* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Actin microfilaments are associated with the migrating nucleus and
Survey
Document related concepts
Signal transduction wikipedia , lookup
Tissue engineering wikipedia , lookup
Programmed cell death wikipedia , lookup
Extracellular matrix wikipedia , lookup
Endomembrane system wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cell growth wikipedia , lookup
Cellular differentiation wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cell culture wikipedia , lookup
Cell nucleus wikipedia , lookup
List of types of proteins wikipedia , lookup
Transcript
1929 Journal of Cell Science 107, 1929-1934 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias Studies on living cells Ursula Meindl1,*, Dahong Zhang2 and Peter K. Hepler3 1Institut für Pflanzenphysiologie, Universität Salzburg, A-5020 Salzburg, Austria 2Department of Zoology, Duke University, Durham, NC 27706, USA 3Department of Biology, University of Massachusetts, Amherst, MA 01003, USA *Author for correspondence SUMMARY Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast. INTRODUCTION cells that have been injected with fluorescently labeled phalloidin and analyzed by confocal laser scanning microscopy (CLSM). Although phalloidin is a poison, when injected at low levels it does not inhibit cell growth, or development, nor does it block cytoplasmic streaming, yet it still stains the MFs and allows these structures and their dynamic transformation to be directly observed in living cells (Cleary et al., 1992; Hepler et al., 1993; Zhang et al., 1993). The results from the present study reveal extensive arrays of actin MFs in the cell cortex and in the form of a cage surrounding the migrating nucleus. Actin microfilaments (MFs) have been associated with polarized tip growth in a variety of plant cells, especially in fungal hyphae and pollen tubes (for references see Heath, 1990). In the desmid Micrasterias, a highly sculptured shape emerges during cell development that consists of numerous growing points positioned in a symmetrical pattern. Here the participation of MFs in the regulation of development derives mainly from studies using the drug cytochalasin (Tippit and Pickett-Heaps, 1974; Noguchi and Ueda, 1981; Lehtonen, 1983). Culture in low concentrations of cytochalasin B, for example, inhibits or retards cytoplasmic streaming and leads to distinctly malformed cells. In addition, nuclear migration in both Micrasterias and the closely related desmid, Euastrum, is altered from the normal condition, although in this regard the effect of cytochalasin is less profound than that caused by microtubule inhibitors (Meindl, 1990; Url et al., 1992). Despite these experimental inferences that MFs are involved in several vital developmental processes, with the exception of two reports (Noguchi and Ueda, 1988; Ueda and Noguchi, 1988), we know remarkably little about the structural distribution of actin in desmids. In the present investigation the spatial localization of MFs in Micrasterias has been examined in living Key words: actin microfilament, confocal laser scanning microscopy, Micrasterias, microinjection, nuclear migration, phalloidin MATERIAL AND METHODS Culture Cells of Micrasterias denticulata Breb. were cultivated under semisterile conditions in a ‘desmid-medium’ with diluted soil extract. The culture flasks were kept at a temperature of 20°C and a light-dark regime of 14 to 10 hours (for detailed method see Kiermayer, 1980; Schlösser, 1982; Meindl, 1990). Preparation for microinjection A small drop of nutrient solution with different developmental stages of Micrasterias denticulata was transferred to a coverslip, which was mounted at the bottom of a small glass chamber. A small amount of 1930 U. Meindl, D. Zhang and P. K. Hepler warm (32-35°C), low temperature gelling agarose (type VII, Sigma) at a concentration of 3% was placed besides the cells. The nutrient solution containing the cells was then mixed with the agarose solution, and the mixture was gently spread over the cover slip and immediately chilled to gel the agarose. The immobilized cells were flooded with nutrient solution. This preparation method enabled the cells to continue their development without any visible change. Microinjection of phalloidin Either FITC- or rhodamine-phalloidin (Molecular Probes Inc.) was used for the microinjection experiments. A stock solution (3.3 µM in methanol) was evaporated almost to dryness with nitrogen gas and then redissolved in 100 mM KCl. The solution, which is never kept longer than 24 hours, was sonicated and centrifuged immediately before use. Microneedles (WPI; Kwik-fil, 1.0 mm OD) were pulled from glass capillaries with a vertical pipette puller (David Kopf Instruments). They were loaded with the phalloidin solution using a thinly drawn plastic tuberculin syringe. The needle was mounted in a Zeiss microneedle holder, connected to a Gilmont micrometer syringe by a water-filled tubing and was maneuvered by a Narishige micromanipulator. Hydraulic pressure was applied to drive the phalloidin solution into the cell. Microinjection was carried out on an inverted microscope (Zeiss) equipped with Normaski optics (for further details of the method see Zhang et al., 1990). Microinjection impalements were made on growing semicells of Micrasterias, preferentially into one of the growing lobes where the cell wall was as thin as possible. This ensured that the wound caused by a shallow injection was minimal. After injection the needle was removed from the cell within 20 to 30 minutes thus allowing the cell to form a plug that healed the wound and guaranteed that no cytoplasm was lost. Confocal laser scanning microscopy Cells injected with phalloidin were examined on a confocal laser scanning microscope (MRC-600, Bio-Rad) with an argon ion laser for up to 2 hours. Observation started immediately after injection when the needle was still in the cell. To prevent damage by the laser the cells were exposed to irradiation at intervals of about 15 minutes for only a few seconds. During this time pictures were taken at different focal planes. Since it was difficult to go back to one particular focal plane after a 15 minute development of this large cell, most emphasis rested on showing the three dimensional MF arrangement rather than to follow the minimal changes that occur in the orientation of the single MFs. Thin optical sections (1-2 µm) where scanned 5 to 10 times (Kalman averaging) to form one image. When FITC-phalloidin was used to label the cells, a red supressor filter was introduced to reduce the autofluorescence of the chloroplast. Pictures where taken directly from the screen after background subtraction and contrast enhancement using either a Kodak T-Max 400 film or an Ilford Pan F film. RESULTS Microinjection of either FITC- or rhodamine-phalloidin into different developmental stages of Micrasterias denticulata results in the appearance of two different patterns of actin MFs a few minutes after injection. Independent from the stage of development a network of actin MFs is present in the cortical cytoplasmic layer directly beneath the plasma membrane (Figs 1A-C, 5A,B). This network covers the entire inner surface of the cell including both the growing and the non-growing semicell and also ensheathes the chloroplast (Fig. 2A-E). It reaches far into the tips of the lobes and continues through the isthmus area. There is no preferential orientation of the microfilament bundles and no visible correlation to the cell pattern. When cell development continues the arrangement of the single filaments changes but their net-like distribution is maintained. A second fluorescent pattern that becomes visible after microinjection of phalloidin is situated around the nucleus. In young developmental stages only the nuclear membrane is stained. As soon as the nucleus starts to leave the isthmus area and migrates into the growing semicell bundles of microfilaments become visible surrounding the isthmus-facing half of the nucleus (Fig. 3A,B). They seem to arise from a knob-like structure that is situated exactly in the center of the isthmus area and exhibits bright fluorescence after rhodamine-phalloidin injection (Fig. 3A). Later in cell development when the nucleus has already moved away from the isthmus the actin cables surrounding the nucleus merge into one thick MF bundle running towards the isthmus and ending exactly in its center (Fig. 4E). From optical serial sections through the area of the nucleus it becomes obvious that the MFs construct a cage or basket around the isthmus-facing half of the nucleus (Fig. 4A-H). When the nucleus moves back to the cell center, the actin MF system seems to be pushed into the non-growing semicell with the microfilament bundles passing the isthmus area and reaching far into the non-growing semicell (Fig. 5A,B). Before the actin MFs vanish (some time after nuclear Fig. 1. (A-C) Different optical planes of a developing semicell of Micrasterias denticulata towards the end of morphogenesis. The cell was injected with rhodamine-phalloidin. A cortical actin network covers the inner surface of the cell and reaches far into the growing tips. A slight background fluorescence results from the autofluorescence of the chloroplast. Actin microfilaments in living Micrasterias cells 1931 Fig. 2. (A-E) Sequence of optical sections through a developing cell of Micrasterias denticulata injected with FITC-phalloidin. A cortical network of actin is visible in the non-growing semicell (right) and in parts of the growing semicell. A dense actin-network also covers the surface of the chloroplast (E). Fig. 3. (A,B) Young bulb of Micrasterias denticulata after injection of rhodamine-phalloidin (two different optical sections). A bundle of actin filaments seems to arise from a bright knob-like structure and surrounds the isthmus-facing half of the nucleus. gsc, growing semicell. migration is finished) their number decreases and the bundle, which reaches into the non-growing semicell, frequently becomes bent (Fig. 6A,B). During the entire course of nuclear migration the nuclear envelope exhibits bright fluorescence. DISCUSSION By microinjection of small amounts of phalloidin into living and growing cells two major sytems of actin MFs have been revealed in the desmid Micrasterias. The first is an extensive array of cortical elements, and the second consists of a cage of clustered MFs that encircle the nucleus and appear to be involved in nuclear migration. Although bundles of cortical MFs similar in thickness to those reported herein had been noted previously in fixed cells of Micrasterias examined by fluorescence and electron microscopy (Ueda and Noguchi, 1988; Noguchi and Ueda, 1988), the prominent nucleus-associated cage has not been reported heretofore. Of added importance is the realization that the current observations have been made on living cells, vitally stained with fluorescent phalloidin, which were continuing their development while under microscopic examination. As in dividing stamen hair cells, where this method has been used successfully to denote the dynamics of actin MFs (Cleary et al., 1992, Zhang et al., 1993), we assert that the MFs visualized in Micrasterias, under conditions in which the cells continue to grow and develop, represent structures that bear a close and meaningful relationship to those in the unperturbed cell. We fully recognize that phalloidin is not an ideal probe and that artifacts cannot be completely excluded; however, the presence of normal growth and development, when compared to control injections, indicates in all likelihood that the actin MFs have not been extensively bundled, stabilized or structurally rearranged. In trying to understand the mechanism of polarized development, the current observations do not provide clear evidence that actin MFs are involved. Not only is the pattern of phalloidin-positive material random and quite unrelated to cell 1932 U. Meindl, D. Zhang and P. K. Hepler Fig. 4. (A-H) Different optical sections through the nuclear region of a developing cell of Micrasterias denticulata. The migrating nucleus (N) is ensheathed by a basket-like arrangement of actin visualized after rhodamine-phalloidin injection. The nucleus is situated within the growing semicell. An actin bundle (E,F) reaches towards the center of the isthmus area (the latter is indicated by arrows). The bright fluorescence at the right upper area of the cell results from a bulk of chloroplast material. Actin microfilaments in living Micrasterias cells 1933 Fig. 5. (A,B) Two different optical levels of a developing Micrasterias cell after injection of FITC phalloidin. A cortical actin network is visible in the growing and the non-growing semicell. The nucleus (N), which is on its way back to the isthmus, is surrounded by actin bundles reaching far into the non-growing semicell. The nuclear membrane exhibits bright fluorescence. Fig. 6. (A,B) Same cell as in Fig. 4. but about 60 minutes later. The nucleus (N) is located close to the isthmus (isthmus indicated by arrows). Only remnants of the actin system that originally surrounded the nucleus (compare Fig. 4) are present. The actin bundle that reaches into the non-growing semicell is bent. shape, but in addition we fail to see stained filaments at the lobe tips where it would be anticipated that some form of growth control must be exerted (Meindl, 1993). Furthermore, the observed MFs are equally distributed between the growing and non-growing semicell, providing additional support against their role in polarized growth, but supporting an involvement in cytoplasmic streaming and chloroplast anchoring. Despite the lack of a visible and specific structural relationship between actin MFs and polarized growth it would be imprudent to suggest that these cytoskeletal elements were not involved. For example, cytochalasin at levels that permit cytoplasmic streaming, has a profound modulating effect on cell shape development (Tippit and Pickett-Heaps, 1974; Noguchi and Ueda, 1981; Lehtonen, 1983). Cytochalasin also disrupts vesicle production and arrangement in the closely related organism, Euastrum (Url et al., 1993). Furthermore, we should recognize that phalloidin might not stain all the actin MFs within the cell; for example, some may be closely linked to membranes in such a way that their phalloidin-binding sites are blocked. We argue therefore that it would be premature to discount actin as a prime cytomorphogenetic factor; further experimentation with other probes such as a fluorescent actin analogue might help resolve the role of actin in polarized growth. The cage of actin MFs that surrounds the nucleus is an interesting new observation. Together with the microtubules, which have been well documented in previous electron microscopic studies (Meindl, 1983, 1992), the actin MFs are thought to contribute to the positioning and migration of the nucleus. In attempting to decipher the relative roles of the two cytoskeletal elements it appears, however, that the MTs are more important since their specific destruction with APM or colchicine causes the nucleus to completely drift away from the isthmus (Meindl and Kiermayer, 1981; Meindl, 1983), whereas treatment with cytochalasin generates only a minor disorientation of nuclear motion (Meindl, 1990). Not only are MFs and MTs co-localized around the nucleus, in addition the position of a brightly fluorescing knob, which serves as the origin of the perinuclear actin system, corresponds exactly to the position of the MT center visualized in earlier studies (Meindl, 1983). Taken together these observations further indicate that MTs and MFs cooperate in regulating the position 1934 U. Meindl, D. Zhang and P. K. Hepler and motion of the nucleus. In conclusion, the observations from living cells injected with phalloidin provide novel information about the localization of actin MFs and help us understand vital processes essential to the growth and development of Micrasterias. Parts of this study were carried out at the Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, Canberra. We are indebted to Professor Brian Gunning for many helpful discussions and for generously providing his laboratory equipment. We also thank Dr Ann Cleary and Dr Geoffrey Wasteneys and other colleagues of the Plant Cell Biology Group for technical suggestions and valuable comments. This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, project 7972 to U.M., and by the US National Science Foundation, grant #s DCB-90-04191 and DCB 93-04953 to P.K.H., and grant # BBS-87-14235 to the Central Microscopy Facilities, University of Massachusetts, Amherst. REFERENCES Cleary, A. L., Gunning, B. E. S., Wasteneys, G. O. and Hepler, P. K. (1992). Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells. J. Cell Sci. 103, 977-988. Heath, I. B. (1990). Tip Growth in Plant and Fungal Cells. San Diego: Academic Press Inc. Hepler, P. K., Cleary, A. L., Gunning, B. E. S., Wadsworth, P., Wasteneys, G. O. and Zhang, D. H. (1993). Cytoskeletal dynamics in living plant cells. Cell Biol. Int. 17, 127-142. Kiermayer, O. (1980). Control of morphogenesis in Micrasterias. In Handbook of Phycological Methods. Developmental and Cytological Methods (ed. E. Gantt), pp. 5-13. Cambridge University Press. Lehtonen, J. (1983). Action of cytochalasin B on cytoplasmic streaming systems and morphogenesis in Micrasterias torreyi (Conjugatophyceae). Nord. J. Bot. 3, 521-531. Meindl, U. and O. Kiermayer (1981). Biologischer Test zur Bestimmung der Antimikrotubuli-Wirkung verschiedener Stoffe mit Hilfe der Grünalge Micrasterias denticulata. Mikroskopie 38, 325-336. Meindl, U. (1983). Cytoskeletal control of nuclear migration and anchoring in developing cells of Micrasterias denticulata and the change caused by the anti-microtubular herbicide amiprophos-methyl (APM). Protoplasma 118, 75-90. Meindl, U. (1990). Effects of temperature on cytomorphogenesis and ultrastructure of Micrasterias. Protoplasma 157, 3-18. Meindl, U. (1992). Cytoskeleton-based nuclear translocation in desmids. In The Cytoskeleton of the Algae (ed. D. Menzel), pp. 138-147. Boca Raton, Ann Arbor, London: CRC Press Inc. Meindl, U. (1993). Micrasterias as a model system for research on morphogenesis. Microbiol. Rev. 57, 415-433. Noguchi, T. and Ueda, K. (1981). Effect of metabolic inhibitors on the formation of cell walls in a green alga, Micrasterias crux-melitensis. Plant Cell Physiol. 22, 1437-1445. Noguchi, T. and Ueda, K. (1988). Cortical microtubules and cortical microfilaments in the green alga Micrasterias pinnatifida. Protoplasma 143, 188-192. Schlösser, U. G. (1982). List of strains. Ber. Deutsch. Bot. Ges. 95, 181-206. Tippit, D. H. and Pickett-Heaps, J. D. (1974). Experimental investigations into morphogenesis in Micrasterias. Protoplasma 81, 271-296. Ueda, K. and Noguchi, T. (1988). Microfilament bundles of F-actin and cytomorphogenesis in the green alga Micrasterias crux melitensis. Eur. J. Cell Biol. 46, 61-67. Url, T., Höftberger, M. and Meindl, U. (1992). Microtubule-microfilament controlled nuclear migration in the desmid Euastrum oblongum. J. Phycol. 28, 537-44. Url, T., Höftberger, M. and Meindl, U. (1993). Cytochalasin B influences dictyosomal vesicle production and morphogenesis in the desmid Euastrum. J. Phycol. 29, 667-674. Zhang, D., Wadsworth, P. and Hepler, P. K. (1990). Microtubule dynamics in living dividing plant cells. Confocal imaging of microinjected fluorescent brain tubulin. Proc. Nat. Acad. Sci. USA 87, 8820-8824. Zhang, D., Wadsworth, P. and Hepler, P. K. (1993). Dynamics of microfilaments are similar, but distinct from microtubules during cytokinesis in living, dividing plant cells. Cell Motil. Cytoskel. 24, 151-155. (Received 30 October 1993 - Accepted, in revised form, 14 March 1994)