Beyond self-assembly: from microtubules to morphogenesis.
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Animals↗
Biomedical subjects
M Kirschner
Publications and source records attributed to M Kirschner.
Purification of tau protein from brain.
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Animals↗
Direct observation of steady-state microtubule dynamics.
Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.
Alkanesulfonates↗
Microtubule dynamics in interphase cells.
The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.
Animals↗
Association of tau protein with microtubules in living cells.
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Adrenal Gland Neoplasms↗
Morphogenesis and the control of microtubule dynamics in cells.
Microtubules show unusual dynamic properties at steady state in vitro. While overall the polymer mass remains stable, individual polymers in the population are either growing or shrinking. This phenomenon called dynamic instability is best explained by the known coupling of polymerization to GTP hydrolysis, and the hypothesis that the stability or instability of the whole polymer is determined by whether GTP or GDP is bound to the terminal subunit. Similar unusual dynamics have now also been found in vivo. By visualizing new subunit assembly after injection of tubulin modified with biotin into living fibroblast cells, we can visualize new growth on individual microtubules with antibody to biotin. Microtubules grow in vivo at about 4 microns min-1 and after rapid and precessive depolymerization old microtubules are replaced by new growth from the centrosome. Some microtubules turn over much more slowly and these stable microtubules have a different spatial distribution from the majority of dynamic ones. The existence of both stable and dynamic microtubules in the same cell suggests a model for morphogenesis of the microtubule cytoskeleton. The rapid turnover of microtubules in the cell provides a complex population upon which selective factors can act. Stability can be generated at the end of the polymer and affects the entire microtubule. This model of selective stabilization at the microtubule ends is discussed in terms of recent experiments on the establishment of kinetochore-pole microtubules during mitosis.
Animals↗
Human atrial natriuretic peptide (ANP) for the treatment of patients with liver cirrhosis and ascites.
The acute effects of human atrial natriuretic peptide (ANP) were investigated in 10 patients with liver cirrhosis and ascites. In all patients, diuresis and natriuresis were stimulated with a wide individual variation (50 to 500%) in response to a bolus injection of 30 micrograms ANP. No side effects of treatment were observed. Continuous infusion of ANP (300 micrograms/10 h/d) in a patient with liver cirrhosis and ascites, resistant to conservative forms of diuretic therapy, resulted in an initial increase of diuresis and natriuresis which subsequently returned to pretreatment levels. After initiation of pulsatile nocturnal treatment (5 pulses of 30 micrograms ANP every 3 h), diuresis increased, leading to a persistent normalization of sodium and chloride excretion. The patient lost 8 kg of weight during 16 days of treatment. Out of 3 additional patients on the same therapeutic regime, only one experienced a weight loss of 5 kg due to increased natriuresis and chloruresis. The remaining 2 patients did not respond during 5 resp. 7 days of therapy.
Adult↗
Influence of the centrosome on the structure of nucleated microtubules.
The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.
Animals↗
M-phase promoting factors from eggs of Xenopus laevis.
When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arrested at the G2/prophase boundary, it induces rapid entry of the cells into M-phase. MPF is present in late G2 and in M-phase of a variety of cell types, such as Xenopus eggs (naturally arrested in M), cleaving embryos, yeast, HeLa, and CHO cultures. MPF has been purified approximately 50-fold from eggs. It is stabilized by gamma-thio-ATP and by phosphoprotein phosphatase inhibitors. It runs as a protein of approximately 100 kd size on gel filtration. Oocytes contain a precursor of MPF, which is activated by post-translational means when a small amount of purified MPF is injected into the cell. Thus, MPF appears to be an auto-activating cytoplasmic trigger of M-phase. At anaphase of the cell cycle, MPF is inactivated due to the appearance of an 'anti-MPF' activity. Monoclonal antibodies have been prepared to partially purified MPF stabilized by gamma-thio-ATP, and several preparations which inactivate MPF were obtained. The antibodies are directed against thio-phosphate groups carried by a set of proteins including MPF. This indicates that MPF is present in our active preparations as a thio-phosphoprotein. These and other data suggest that MPF is normally activated in the cell cycle by a phosphorylation reaction.
Animals↗
Temporal and spatial regulation of fibronectin in early Xenopus development.
Pattern formation and temporal control of gene expression in Xenopus development were investigated using fibronectin as a biochemical marker. We determined the spatial localization of fibronectin in the embryo by immunofluorescence and the temporal program of its expression by biosynthesis studies and Western blotting techniques. At the start of gastrulation, fibronectin is localized on the roof of the blastocoel which serves as the surface upon which mesodermal cells will migrate. However, since we find fibronectin secreted by all parts of the embryo, localization is probably achieved through spatially localized receptors that bind secreted fibronectin. Fibronectin levels and fibronectin synthesis rates increase following the midblastula stage. This increase is independent of transcription and therefore involves activation of maternal RNA for fibronectin. Since this message mobilization also occurs in activated but unfertilized eggs, this event must be regulated separately from the midblastula transition.
Animals↗
Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.
We have examined the regulation of maturation-promoting factor (MPF) activity in the mitotic and meiotic cell cycles of Xenopus laevis eggs and oocytes. To this end, we developed a method for the small scale extraction of eggs and oocytes and measured MPF activity in extracts by a dilution end point assay. We find that in oocytes, MPF activity appears before germinal vesicle breakdown and then disappears rapidly at the end of the first meiotic cycle. In the second meiotic cycle, MPF reappears before second metaphase, when maturation arrests. Thus, MPF cycling coincides with the abbreviated cycles of meiosis. When oocytes are induced to mature by low levels of injected MPF, cycloheximide does not prevent the appearance of MPF at high levels in the first cycle. This amplification indicates that an MPF precursor is present in the oocyte and activated by posttranslational means, triggered by the low level of injected MPF. Furthermore, MPF disappears approximately on time in such oocytes, indicating that the agent for MPF inactivation is also activated by posttranslational means. However, in the absence of protein synthesis, MPF never reappears in the second meiotic cycle. Upon fertilization or artificial activation of normal eggs, MPF disappears from the cytoplasm within 8 min. For a period thereafter, the inactivating agent remains able to destroy large amounts of MPF injected into the egg. It loses activity just as endogenous MPF appears at prophase of the first mitotic cycle. The repeated reciprocal cycling of MPF and the inactivating agent during cleavage stages is unaffected by colchicine and nocodazole and therefore does not require the effective completion of spindle formation, mitosis, or cytokinesis. However, MPF appearance is blocked by cycloheximide applied before mitosis; and MPF disappearance is blocked by cytostatic factor. In all these respects, MPF and the inactivating agent seem to be tightly linked to, and perhaps participate in, the cell cycle oscillator previously described for cleaving eggs of Xenopus laevis (Hara, K., P. Tydeman, and M. Kirschner, 1980, Proc. Natl. Acad. Sci. USA, 77:462-466).
Animals↗
Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs.
We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.
Animals↗
Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes.
To study the role of the centrosome in microtubule organization in interphase cells, we developed a method for obtaining cytoplasts (cells lacking a nucleus) that did or did not contain centrosomes. After drug-induced microtubule depolymerization, cytoplasts with centrosomes made from sparsely plated cells reconstituted a microtubule array typical of normal cells. Under these conditions cytoplasts without centrosomes formed only a few scattered microtubules. This difference in degree of polymerization suggests that centrosomes affect not only the distribution but the amount of microtubules in cells. To our surprise, the extent of microtubules assembled increased with the cell density of the original culture. At confluent density, cytoplasts without centrosomes had many microtubules, equivalent to cytoplasts with centrosomes. The additional microtubules were arranged peripherally and differed from the centrosomal microtubules in their sensitivity to nocodazole. These and other results suggest that the centrosome stabilizes microtubules in the cell, perhaps by capping one end. Microtubules with greater sensitivity to nocodazole arise by virtue of change in the growth state of the cell and may represent free or uncapped polymers. These experiments suggest that the spatial arrangement of microtubules may change by shifting the total tubulin concentration or the critical concentration for assembly.
Animals↗
Respective roles of centrosomes and chromatin in the conversion of microtubule arrays from interphase to metaphase.
We report the results of studies in which partially purified centrosomes, nuclei, and DNA were injected into frog's eggs, which are naturally arrested in metaphase or interphase. These results have led to an independent assessment of the contributions of the centrosome and the chromatin to the formation of the mitotic spindle and suggest a simple explanation for the transition from interphase to metaphase microtubule arrays.
Animals↗
Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material.
A human autoantibody from a schleroderma patient was found to immunostain interphase and mitotic centrosomes in a variety of vertebrate cells. Electron microscopic immunocytochemistry localized this antigen in dense pericentriolar material (PCM) surrounding the centrioles. The meiotic spindle of the mouse egg has no centriole but it exhibited a broad PCM band at each pole. This pattern was also found from the first through fourth mitotic divisions. During this time PCM was found assembled at a single locus in the cell and exclusively in mitotic cells; it was not observable in interphase cells. In the blastocyst, only polar trophoblast cells had characteristic centrosomes throughout the cell cycle. Results suggest PCM can exist, disperse, and reorganize during the cell cycle independently of the centriole, and its distribution in the embryo differs in cells having different fates.
Animals↗
Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis.
We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.
Animals↗
Anticentromere and anticentriole antibodies in the scleroderma spectrum.
We studied serum samples from 106 patients, including 80 in the scleroderma spectrum, by indirect immunofluorescent microscopy, using PtK1 rat kangaroo tissue culture cells as substrate. Anticentromere (Kinetochore) antibodies were present in 28 patients, and anticentriole antibodies were present in four patients. Anticentromere antibodies were usually present in patients with a benign, chronic form of systemic scleroderma, which has been termed the CREST (Calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactyly, and telangiectasia) syndrome. The four patients with a previously undescribed anticentriole antibody were all in the scleroderma spectrum. Possibly, these antibodies may have diagnostic and prognostic importance. Further, they will be useful in studying the structure and function of these cellular organelles.
Antibodies↗
A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.
The Xenopus embryo undergoes 12 rapid synchronous cleavages followed by a period of slower asynchronous divisions more typical of somatic cells. This change in cell cleavage has been termed the midblastula transition (MBT). We show that at the MBT the blastomeres become motile and transcriptionally active for the first time. We have investigated the timing of the MBT and found that it does not depend on cell division, on time since fertilization or on a counting mechanism involving the sequential modification of DNA. Rather, the timing of the MBT depends on reaching a critical ratio of nucleus to cytoplasm. We view the MBT as a consequence of the titration of some substance, originally present in the egg, by the exponentially increasing nuclear material. When this substance is exhausted a new cell program is engaged, leading to the acquisition of several new cell properties.
Age Factors↗