Centrosome-intrinsic mechanisms modulate centrosome integrity during fever

Abstract

The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted.

FIGURE 1:

Elevated temperature in human febrile patients, primary mouse macrophages, and RPE cells leads to centrosome damage. (A) Comparison of γ-tubulin levels (red) in leukocyte centrosomes of febrile individuals (left, as indicated) and controls (left, top; cntrl); bar, 10 μm. Right, semiquantitative analysis of γ-tubulin signal intensity (×10⁵ arbitrary units, n = 60–120 cells/sample, mean ± SEM, t test). (B) Images (cntrl and treated as indicated; inset, centrosomes), maximum projections; bar, 10 μm. Graphs, comparison of γ-tubulin at centrosomes in hRPE cells after exposure to the febrile patient temperature regimen (bottom, left) or HS (bottom, right), analyzed as in A. (C) Sucrose gradients of centrosomes from RPE cell control and HS lysates, separated by SDS–PAGE and immunoblotted for γ-tubulin. Fractions 4 and 5 indicate loss of centrosomal γ-tubulin from HS cells, whereas total (In, input) γ-tubulin is not decreased. (D) Images of γ-tubulin staining in primary mouse macrophages treated with HS, inflammatory cytokine interleukin-1 (IL-1), LPS, or control (cntrl, inset) centrosomes (bar, 5 μm). Right graph, semiquantitative analysis (×10⁵ arbitrary units, n = 25–30 cells/sample, mean ± SEM).

FIGURE 2:

Centrosome defects during exposure to elevated temperature. (A) Maximum projections from confocal images of control and HS cells showing disruption of pericentriolar material marker PCM1; bar, 10 μm. Changes in PCNT are shown as semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM). (B) Centrin2 (centriole marker, Cent2; left) and ninein (appendage marker, right) staining in RPE cells after HS (integrated intensity, ×10⁵ arbitrary units; n = 40–60 centrosomes/sample, mean ± SEM). Middle, maximum projections of centrosomes (Cent2, green; ninein, red). (C) Centriole integrity after HS based on intensity profiles of acetylated tubulin (left), glutamilated tubulin semiquantitative analysis of integrated signal intensity (middle left; ×10⁶ arbitrary units, mean ± SEM), SAS6 (middle right; ×10⁵ arbitrary units), and Cep120 (right; ×10⁶ arbitrary units); bar, 10 μm. (D) Signals of mother centriole markers p150, cenexin, and Cep83, respectively, are decreased in stressed cells; maximum projections of confocal microscope images of centrosomes (bar, 10 μm) and semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM). (E) Changes in rootletin are shown as semiquantitative analysis of integrated intensity (×10⁶ arbitrary units, mean ± SEM) and maximum projections of centrosomes from control and HS cells; bar, 10 μm. (F) Daughter centriole marker centrobin signal is decreased in stressed cells; maximum projections of confocal microscope images of centrosomes and semiquantitative analysis of integrated intensity (×10⁶ arbitrary units, mean ± SEM). (G) Maximum projections from confocal microscopy images of growing ends of MTs from control and HS cells (DNA in blue, EB1 in red; bar, 10 μm) and maximum projections from confocal microscopy images of MTs from control and HS cells (DNA in blue, α-tubulin in red).

FIGURE 3:

Consequences of centrosome damage. (A) MT nucleation 1 or 5 min after release from nocodazole treatment (α-tubulin, red; γ-tubulin, green) in control and HS RPE cells (bar, 10 μm). Right, percentage of cells with detectable MT nucleation at the centrosome at 1 min time point (three experiments, mean ± SD, t test). (B) Cilia formation is compromised after HS treatment (maximum projections; glutamylated tubulin, red; γ-tubulin, green); bar, 10 μm. Graph, percentage of ciliated cells (400–600 cells/experiment, three experiments, mean ± SD, t test). (C) Mitotic defects. Top left, percentage of cells in mitosis (three experiments, mean ± SD, t test). Top right, percentage of cells at different stages of mitosis (three experiments, mean ± SD, t test). Images, maximum projections of prometaphase cells, cntrl and HS, as indicated; bar, 10 μm. (D) HS leads to immune synapse (IS) defects. Left, γ-tubulin intensity is reduced at centrosome in HS-exposed Jurkat cell-CD3/28 bead conjugates (×10⁵ arbitrary units, 20–40 centrosomes/sample, mean ± SEM, t test). Right, centrosome polarization toward IS is disrupted after HS exposure (distance between centrosome and IS [target bead] in micrometers). Images, maximum projections of Jurkat cell-CD3/28 bead conjugates stained with α-tubulin (green) and γ-tubulin (red) before and after HS show disruption in MT organization and centrosome damage; bar, 10 μm.

FIGURE 4:

Heat stress triggers proteasome degradation of the centrosome. (A) Pretreatment with the proteasome inhibitor LA prevents γ-tubulin disruption upon HS; semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM). (B) Loss of centrosome integrity based on 5051 marker, an autoimmune serum against centrosome that recognizes multiple centrosome proteins (Calarco-Gillam et al., 1983), after HS is prevented by LA pretreatment. Semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM). (C) Inhibition of the proteasome by MG132 prevents HS-induced disruption of the PCNT signal at the centrosome: semiquantitative intensity profiles of PCNT in HS cells or HS cells pretreated with MG132 for 1 h. (D) The ubiquitin signal (Ubi) is increased in HS centrosomes; maximum projections of microscope images of centrosomes (bar, 5 μm) and semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM). (E) Proteasome activity at the centrosome after HS is increased threefold relative to activity at the centrosomes of control cells. Cells expressing the centrosome-targeted proteasome activity reporter Ub-G76VGFP-PACT were counted in cntrl and HS conditions; mean ± SD. (F) Maximum projections of confocal microscopic images of HS cells expressing either the dominant-negative mutant (GFP-S5aC-PACT) or GFP-PACT and immunostained for γ-tubulin (red). After HS, the centrosome is less disrupted in cells expressing GFP-S5aC-PACT than in GFP-PACT–expressing cells; bar, 10 μm.

FIGURE 5:

Centrosome degradation is specific. (A) Mitochondria are not degraded during HS. Maximum projections from confocal microscopy images show representative images of cntrl and HS cells immunostained with ATP5A (left; bar,10 μm) and mitochondrial Hsp70 (green) and γ- tubulin (red); middle and right, inset, centrosomes (bar, 10 μm). (B) Kinetochores are not degraded during HS. Maximum projections from confocal microscopy images show representative images of prometaphase cells from cntrl and HS cells immunostained with CenpE (green) and CREST (red); bar 10 μm. (C) Semiquantitative analysis of integrated intensity of the midbody marker RacGap (×10⁵ arbitrary units, mean ± SEM) demonstrates no significant difference in signal intensity in HS cells. (D) Semiquantitative analysis of integrated intensity of the midbody marker mitotic kinesin-like protein (×10⁵ arbitrary units, mean ± SEM) demonstrates no significant difference in signal intensity in HS cells. (E) Maximum projections from confocal microscopy images show representative images of cntrl and HS cells immunostained with Sept7 (red, decorates midbody, MB, and centrosomes, Cen) and Centrin2 (green, as centrosome marker); bar, 5 μm. HS results in loss of Septin7 from centrosomes but not from midbodies in the same cell. (F) Disruption of Septin7 signal after HS is shown at the centrosome but not at the midbody by semiquantitative analysis of integrated intensity (×10⁵ arbitrary units, mean ± SEM).

FIGURE 6:

The centrosome is a substrate organelle for molecular chaperone Hsp70. (See also Supplemental Figures S1–S3.) (A) Left, images of maximum projections from cntrl and HS cells as indicated show that upon HS, Hsp70 accumulates at the centrosome and in the nucleus (inset, arrow points at the centrosome); bar, 10 μm. Semiquantitative analysis of integrated intensity (×10⁵ arbitrary units) of centrosomal Hsp70 before and after HS (15–20 centrosomes/sample, mean ± SEM). (B) Superresolution images (OMX) demonstrate loss of γ-tubulin (red) from HS cells and recruitment of Hsp70 (green) as an outer layer in stressed cells; bar, 0.2 μm. (C) Immunoprecipitation (IP) of Hsp70 pulls down PCNT. Immunoglobulin G (IgG), control. (D) Centrosome-targeted Hsp70 (cHsp70) protects γ-tubulin from HS, whereas membrane-targeted Hsp70 (mHsp70) does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosomal γ-tubulin after HS in stably expressing centrosome-(cHsp70) or membrane-(mHsp70) targeted Hsp70-expressing cells (four experiments, 500–600 cells/sample, mean ± SD, one-way analysis of variance [ANOVA] combined with Tukey’s multiple comparison test). (E) cHsp70, but not the chaperone-negative mutant cHsp70D10S, protects γ-tubulin signal at the centrosome; semiquantitative analysis of integrated γ-tubulin signal intensity (×10⁵ arbitrary units, mean ± SEM). (F) cHsp70 protects centrosomal Centrin2 from HS, whereas mHsp70 does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosome-localized Centrin2 after HS in stably expressing cHsp70- or mHsp70-targeted Hsp70 cells (three experiments, 500–600 cells/sample, mean ± SD, one-way ANOVA combined with Tukey’s multiple comparison test).

FIGURE 7:

Hsp70 enables centrosome recovery, and protects centrosome functions after HS. (A) Semiquantitative analysis (×10⁵ arbitrary units) of Centrin2 signal intensity shows centrosome disruption and recovery after HS during 24 h. (B) Semiquantitative intensity profiles of PCNT from cells recovered for 24 h at 37˚C after HS demonstrate that Hsp70 depletion impairs centrosome recovery. (C) Confocal microscopy images demonstrating rescue of MT regrowth early after microtubule depolymerization (α-tubulin, 1 min of regrowth) after HS in cells expressing the centrosome targeting protein cHsp70 but not the membrane-targeting protein mHsp70. Percentage of the cells positive for detectable MT regrowth 1 min after HS exposure in RPE cell lines expressing cHsp70 or mHsp70 (five experiments, 400–500 cells/sample, mean ± SD). (D) Percentage of the cells with cilia after HS in cells expressing cHsp70 or mHsp70 (three experiments, 400–500 cells/sample, mean ± SD). Maximum projections of ciliated cells (cilia marker, glutamylated tubulin, red; centrosome marker, γ-tubulin, green) after HS exposure in cells expressing cHsp70 or mHsp70. (E) Centrosomal Hsp70 protects centrosome from HS-induced damage in IS conjugates; left, ×10⁵ arbitrary units, 10–40 centrosomes/sample, mean ±S EM; middle, distance between centrosome and IS, micrometers. Right, γ-tubulin, red, over DIC images; bar, 5 μm.