Analysis Of Variance ◽  
Light Microscope ◽  
Egg Yolk ◽  
Live Cells ◽  
Intact Cells ◽  
Uht Milk ◽  
Liquid Storage ◽  
Gelatin Concentration ◽  
Semen Extender

The aim of our study was to examine how different gelatin concentrations affect ram semens viability in liquid storage at 5 oC for five days. Our hypothesis was if we add gelatin to the semen extender, than the viability of ram semen will be better in the extenders containing gelatin, than the control. We used two different semen extenders:1.5% UHT milk and 1.5% UHT milk + 5% egg yolk. We added 0; 0.5; 1.0; 1.5; 2.0% Dr. Oetker gelatin to the semen extenders. We stored the semen for five days at 5 oC and in every 24 hour we made sampling.We stained the smears with Kovács-Foote staining and evaluated them with light-microscope. We categorized the cells in five groups like: live and intact cells, live cells with injured acrosome, dead cells, live head with dead tail and live tail with dead head. We used one-way analysis of variance (ANOVA) to assign how gelatin concentration affects the number of the categorized cells. On the fifth day, the viability was the best in the following semen extenders: 1.5% fat UHT milk + 1.0% gelatin and 1.5% fat UHT milk + 1.5% gelatin, but it was not significant (p>0.05).

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