Microtubule Dynamics Investigated by Microinjection of Paramecium Axonemal Tubulin: Lack of Nucleation but Proximal Assembly of Microtubules at the Kinetochore during Prometaphase

• Published in: The Journal of cell biology Vol. 108; no. 3; pp. 939 - 953
• Main Authors: Geuens, G., Hill, A. M., Levilliers, N., Adoutte, A., DeBrabander, M.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.03.1989; The Rockefeller University Press
• Subjects: Animals; Antibodies; Biological and medical sciences; Cell Line; Cell structures and functions; Cells; Centromere - metabolism; Centromere - ultrastructure; Centrosomes; Chromosomes; Chromosomes - metabolism; Cytoskeleton, cytoplasm. Intracellular movements; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Immunohistochemistry; Interphase; Kinetochores; Metaphase; Microinjections; Microscopy; Microscopy, Electron; Microtubules; Microtubules - metabolism; Microtubules - ultrastructure; Molecular and cellular biology; Nucleation; Paramecium; Prophase; Spindle Apparatus - ultrastructure; Tubulin - metabolism; Immunogold technique; Protozoa; Cell culture; Incorporation; Immunocytochemistry; Interphase; Mitosis; Paramecium tetraurelia; Contractile protein; Electron microscopy; Axoneme; Tubulin; Molecular assembly; Ultrastructure; Metaphase; Cytoskeleton; Ciliata; Microtubule; Localization
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.108.3.939

Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin, followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types. In agreement with the dynamic instability model, during interphase, fast (2.6 μm/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (∼6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.