The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules - PubMed (original) (raw)

The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules

A Khodjakov et al. J Cell Biol. 1999.

Abstract

gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.

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Figures

Figure 1

DIC (A) and corresponding epifluorescence (B) micrographs of living PKG cells. Note that the centrosomes in interphase cells consist of two dots that are either adjacent or well separated from one another.

Figure 2

A–I, Selected frames from a time-lapse sequence of a PtKG cell progressing through mitosis. The top half of each frame presents the DIC image of the cell, while the bottom half represents the corresponding epifluorescence image. In this cell, the two centrosomes were already well separated in early prophase (A), and their γTGFP intensity remained relatively constant during the next hour (B). The γTGFP content then increased rapidly during spindle formation (C and D) and decreased as the cell exited mitosis (G–I). Time is in minutes.

Figure 6

Centrosome-associated γTGFP intensity versus time in the cell shown in Fig. 5. A comparison of this plot from a nocodazole treated cell, with that of control cells containing Mts (Fig. 3), reveals that the γTGFP content of the centrosome increased with approximately the same kinetics during early mitosis, regardless of whether Mts were present.

Figure 3

Centrosome-associated γTGFP intensity versus time in the cell shown in Fig. 2. Note that the two centrosomes displayed roughly the same intensity, which remained relatively constant. At the 55 min time point, the γTGFP content of both centrosomes suddenly began to increase, and reached a maximum ∼30 min later. It then remained at this level for ∼100 min, after which time the cell entered anaphase and the γTGFP intensity dropped. Note that the residual γTGFP associated with the early G1 centrosome (time = 295 min) was ∼50% that associated with the same centrosome before its mitotic activation.

Figure 4

A pseudocolor map of centrosome-associated γTGFP intensity in the cell pictured in Fig. 2 and Fig. 3. A, Depicts the two centrosomes, highly magnified, as they proceed from early prophase (00 min) to the next G1 (298 min). Note that the γTGFP content of each centrosome suddenly increased in late prophase, and even after reaching its maxim intensity in its center (82) it continued to acquire additional γTGFP around its periphery. As a result, the apparent size of the centrosome during the later stages of spindle formation (122–172 min) was several times larger than in early prophase. B, Depicts the incorporation and redistribution of γTGFP within the spindle as it matured (92–172 min), and its rapid decrease after anaphase onset (172–179 min). Note that γTGFP became concentrated in each half-spindle only after the centrosome had reached its full intensity, which occurred during true metaphase (122–172 min).

Figure 5

The sudden incorporation of γTGFP into the centrosome during the early stages of mitosis does not depend on the presence of Mts. In this example, a PtKG cell entered mitosis (A–C) in the presence of 4 μM nocodazole. Note that the γTGFP intensity of both centrosomes remained relatively constant between A and B, and then suddenly increased in late prophase (C) and remained high during the mitotic arrest (D–E). The pseudocolored inserts in each frame use the same color map as in Fig. 4, and the centrosomes are magnified two times from the black and white images.

Figure 7

The γTGFP content of the prophase centrosome can be downregulated by inducing the cell to return to interphase. A–E are selected frames depicting a prophase PtKG as it progressively returned to G2 in response to excessive illumination (see text for details). In this example, the elevated γTGFP content of the two separated centrosomes progressively decreased as the chromosomes decondensed (A–D). It then remained constant until filming was terminated ∼18 h later (D–E).

Figure 8

Centrosome-associated γTGFP intensity versus time in the cell shown in Fig. 7. In response to a radiation-induced reversal of the cell cycle, the γTGFP content of both separated prophase centrosome steadily decreased over a three hour period. It then remained at interphase levels for the next 18 h.

Figure 9

Photobleaching centrosome-associated γTGFP does not affect the Mt-nucleation potential of the centrosome. In this experiment, CVG cells were incubated in 4 μM nocodazole for two hours to disassemble the Mts. (A and B) DIC/fluorescence images of a cell before (A) and several seconds after (B) one of its two separated centrosome was photobleached by 488-nm laser light. Immediately after photobleaching, nocodazole was washed out and the cell was fixed (∼3 min after B) as new Mts were polymerizing. C–F shows the same cell after fixation and staining with Hoechst 33342 (D), anti–γ-tubulin (E), and anti–α-tubulin (F) antibodies. Note that the photobleached centrosome has a normal content of γ-tubulin (E) and that it has nucleated numbers of Mt similar to that of the nonirradiated centrosome (F).

Figure 10

The γTGFP associated with interphase centrosomes is in dynamic exchange with a cytoplasmic pool. A–F, Selected frames from a time-lapse recording depicting the recovery of fluorescence after photobleaching one of the centrosomes in a PtKG cell. In this example, the top centrosome was photobleached at the 32 min time point and its recovery was followed for eight hours.

Figure 12

FRAP experiment in a PtKG cell arrested in mitosis by 4 μM nocodazole. In this cell, one of two separated centrosomes was photobleached (B and C) ∼40 min after nuclear envelope breakdown. The γTGFP signal recovered after photobleaching, indicating that the centrosome-associated γTGFP continued to turn over during mitosis and that this process does not require Mts.

Figure 11

Centrosome-associated γTGFP intensity versus time in the cell shown in Fig. 10. During the first 60 min after photobleaching, the centrosome recovered ∼50–60% of its original intensity and it then remained at this level for the next six hours.

Figure 13

Centrosome-associated γTGFP intensity versus time in the cell shown in Fig. 12. Note that in contrast to interphase cells, the irradiated centrosome recovered its full intensity over a period of 90 min and then remained at this level.

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The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules - PubMed (original) (raw)