Protoplasmic streaming inAcetabularia

Summary A compilation of characteristics of the two different systems of intracellular transport inAcetabularia (Koop andKiermayer 1980 a and b) is given.The presence of microfilaments-presumably F-actin-in the cytoplasm ofAcetabularia is demonstrated by electron microscopy.The evidence for an involvement of microtubules in streaming is strengthened by the induction of birefringent vinblastine crystals in the stalk of vegetative cells.Isolated portions of cytoplasm formin vitro more than 100 m long filopodium-like processes, which are highly birefringent. The processes show intensive immunofluorescent staining with both, anti-actin and anti-tubulin as a primary antibody.A perfusion buffer is presented, which after replacing the vacuolar sap does not lead to a change in cytoplasmic morphology or streaming pattern and velocities.     

Transient reduction of responsiveness of blue-light-mediated hair-whorl morphogenesis inAcetabularia mediterranea induced by blue light

After a prolonged period of red light the formation of a new whorl of lateral hairs can be induced inAcetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) by a pulse of blue light. It has previously been shown that the response to blue light obeys the law of reciprocity. In this paper we demonstrate that the responses to blue light are additive only within 10 min after the onset of blue-light treatment, since the responsiveness of the cells is also affected by blue light. One hour after a short blue-light pulse the response to a second blue-light pulse has come to a minimum. After that, the responsiveness is restored in a refractory period of several hours. The fluenceresponse curves for hair-whorl formation at the time of minimum responsiveness are shifted parallel to the original fluence-response curves without preirradiation. Again, the law of reciprocity applies. This indicates an increased light requirement only for the same degree of hair-formation response. The sensitivity to blue light of the reduction of responsiveness response is higher by a factor of about 50 than the induction of hairformation response.     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Studying the effects of actin cytoskeletal destabilization on cell cycle by cofilin overexpression

The significance of actin cytoskeleton on cell growth was historically studied using toxic drugs, such as cytochalasin. However, it is possible that unpredictable effects of these agents may have influenced the reported observations. In our study, we have established a drug-free system using cofilin overexpression to investigate the relationship between actin filaments and cell cycle progression. Cofilin is a member of the actin depolymerization factor (ADF)/cofilin family, cofilin cDNA was cloned to a tetracycline-inducible gene expression vector and stably transfected to human lung cancer H1299 epithelial cells. Destabilization of actin filaments and morphological change was detected in cofilin overexpressing cells by actin analysis and microscopy, respectively. Measurements of growth rates showed that cell proliferation was retarded in cells with overexpressed cofilin. Also, cell cycle analysis showed that approx 90% of cofilin overexpressing cells were arrested in G1 phase, which is consistent with previous reports that drug-mediated disruption of actin filaments can cause G1 phase arrest. Taken together, cofilin overexpression cell model provides evidence that the effects of actin cytoskeletal destabilization on cell cycle progression can be studied using molecular approach instead of drug.     

Hair morphogenesis inAcetabularia mediterranea: Temperature-dependent spacing and models of morphogen waves

Summary Whorls of sterile hairs inA. mediterranea show, at the moment of first appearance of hair initials, a spacing independent of number of hairs in the whorl but dependent on temperature. By changing the temperature at various times before appearance of hair initials, the pattern-forming event can be located at about 3–4 hours before initials become visible.The temperature dependence of spacing is like that of a chemical rate parameter: In (spacing)versus 1/T is linear. This suggests that the spacing is controlled by kinetic rather than structural factors, and correlates well with reaction-diffusion theory.Mathematical analysis and computer simulation have been used to show that the observed sequence of tip-flattening followed by whorl initiation can be interpreted in terms of published models for generation of dissipative structures by reaction and diffusion, and that at least two sequential processes must occur, the first of which shifts growth activity from extremity to circumference of the growing tip, permitting the second to operate around the circumference.Submitted to workshop on Morphogenesis inAcetabularia, Berlin (West), September 1980.     

Nuclear division in the cyst,and white spot nuclei preceding cyst formation,inAcetabularia wettsteinii

Summary Electron microscopy of nuclear division in young cysts ofAcetabularia wettsteinii shows that the dividing nucleus hat two additional cisternae of endoplasmic reticulum immediately outside the nuclear envelope. These additional cisternae are attached to, and apparently formed from a membrane body which develops outside the nucleus in early prophase. The interphase nucleus does not have the additional cisternae. The nucleoli are extruded from the nucleus at anaphase, the nucleolar bodies remaining in the peri-nuclear cytoplasm. The chromosomes have localized centromeres; the stratified ultrastructure characteristic of some chlorophycean and animal kinetochores has not been found inAcetabularia, although the kinetochore appears distinct, projecting from the chromatid, and has attached microtubules. The condensed bodies of the white spot nucleus are discussed.     

Multinucleate cyst formation in Acetabularia mediterranea after x-irradiation

Cells of Acetabularia mediterranea were irradiated with increasing doses of X-rays (64.5–258·10^-4 kC kg^-1). The cells are radioresistant up to 193.5·10^-4 kC kg^-1 in terms of growth and progression through he life cycle but the morphogenesis of whorls, caps, and cysts is accompanied by morphological alterations. Microscopical examination of cyst bearing caps in irradiated cells has shown the presence of giant cysts neighboring particularly small ones. Photographic recording of cyst development showed that the multinucleate cap cytoplasm partitions into multinucleate portions rather than uninucleate ones as in the control cells. After complete cleavage a cyst wall is deposited onto the multinucleate cytoplasm. In contrast to uninucleate cysts with one lid the wall contains multiple lids. Their number appears to correspond to the number of nuclei in the cytoplasm compartment during cleavage. The results indicate that X-rays preferentially inhibit the synthesis of a factor which plays a role in establishing the normal spatial morphogenetic pattern necessary for cyst formation.     

Ultrastructural characterization of the locomotory cytoskeletal system of the spermatozoid inGinkgo biloba

Summary The development of the locomotory cytoskeletal system of sperm is carefully coordinated with the development of the sperm inGinkgo biloba. Here we report further ultrastructural characterization of the locomotory cytoskeletal system in the developing spermatid and mature spermatozoid, particularly with respect to the initiation and early development of the flagellar apparatus. A multilayered structure (MLS) assembles from an electron-dense matrix that self-organizes after blepharoplast breakup and then further elongates. At the tail of the assembling MLS, the spline microtubules connect to an anterior beak of the nuclear envelope. Nuclear-pore complexes are found on the nuclear envelope close to this beak. The mitochondria which elongate and line up one behind the other are tightly associated with the MLS. The MLS ofG. biloba is composed of an upper layer of parallel spline microtubules and a lower layer consisting of a fibrous lamellar strip composed of paralled fibers about 9 nm in diameter. Higher-magnification images show that the fully assembled fibers of the lamellar strip consist of subunits which suggest that protofilaments are involved in the assembly processes. A unique cytoskeletal system of the spermatozoid inG. biloba is given by the anterior bundle of microtubules. This bundle, in which microtubules are arranged parallel to each other, forms between the plasmalemma and the MLS and is about 214–392 nm in cross section. These microtubules expand spirally along the MLS band. Other details of cellular fine structure of the mature spermatozoid are described.     

The perinuclear microtubule system in the green algaAcetabularia: anchor or motility device?

Summary Migrating secondary nuclei inAcetabularia are tightly associated with actin bundles and possess a comet-like tail composed of microtubules. When secondary nuclei begin to settle in preparation for cyst morphogenesis, the tails expand into radially symmetrical arrays of microtubules. Concomitantly, nuclei become gradually dissociated from the actin bundles and eventually stop moving, even though the actin bundles remain intact and persist through this stage. If, however, the radial perinuclear microtubule arrays are destroyed by inhibitors, the nuclei reassociate with the actin bundles and regain their motile activity. Because this movement is sensitive to Cytochalasin D, we propose that actin is required for nuclear movements, whereas microtubules most likely function as a trailing anchor that begins to act as a braking device above a certain threshold in the number and length of perinuclear microtubules.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

An interconnected plastidom inAcetabularia: Implications for the mechanism of chloroplast motility

Summary In the unicellular green algaAcetabularia, the vital fluorochrome 3,3′-dihexyloxacarbocyanine (DiOC₆) readily accumulates in chloroplasts and mitochondria at low concentrations, suboptimal for the visualization of the endoplasmic reticulum (ER). These organelles align along motility tracks and partially obscure each other, resulting in the loss of image information in conventional fluorescence microscopy. However, superior imaging of organelles was achieved by confocal laser scanning microscopy, which was particularly evident in areas where mitochondrial profiles overlap with chloroplasts. In addition to the tubular mitochondria, a new type of tubular membrane profiles was discovered inAcetabularia which connects the chloroplasts with each other. These tubules may either form short bridges or may stretch over hundreds of micrometers before connecting to the next chloroplast. Because staining intensity, size and overall shape of mitochondria and the connecting membrane tubules were very similar, pharmacological treatments have been applied to differentiate more clearly between the two compartments. Inhibitors of mitochondrial function are shown here to affect mitochondrial shape but not that of the chloroplast tubules. Finally, electron microscopic analysis of thin sectioned materials revealed long tubular emanations from the chloroplasts proving their plastidal origin. The function of these hitherto unknown plastidal membrane tubules is not known, but their behaviour suggests that they interact with the cytoskeleton and effectively modify chloroplast behaviour.     

The life cycle ofAcetabularia (Dasycladales,Chlorophyceae): A compilation of evidence for meiosis in the primary nucleus

Summary The life cycle ofAcetabularia is described with special reference to nuclear divisions. Recent arguments, derived from the fields of cytology, genetics and systematics are in favour of the hypothesis, that meiosis occurs during the division of the primary nucleus. This hypothesis is summarized in a schematical representation of the whole life cycle.     

Action potentials inAcetabularia: Measurement and simulation of voltage-gated fluxes

Summary Amounts and temporal changes of the release of the tracer ions K⁺ (⁸⁶Rb⁺),²²Na⁺, and³⁶Cl^– as well as of H⁺ in the course of action potentials inAcetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systemsX in the plasmalemma: Cl^– pumps, K⁺ channels, and Cl^– channels (which are marked in the following by the prefixes,P, K andC) with their equilibrium voltages _X V _e and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (_P L, _KL,_C L=1) but voltage gating be different: the pump ( _{p V e} about –200 mV) being inactivated (open,oclosed,c) at positive going transmembrane voltages,V _m; the K⁺ channels (_K V _e about –100 mV) are inactivated at negative goingV _m; and the Cl^– channels (_C V _e: around 0 mV), which are normally closed (c) at a restingV _m (near_PV_e) go through an intermediate open (o) state at more positiveV _m before they enter a third shut state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factorf=exp(V _mF/(2RT)), simulate, the action potential fairly well with the following parameters (_PK_co10/f ks^–1,_PK_oc1000·f ks^–1,_KK_co200·f ks^–1,_Kk_oc2/f ks^–1,_cK_co500·f ks^–1,_CK_oc5/f ks^–1,_CK_so0.1/f ks^–1,_Ck_os20·f ks^–1). It is also shown that the charge balance for the huge transient Cl^– efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na⁺.     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

Role of red light in hair-whorl formation induced by blue light in Acetabularia mediterranea

After a pre-treatment with red light, hair formation at the growing tip of the siphonaceous green alga Acetabularia mediterranea Lamour. (= A. acetabulum (L.) Silva) can be induced by a pulse of blue light. Red light is needed again after the inductive blue-light pulse if the new whorl of hairs is to develop within the next 24 h. In order to investigate the role of this red light, the duration of the red irradiation was varied and combined with periods of darkness. The response of hair-whorl formation was dependent on the total amount of red light, regardless of whether the red irradiation followed the blue pulse immediately or was separated from it by a period of darkness. Furthermore, periods of exposure to the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1-1dimethylurea had a similar effect to darkness. Both observations indicate that this red irradiation acts as a light source for photosynthesis. Whether or not the red light had an additional effect via phytochrome was tested in another type of experiment. The dependence of hair-whorl formation on red-light irradiance in the presence of simultaneous far-red irradiation was determined for the pre-irradiation period as well as for the irradiation period after the blue pulse. In both experiments, far-red light caused a small promotion of hair-whorl formation when low irradiances of red light were used. However, these differences were attributable to a low level of photosynthetic activity (which in fact was measurable) caused by red light reflected in the growth chamber. Furthermore, lowering the proportion of active phytochrome by far-red light would be expected to suppress hair-whorl formation. The influence of far-red light was also tested in a strain of Acetabularia mediterranea that developed hair whorls in about 20% of cells even when kept in complete darkness after the blue-light pulse. Far-red irradiation had no effect. These results strongly indicate that phytochrome is not involved in hair-whorl formation. Rather it is concluded that the effects of red light are caused by photosynthesis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Microtubule formation on the nuclear surface during meiosis inAcetabularia acetabulum

Summary Microtubules (MT) are a feature of all eukaryotic cells. However, they have not been observed in the cytoplasm of the vegetative phase ofAcetabularia acetabulum. Previous investigators have reported that, in the propagative phase, MTs function as anchors in the transport of secondary nuclei to the cap. They also form elaborate arrays around nuclei during cyst formation. The life history ofA. acetabulum is marked by changes in chromatin, the nucleolus, and the perinuclear cytoplasm. In this study light microscopical features of the nucleolus and changes in chromatin, labelled with anti-histon antibodies, were used to define the developmental stages. Anti-tubulin antibodies have been used to trace the origin and development of MTs, MTs are formed on the surface of the primary nucleus. They are organized first into short thick sticks and then later elongate into thinner strands which enclose the nucleus in a dense network. Following these events on the surface of the nucleus, the spindle develops inside the nuclear membrane which remains intact throughout the mitotic division.     

The differentiation of cytoskeletal structures in nematocytes of Hydra

Nematocytes of hydra feature a complex cytoskeleton consisting mainly of several bundles of actin filaments and a basket-like structure formed by microtubules. The aim of this study was to establish the sequence of appearance of cytoskeletal elements during nematocyte development using immuno-fluorescence and electron microscopical techniques. Our results are a first step in trying to understand developmental hierarchies and mechanisms which govern the synthesis and assembly of the cytoskeleton in nematocytes. The finger-shaped rods around the apex of the capsule are the first detectable elements of the cytoskeleton. Microtubules of the basket structure then follow and later, the actin filaments of microvilli which support the cnidocil. The actin filaments, however, do not show the highly ordered bundling pattern characteristic of filaments in functional nematocytes.     

Probable involvement of cytoskeleton in stomatal-pore formation inAsplenium nidus L.

Summary Stomatal-pore formation in the fernAsplenium nidus L. commences in postcytokinetic guard cells at the mid-region of the ventral wall, before the deposition of any cellulosic wall material on it, by the local movement of the adjacent plasmalemmata apart from each other. In this way a rudimentary internal stomatal pore is formed. At this stage the ventral wall exhibits an undulated appearance and gives a positive reaction to aniline blue. Detailed study of postcytokinetic guard cells by electron microscopy, as well as after tubulin immunolabeling and actin staining, shows that stomatal pore initiation coincides with the initiation of the organization of the anticlinal microtubule bundles along the middle of the ventral wall and the colocalization of actin filaments at the same sites. Afterwards, the stomatal pore broadens towards the periclinal walls, a phenomenon keeping pace with the further bundling of the cytoskeletal elements beneath the plasmalemmata lining the middle of the ventral wall. At this stage the anticlinal microtubule bundles lining the stomatal pore are very prominent. The above findings, as well as the fact that treatments with antimicrotubule drugs inhibit the internal stomatal-pore formation, denote that the cortical cytoskeleton lining the ventral wall and particularly the microtubules are involved in this process. Afterwards, distinct local wall thickenings are deposited at the sites of junction of the mid-region of the ventral wall with the periclinal walls as well as at the junctions of the polar ventral-wall ends with the external periclinal wall. Along the middle-lamella region of the former wall thickenings the fore- and rear-chambers of the stomatal pore are formed. The final stomatal-pore opening is achieved by disruption of the expanded thin median periclinal wall region inherited from the guard cell mother cell and of the overlying cuticle, which covers the stomatal pore externally and internally. At the same time the fore-chamber of the stomatal pore broadens by a schizogenous opening towards the polar ventral-wall ends. The observations show that the stomatal-pore formation inA. nidus is a unique process, which is probably restricted to ferns.Abbreviations Af actin filament - GC guard cell - Mt microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - VW ventral wall     

Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) I. Pre-division development and the formation of spine-scales and body scales

Summary Specific events preceding cell division in the unicellular phytoflagellateApedinella radians (Pedinellophyceae) were investigated ultrastructurally and by immunofluorescence microscopy. These events include the sequential formation and secretion of a duplicate set of elongate spine-scales, the duplication and development of basal bodies/flagella, the generation of two new dictyosomes at the future sites of spindle poles, and the constriction (not division) of chloroplasts. Actin and microtubules are involved in spine-scale formation, while actin alone appears to play a role during body scale formation. Both scale types are formed intracellularly within specialized vesicles located adjacent to the posterior cell membrane. The length of spine-scales is not limited by cell size, as they form within cell extensions. Following secretion, spine-scales remain attached below the equator of the cell, and are not deployed to their interphase positions until after the completion of cell division.Abbreviations 08/28 polyclonal anti-centrin antibody - 17E10 monoclonal anti-centrin antibody - 26/14-1 polyclonal anti-centrin antibody - BSA bovine serum albumin - BSFV body scale forming vesicle - FITC fluorescein isothiocyanate - Ssc polyclonal anti-centrin antibody - SSFV spine-scale forming vesicle     

A cytoskeletal system predicts division plane in meiosis ofSelaginella

Summary An ultrastructural investigation of the monoplastidic microsporocytes ofSelaginella arenicola revealed a unique cytoskeletal array that predicts the future division plane before nuclear division takes place. By midprophase of the first meiotic division, the single plastid has divided once and the two plastids lie on opposite sides of the nucleus which is elongated in the plane of the incipient metaphase I spindle. A cytoplasmic structure, the procytokinetic plate (PCP), predicts the division plane of of both plastid and cytoplasm. The PCP consists of a distinct concentration of vesicles lying in the future division plane and an elaborate system of microtubules aligned parallel to the long axis of plastids and nucleus. Microtubules of the axially aligned system appear to terminate in clusters of vesicles in the central zone of the PCP. The PCP with axially aligned microtubules is as predictive of the division plane in these meiotic cells as is the girdling preprophase band of microtubules in mitotic cells.