A Comparison of the Organization of Chloroplast-Associated and Chloroplast-Free Action Bundles in Nitella Internodal Cells
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University of the Pacific Scholarly Commons University of the Pacific Theses and Dissertations Graduate School 1987 A comparison of the organization of chloroplast-associated and chloroplast-free action bundles in Nitella internodal cells Kelly Tennyson Dineley University of the Pacific Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Life Sciences Commons Recommended Citation Dineley, Kelly Tennyson. (1987). A comparison of the organization of chloroplast-associated and chloroplast-free action bundles in Nitella internodal cells. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/2142 This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. A Comparison of the Organization of Chloroplast-Associated and Chloroplast-Free Actin Bundles in Nitella Internodal Cells In Partial Fulfillment of the Requirements for the Degree Master of Science by Kelly T. Dineley May 1987 This thesis, written and submitted by is approved for recommendation to the Committee on Graduate Studies, University of the Pacific. Department Chairman or Dean: Thesis Committee: /~d/~ Chairman ~SJ44(L . ACKNOWLEDGEMENTS I would like to express sincere gratitude to Dr. Paul A. Richmond for the many hours he has contributed to the completion of this work. His skills and experience as a researcher were invaluable in outlining the goals and scope of this investigation as well as in guiding its execution. Many thanks must also be extended to Cindy Custer-Coon for her technical expertise in all aspects of scanning and transmission electron microscopy without which, many of the micrographs presented in this paper would not have been possible. Finally, but most importantly, I wish to express appreciation for my family and friends whose support and love made the hard work these past three years easier and more worthwhile. iii CONTENTS Page ACKNOWLEDGEMEl<TS ••••.•.••••...••..•••••••••••.••..•••••• iii ABSTRACT . .••....••........•.........•...•...•....••...•... v LIST OF TABLES •••••.•••••••.•••••••.•••••.•••.••••••••.•. vi LIST OF FIGURES •.•.•.••..••.•••••••.•.••••.•••••••••.••• vii INTRODUCTION . ....•...........•...................••.....• . 1 MATERIALS AND METHODS •....•.•.•••.•.•....••.••••••••••.•• 16 Transmission Electron Microscopy ••••••••.••••••••••••.. l6 Scanning Electron Microscopy ..••.••••••...•••••••••••.. 19 Window Technique . ...................................... 21 RESULTS •••••••.••...•••.•.•.•.•.••.••••••.•.•••••••••••.• 23 Sidewall ............................................... 23 Endwall .•••.••..•..••..••..••••.••••.••..•••••••••.•... 26 Irradiated Windows ..................................... 28 DISCUSSION ••.•••..•.•••••••••..•..•••••..•••.•••••••.••.• 30 REFERENCES .•.•.••...••....••..•........•••...••...••..•.. 7 4 iv ABSTRACT The actin fibrils found at the ectoplasm-endoplasm interface in Nitella internodal cells are a major component of the mechanism that is responsible for cytoplasmic streaming in these giant algal cells. The fibrils have been shown to attach to the inner surface of internodal chloroplasts which are embedded in long files within the stationary ectoplasm along the length of the cell. The existence of actin bundles at the ends of the cell, where chloroplast files are absent, has not been examined. Through the use of scanning and transmission electron microscopy, the present work shows that actin bundles are continuous throughout the cell and that those bundles in the chloroplast-free endwall region have a distinct distribution from those associated with chloroplast files. Additionally, the organization of regenerated actin bundles in blue light-irradiated areas of cells {in which an area of the cell cortex is stripped of its chloroplasts and associated actin fibrils) is compared to untreated regions of the cell. These morphological observations are quantified and discussed in terms of their implications towards the nature of actin bundle immobilization and bundle organization during cell ontogeny. v TABLES Table Page I. Character and Dimensions of Chloroplast- Associated Actin Bundles ...................... 56 II. Bundle Spacing Measurements ...•••............... 57 III. Bundle Spacing in Irradiated Windows ............ 58 vi FIGURES Figure Page 1. Nitella Plant Shoot Diagram •.•................ 59 2. Diagram of Nitella Internodal Cell Cortex and Subcortical Actin Bundle Organization ..... 59 3. SEM Micrograph of the Interior of an Internodal cell ............................... 60 4. TEM ~licrograph of Cortical Chloroplasts and Subcortical Actin Bundles ..................... 61 5. TEM Micrograph of Mitochondria in Region of Streaming Endoplasm .................••......•. 61 6. SEM Micrographs Illustrating Effects of Not Centrifuging Specimens Prior to Fixation and Freeze-Drying .•..•...........•.••....•.•.. 6 2 7. SEM Micrograph Illustrating Effect of Centrifuging During Specimen Preparation ••.... 63 8. SEM Micrograph Exhibiting Microtubules on the Plasma Membrane Near the Internodal Cell Cortex .............•......•................... 64 9. SEM Micrograph of Cortical Chloroplasts and Associated Actin Bundles ...................... 64 10. SEM Micrograph of Branching Chloroplast- associated Actin Bundles ...................... 65 11. SEM Micrographs Exhibiting Mitochondria Under Chloroplast Periphery .•..•.................... 66 12. SEM Micrograph of the Null Zone in an Internodal Cell ..••...........•............... 6 7 13. SEM Micrographs of the Corner Aspect of an Internodal Cell .••...........•••.......•...... 68 14. SEM Micrograph of the Internodal Cell Transition Zone ..•...................•.....•.. 69 15. TEM Cross Sections Through the Tip of an Internodal Cell .....•.•............•..••...... 70 vii 16. TEM Micrograph of Actin Bundles Turning the Corner of an Internodal Cell. ................ 70 17. TEM Longitudinal Section Illustrating the Continuity of Actin Bundles Beyond Chloroplast Files ............................. 70 18. TE~l Micrographs of Actin Bundles Near and AT the End of the Internodal Cell ................ 71 19. SEM Micrograph of Regenerated Actin Bundles an Internodal Cell Irradiated Window .......... 72 20. SEM Micrograph of Actin Bundle Organization at the Downstream End of an Irradiated Window in an Internodal Cell. ................. 73 viii INTRODUCTION Intracellular motility is a common feature of eukaryotic cells. It is necessitated by the dynamic relationships amoung the complex systems of cytoplasmic organelles that are found in these cells. Two main systems of cell motility exist. They are defined by the cytoskeletal elements of which they are composed and upon which they depend for force generation. One is based on the protein tubulin and functions in the form of microtubules. The principal component of the other is actin containing microfilaments. Microtubule and microfilament systems form the basis for virtually all intracellular motile activities. A third cyt~skeletal component, intermediate filaments, has a structural or tension-bearing role in some cells and does not appear to have more than a supporting role in cell dynamics. Each motility system is a unique complex of tubulin and/or actin that is complemented by associated proteins which act as connectors, supporters, or ATPases in either a labile or stable arrangement. Although the two motility systems are morphologically and biochemically distinct, their underlying molecular mechanisms are similar. Each can produce movement in two ways: through a sliding mechanism or by the assembly and disassembly of filaments. The assembly and disassembly of filaments effect movement as a consequence of polymerization/depolymerization of either actin or tubulin in response to certain cell stimuli. By changing the 1 2 chemical (ions, drugs) and/or the thermodynamic (temperature, pressure, subunit concentration) conditions in a cell, assembly and disassembly of microfilaments and microtubules can be controlled. Both actin and tubulin exist in two functional states: as soluble subunits and as assembled filaments (for reviews of actin and tubulin structure, biochemistry and function in cell motility see Shay, 1984; Stebbings and Hyams, 1979; Fulton, 1984). Actin polymers are formed by the assembly of the 42 KD globular actin (G-actin) monomers into a double-stranded filament (F-actin) that is twisted into a helix 7 nm wide with a half-pitch of 34 nm. On the other hand, microtubules are cylindrical structures approximately 24 nm in diameter with a central lumen 15 nm wide. The microtubular shell is made up of 13 longitudinal protofilaments of tubulin, a dimeric globular polypeptide of 100 KD. Each dimer is composed of two closely related, but nonidentical, polypeptides called alpha-tubulin and beta-tubulin. Actin monomers and tubulin dimers are asymmetric molecules in that there is a difference in the affinities of free molecules over their surfaces. This inherent polarity is expressed in polymers of actin and tubulin as a difference in the rate of growth and depolymerization at their ends. The end that tends to add subunits is referred to as the positive (+) end and the end that loses subunits is called the negative (-) end. 3 The polarity of intact microfilaments and microtubules can be visualized experimentally. Microfilaments