Progress 10 in Molecular and Subcellular Biology With Contributions by P. S. Agutter, K. Dose, H. Stebbings Edited by 1. Jeanteur, Y. Kuchino, W. E. G. Muller (Managing Editor), P.L. Paine With 18 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Professor Dr. WERNER E. G. MULLER Physiologisch -Chemisches Institut Universitat Mainz Duesbergweg 6 6500 Mainz, FRG ISBN-13 :978-3-642-73601-8 e-ISBN-13 :978-3-642-73599-8 DOl: lO.1007/978-3-642-73599-8 Library of Congress Catalog Card Number 75-79748 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broad casting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its version of June 24, 1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1988 Softcover reprint of the hardcover 1st edition 1988 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Fotosatz & Design, Berchtesgaden 2131/3130-543210 Contents H. STEBBINGS: Translocation Along Microtubules in Insect Ovaries (With 6 Figures) A. Introduction . . . . . . . . . . . . . . . . . . . . . . I B. Insect Ovaries, Their Morphology and Development .... I C. Translocation of Materials Between Nutritive Cells and Oocytes 2 D. Ultrastructure of Nutritive Tubes . . . . . . . 5 E. Mechanisms of Microtubule-Associated Transport Along Nutritive Tubes . . . . . . . . . . . . 6 F. The Motor(s) for Microtubule-Based Translocation II References . . . . . . . . . . . . . . . . . . . 12 P. S. AGUTTER: Nucleo-Cytoplasmic Transport ofmRNA: Its Relationship to RNA Metabolism, Subcellular Structures and Other Nucleocytoplasmic Exchanges (With 9 Figures) A. Introduction . . . . . . . . . . . . . . . . . 15 B. An Overview of Nucleocytoplasmic Transport 21 C. RNA Metabolism and Ribonucleoprotein Structure 36 D. Messenger RNA Transport . . . . 58 E. The Control of Messenger Transport 69 F. Conclusions 77 References . . . . . . . . . . . . 80 K. DOSE: Prebiotic Evolution and the Origin of Life: Chemical and Biochemical Aspects (With 3 Figures) A. The Object and Its History ............ 97 B. Modern Hypotheses, General Objections and Supports 99 C. The Oparin Hypothesis and the Simulation Experiments 101 D. The Origin of Genetic Information 103 E. Conclusions 110 References . 110 Subject Index 113 Contributors AGUTTER, P. S., Department of Biological Sciences, Napier College, Colin ton Road, Edinburgh EHlO 5DT, Great Britain DOSE, K., Institut fUr Biochemie, Johannes Gutenberg-Universitat, Becherweg 30, 6500 Mainz, FRG STEBBINGS, H., Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 ISH, Great Britain Translocation Along Microtubules in Insect Ovaries H. STEBBINGS1 A. Introduction The study of microtubule-associated intracellular translocation is, at present, one of the most exciting areas in cell biology. Most of the research has focussed on the trans location which occurs along nerve axons, but other asymmetric cells, such as chromatophores and various protozoa, have also been studied extensively, and the phenomenon probably occurs to some extent in all cells. It is particularly emphasized too in the ovaries of certain insects, and this is the subject of the present chapter. B. Insect Ovaries, Their Morphology and Development Whereas in some orders of insects the oogonia simply develop into oocytes (panoistic ovaries), during oogenesis in other orders the oogonia generate not only oocytes, but also nutritive cells (meroistic ovaries). The way in which this happens has been the subject of much study (see King and Biining 1984) but will not be considered here. Where nutritive cells do occur they show one of two arrangements with respect to the oocytes and it is this aspect which is of particular significance to the topic of intracellu lar translocation. In some instances the nutritive cells alternate with the oocytes, with which they retain cytoplasmic continuity down the length of the egg tube or ovariole (polytrophic). In other cases, all of the nutritive cells remain confined within an an terior trophic end of the ovary (telotrophic). In the latter, which occur notably in the hemiptera, the nutritive cells are connected with the oocytes, at least during the early previtellogenic stage of oogenesis, by way of cytoplasmic bridges or channels called nutritive tubes. One nutritive tube connects to each oocyte, which means that there may be some 20 nutritive tubes in a single ovariole of some species. Nutritive tubes are usually in the order of 20 ,urn in diameter and each tube gradually elongates as the oocyte with which it connects passes down the ovariole, so that it may extend many millimetres in some species (Fig. 1). IDepartment of Biological Sciences, University of Exeter,Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, Great Britain 2 H. Stebbings 1 Fig. 1. Anterior portion of ovariole of Notonecta glauca viewed in polarized light. The massive array of aligned microtubules within the trophic core and nutritive tubes (arrows) renders them birefringent. Bar = 0.4 mm Nutritive tubes contain massive numbers of aligned microtubules and hence the sys tem has proved, and is continuing to be, extremely valuable for studying microtubule associated translocation in cells. Moreover, the system of aligned microtubules is a highly dynamic one. Apart from elongating while translocation is occurring and the oocyte is passing down the ovariole, there comes a point at which the oocyte which it supplies starts to accumulate yolk, and in most species at this onset of vitellogenesis the nutritive tube connection is discontinued and the microtubules comprising it dis appear. C. Translocation of Materials Between Nutritive Cells and Oocytes The concept that materials pass from nutritive cells to the developing oocytes in meroistic insect ovaries is a longstanding one (see Lubbock 1859). Indeed there have been many morphological and histochemical studies of hemipteran telotrophic ovaries which have supported this supposition (see Stebbings 1986). Translocation along nutritive tubes has, however, been confirmed by means of radioactive labelling followed primarily by autoradiography in a number of studies using a variety of in sects (see Gutzeit 1986). There have also been reports of the direct observation of movement of components between nutritive cells and oocytes using time-lapse cinematography (Adams and Eide 1972). This has been made considerably more feas- Translocation Along Microtubules in Insect Ovaries 3 ible by the development over the past few years of video-enhanced microscopy (see Inoue 1986) and its application to telotrophic ovarioles amongst other systems has al lowed the visualization of the movement of particulate material, notably mitochon dria, along nutritive tubes (Dittmann etal. 1987). 1. Components Which lranslocate Along Nutritive Tubes Histochemical studies showed the nutritive tubes to be strongly basophylic and au toradiographic data has confirmed that large amounts of RNA synthesized in the nu tritive cells pass along the nutritive tubes to the oocytes. Most of the RNA is ribosomal, but mRNA and tRNA also pass (Davenport 1976). The application of labelled precursors, followed by gel electrophoretic analysis of the contents of nutritive tubes and fluorography has shown label, not only in ribosomal proteins, but in many other polypeptide components of the nutritive tubes (Stebbings etal. 1985). Also, analysis of the polypeptide compositions of nutritive cells, nutritive tubes and oocytes from the same ovary has demonstrated them to be comparable, suggestive of their passage through the system (Sharma and Stebbings 1985). The question of protein transport has also been addressed by introducing fluorescently labelled proteins into the different compartments of the ovary by mi croinjection, and then monitoring their passage, but the results from this approach are equivocal (Woodruff and Anderson 1984). With regards the translocation of organelles, apart from microtubules, ribosomes are the most conspicuous components, and in many instances fill the remainder of the nutritive tubes. Such observations, combined with the autoradiographic data, and the fact that nutritive cells have extremely prominent nucleoli, mean that there is little doubt that ribosomes assembled in the nutritive cells pass along the nutritive tubes to the oocytes. Indeed it is a feature of oocytes generally that they accumulate ribo somes during oogenesis. Mitochondria are similarly accumulated by oocytes and in some hemipterans this is accomplished, at least in part, by importation of mitochon dria from the nutritive cells via nutritive tubes. The study of a limited number of species suggests that this importation of mitochondria occurs in insects which have rapid oogenesis, but not to any extent in those where oogenesis is protracted (Hyams and Stebbings 1977). 11. Characterization of Movement Estimations of the rates of movement of RNA down nutritive tubes, from autoradio graphs show movement to be slow. In Notonecta (Fig. 2) this was assessed at 0.5 mml day (Macgregor and Stebbings 1970), and a similar rate was recorded in the same way for Pyrrhocoris (Mays 1972). Interestingly, in the latter case a much faster component (200 jLm/h) superimposed on the slower was also recorded. 4 H. Stebbings Fig. 2. Autoradiograph of a longitudinal section through a row of oocytes within an ovariole. Twenty-four h after the application of 3H-uridine there is heavy labelling over the nutritive tubes (arrows) indicating the presence of newly synth esized RNA. Bar = 40,urn (MacGregor and Steb bings 1970) The rates of movement of mitochondria along nutritive tubes appear to be consider ably greater than the bulk movement of RNA. From direct observations these have been estimated at approximately 3 ,umlmin in Dysdercus (Dittmann et al., 1987) or comparable to the faster movement recorded by autoradiography. Surprisingly perhaps, the movement of mitochondria was seen in the latter report to be bidirec tional. Evidence of different rates of movement along individual nutritive tubes clearly ar gues against a cohesive flow of all components along these channels. This is supported by the fact that different polypeptide components of nutritive tubes incorporate label led precursors at different rates and independently of the proportions in which they exist in the tubes (Stebbings etal. 1985). By the same token, selectivity over what translocates also appears to exist, and suggestions that the entire contents of the nutri tive cells pass down nutritive tubes to the oocytes are patently incorrect. Indeed it has already been discussed that mitochondria translocate in some species, but not in others, and plainly in no cases do the nutritive cell nuclei pass to the haploid gametes. Translocation Along Microtubules in Insect Ovaries 5 D. Ultrastructure of Nutritive Thbes Electron microscopy of nutritive tubes in telotrophic ovaries of all hemipterans so far studied has shown them to be packed with longitudinally orientated microtubules (Brunt 1970; Huebner and Anderson 1970; Macgregor and Stebbings 1970). This is not the case, however, for the much less extensive nutritive tubes of polyphagous cole opterans (Biining 1979; Stebbings 1981), where microtubules do occur but not in such large numbers or any conspicuous orientation. In hemipteran nutritive tubes the mic rotubule system may be very extensive. In Notonecta, for example, a transverse sec tion of a single tube shows in the order of 30,000 microtubule profiles. The dynamics of the microtubule system match those of the nutritive tubes they comprise. This has been illustrated by investigations into the way t~e microtubule sys tem is assembled and elaborated in Dysdercus (Hyams and Stebbings 1979a) as the nutritive tubes themselves widen and lengthen. These studies have also shown that the microtubules within a nutritive tube adopt an altered arrangement and rapidly de polymerize when the nutritive cell contribution to an oocyte ceases and the nutritive tube becomes redundant in late oogenesis. In transverse sections of functional nutritive tubes, the microtubules can be seen to be evenly spaced, and while the spacing is the same in all of the functional tubes of a particular species, the spacing varies considerably from species to species (Hyams and Stebbings 1977). Interestingly too, the separation of the microtubules in the limited number of species looked at shows a correlation with the size of components which pass between them along the tubes. In Corixa, for example, where the microtubules ar~ very closely packed, mitochondria which are larger than the distances between ad jacent microtubules do not pass down the tubes. In Oncopeltus, on the other hand, the microtubules show a much greater separation and mitochondria do pass down the nutritive tubes of this species (cf. Figs. 3 and 4). An obvious feature of nutritive tube microtubules is that they each appear sur rounded by an electron-clear zone into which other organelles do not encroach. The basis behind the separation is not known for certain, and while one view is that the zone is maintained by invisible microtubule-associated proteinaceous structure (Amos 1979), there is some evidence that it results from electrostatic repulsion be tween adjacent microtubules, and microtubules and surrounding organelles (Steb bings and Hunt 1982). Moreover, while nutritive tube microtubules do possess mic rotubule-associated proteins (MAPs) (Stebbings etal. 1986) distinct regular projec tions have not been observed from nutritive tube microtubules by any technique used so far, although linkages between microtubules and translocating mitochondria can not be ruled out (Stebbings and Hunt, 1987). As well as being parallel, it appears that the microtubules which pack nutritive tubes are all of a single polarity. This has been shown (Stebbings and Hunt 1983) using the tubulin hook-decoration technique which has been applied to· a number of mi crotubule systems. The technique also reveals that the plus or fast growing ends of the microtubules occur at the ends of the nutritive tubes adjacent to the trophic region,