Hsp70 protects mitotic cells against heat-induced centrosome damage and division abnormalities

Abstract

The effect of heat shock on centrosomes has been mainly studied in interphase cells. Centrosomes play a key role in proper segregation of DNA during mitosis. However, the direct effect and consequences of heat shock on mitotic cells and a possible cellular defense system against proteotoxic stress during mitosis have not been described in detail. Here, we show that mild heat shock, applied during mitosis, causes loss of dynamitin/p50 antibody staining from centrosomes and kinetochores. In addition, it induces division errors in most cells and in the remaining cells progression through mitosis is delayed. Expression of heat shock protein (Hsp)70 protects against most heat-induced division abnormalities. On heat shock, Hsp70 is rapidly recruited to mitotic centrosomes and normal progression through mitosis is observed immediately after release of Hsp70 from centrosomes. In addition, Hsp70 expression coincides with restoration of dynamitin/p50 antibody staining at centrosomes but not at kinetochores. Our data show that during mitosis, centrosomes are particularly affected resulting in abnormal mitosis. Hsp70 is sufficient to protect against most division abnormalities, demonstrating the involvement of Hsp70 in a repair mechanism of heat-damaged mitotic centrosomes.

Figure 1.

Heat shock during mitosis causes division abnormalities. Cells expressing H2B-GFP under control conditions (A) or after heat shock for 15′ at 43°C (B and C). Time is given in minutes after heat shock. Under control conditions mitotic progression occurred normally (A). After heat shock, mitotic abnormalities were observed such as the formation of two daughter cells with micronuclei (B) or premature decondensation of chromatin (C). Bar, 10 μm. For movies, see http://coo.med.rug.nl/sscb/video.htm, Figures 1, 2, 3.

Figure 2.

Heat shock during mitosis leads to a heat-dose–dependent decrease in normal mitoses and to an increase in mitotic delay. Mitotic progression of cells expressing H2B-GFP was recorded by time-lapse confocal microscopy. The percentage of normal mitoses (A) and the time in minutes from early metaphase to anaphase onset (B) is given in control cells (no HS), after 15′ heat shock at 42°C (15′ 42°C), after 15′ heat shock at 43°C (15′ 43°C) and after 15′ heat shock at 44°C (15′ 44°C). For each condition, >20 mitoses were recorded. Data represent mean ± SD.

Figure 3.

Severe, but not mild, heat shock affects antibody staining of centrosome proteins and organization of microtubules, whereas both severe and mild heat shock affects antibody staining of dynamitin/p50. Cells were untreated (no HS), mildly heat shocked (15′ 42°C), or severely heat shocked (15′ 45°C). Cells were fixed and labeled with antibodies to γ-tubulin (A–C, red), to pericentrin (D–F, red), or to α-tubulin (G–I, red), with CREST antiserum (J–L, green), or with antibodies to dynamitin/p50 (M–P, green). DNA was stained by DAPI (A–P, blue).

Figure 4.

Thermotolerance protects against heat-induced mitotic abnormalities. Cells were left untreated (no HS) or preconditioned to induce thermotolerance (TT). Expression of Hsp27, Hsp40, and Hsp70 was analyzed by Western blotting (A), demonstrating that in TT cells Hsp27, Hsp40, and Hsp70 are induced. Heat shock was given to H2B-GFP–expressing cells (15′ 42°C) or H2B-GFP–expressing TT cells (TT + 15′ 42°C), and mitotic progression was recorded by time-lapse confocal microscopy. Per condition, >20 cells were recorded. The percentage of normal mitoses (B) and mean time ± SD in minutes from early metaphase to anaphase onset (C) are given. After the indicated treatments, cells were methanol/acetone fixed and labeled with monoclonal antibody to Hsp70 (D–G, red) and polyclonal antibody to γ-tubulin (H–K, green). DNA was stained by DAPI (D–O, blue). Overlays of the images are shown in L–O. Bar, 10 μm.

Figure 5.

Hsp70 localizes transiently to centrosomes of heat shocked mitotic cells. Cells expressing Hsp70-EYFP were left untreated (E) or treated with heat shock (15′ 42°C; A–D, F). Immediately after heat shock, cells were fixed with methanol/acetone (A–D) and labeled with monoclonal anti-γ-tubulin (B). DNA was stained with DAPI (C). Hsp70-EYFP (A) colocalizes with γ-tubulin in heat-shocked mitotic cells (D). Mitotic cells expressing Hsp70-EYFP were selected for time-lapse analysis under control conditions (E) or after heat shock (F). Time is given in minutes after heat shock. Note that Hsp70-EYFP only localizes to centrosomes of heat-shocked cells (F, 8′) and disappears from centrosomes just before anaphase onset in mitotic cells (F, 16′) and later in interphase cells (F, 66′). Bar, 10 μm. For movies of the time-lapse data in E and F, see http://coo.med.rug.nl/sscb/video.htm, Figures 4 and 5.

Figure 6.

Hsp70 protects against heat-induced mitotic abnormalities and reduces heat-induced delay in mitotic progression. Mitotic progression of cells expressing H2B-GFP (control) or Hsp70-EYFP (Hsp70-EYFP) was recorded by time-lapse confocal microscopy. The percentage of normal mitoses (A) and the time in minutes from early metaphase to anaphase onset (B) are given in control cells (no HS), after 15′ heat shock at 42°C (15′ 42°C), and after 15′ heat shock at 43°C (15′ 43°C). For each condition, >20 mitoses were recorded. Data represent mean ± SD.

Figure 7.

Hsp70-expressing cells show dynamitin/p50 antibody labeling at centrosomes but not at kinetochores after mild heat shock. Cells expressing Hsp70-EYFP (A and E) were mildly heat shocked (15′ 42°C). Cells were fixed and labeled with antibodies to dynamitin/p50 (B and F), and DNA was stained with DAPI (C and G). Overlays of the images are shown in D and H.