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. 2016 May 24;7(21):30820-30.
doi: 10.18632/oncotarget.9003.

Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

Siamak A Kamranvar  1 , Deepesh Kumar Gupta  1 , Ying Huang  1 , Rajesh Kumar Gupta  2 , Staffan Johansson  1
Affiliations

Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

Siamak A Kamranvar et al. Oncotarget. .

Abstract

Adhesion to extracellular matrix is required for cell cycle progression through the G1 phase and for the completion of cytokinesis in normal adherent cells. Cancer cells acquire the ability to proliferate anchorage-independently, a characteristic feature of malignantly transformed cells. However, the molecular mechanisms underlying this escape of the normal control mechanisms remain unclear. The current study aimed to identify adhesion-induced reactions regulating the cytokinesis of non-transformed human fibroblasts.The adhesion-dependent control of cytokinesis was found to occur at a late stage close to the abscission, during which the endosomal sorting complex required for transport (ESCRT) severs the thin intercellular bridge connecting two nascent daughter cells. CEP55, a key protein involved in the abscission process, was localized at the midbody in both adherent and non-adherent fibroblasts, but it was unable to efficiently recruit ALIX, TSG101, and consequently the ESCRT-III subunit CHMP4B was missing in the non-adherent cells. PLK1, a kinase that prevents premature recruitment of CEP55 to the midbody, disappeared from this site more rapidly in the non-adherent cells. A FAK-Src signaling pathway downstream of integrin-mediated cell adhesion was found to decelerate both PLK1 degradation and CEP55 accumulation at the midbody. These data identify the regulation of PLK1 and CEP55 as steps where integrins exert control over the cytokinetic abscission.

Keywords: CEP55; FAK; PLK1; cytokinesis; integrin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Adhesion-dependent and –independent stages of cytokinesis in non-transformed fibroblasts
(A) Representative bright field micrographs of a single BJ fibroblast progressing through cytokinesis in the presence (adhesion) and the absence (suspension) of fibronectin contacts, respectively. (B) Mean % ± SD of the number of mitotic BJ fibroblasts that completed cytokinesis during the specified time periods after the formation of cleavage furrow. (C) Representative fluorescence images illustrating an early and a late cytokinetic stage of BJ cells. The status is based on fluorescence signal distribution of Aurora B as indicator of cleavage furrow width and the presence of CEP55 in the cleavage furrow region. Nuclei were stained with DAPI (blue). (D) Mean % ± SD of the number of mitotic BJ cells in the different stages of cytokinesis after the indicated incubation time periods. (E) Representative bright field micrographs showing the progression of the cleavage furrow ingression in the mitotic BJ cells adhering to fibronectin or kept in suspension for the indicated time periods. The bars mark the distance between the opposite membrane borders. (F) Mean ± SD of the cleavage furrow width. The square frames show the midbody region at higher magnification. ***P-value less than 0.001.
Figure 2
Figure 2. Cell adhesion is dispensable for the recruitment of the MKLP1 and CEP55 but required for the ESCRT recruitment to the midbody
(A, B, F) Representative immunofluorescence micrographs illustrating the distribution of selected proteins in the F/M region (labeled with Aurora B) at the beginning of cytokinesis and after 60 minutes incubation of BJ cells adhering to fibronectin or kept in suspension. (A) MKLP1 (green, upper left panel) and CEP55 (green, upper right panel), (B) TSG101 (green, lower left panel) and ALIX (red, lower right panel), (F) CHMP4B (green). The square frames show the midbody area at higher magnification. Arrows indicate midbody regions in the merged pictures. Nuclei were stained with DAPI (blue). Mean % ± SD of the number of mitotic cells in cytokinesis lacking (C) MKLP1 and CEP55, (D) TSG101 and ALIX and (G) CHMP4B in the F/M regions. ***, **, and * represent P-value less than 0.001, 0.01 and 0.05, respectively. (E) Representative western blot of cell lysates illustrating the level of ALIX and TSG101 in exponentially proliferating cells (Control) and synchronized mitotic cells directly after isolation (Zero) and following two hours incubation under the adhesion or suspension conditions.
Figure 3
Figure 3. Cell adhesion is involved in the regulation of the presence of PLK1 and CEP55 at the midbody
(A) Representative immunofluorescence images illustrating the presence of PLK1 (green) in the F/M regions stained for Aurora B (red) at the beginning of cytokinesis and after 60 minutes incubation of BJ cells adhering to fibronectin or kept in suspension. Nuclei were stained with DAPI (blue). Red squares indicate midbody areas used for the quantification of PLK1 fluorescence signal intensity. (B) Mean % ± SD of the number of mitotic cells in cytokinesis lacking PLK1 fluorescence signal in the F/M regions. *P-value less than 0.05. (C) Representative immunofluorescence micrographs showing the variation of PLK1 (green) and CEP55 (red) fluorescence signal intensity in the indicated F/M regions at the beginning of cytokinesis and in the indicated time points after the incubation of BJ cells adhering to fibronectin (upper panel) or kept in suspension (lower panel). The corresponding PLK1 and CEP55 signal intensity profiles along the hatched line in each interest area were drawn in the right panel. (D, E) Mean intensity ± SE of the PLK1 (D) and CEP55 (E) fluorescence signal intensity in the marked F/M regions 0, 30, 45 and 60 minutes after incubation of the cells adhering to fibronectin or kept in suspension (the background signal in each cell was subtracted). The fluorescence signal intensity of PLK1 and CEP55 were analyzed in 50 mitotic cells for each time point from each of three independent experiments.
Figure 4
Figure 4. FAK-Src signaling is involved in abscission timing through PLK1 controlling the recruitment of CEP55
(A) Representative immunofluorescence images showing the PLK1 (green) and CEP55 (red) signals at the midbody of mitotic BJ cells in cytokinesis replated on fibronectin and treated with DMSO, FAK inhibitor (PF) and Src inhibitor (SU) for 1 hour. (B) Quantification of PLK1 and CEP55 signals at the midbody of cells shown as mean ± SD. (C) The variation in the PLK1 and CEP55 fluorescence signal intensities presented as fold change relative to control values after the treatment of the cells with the indicated inhibitors. (D) Representative western blot and quantification analysis (mean ± SD; n = 2) showing the expression level of PLK1 in the mitotic cells treated as described above. (E) Representative bright field micrograph illustrating the progression of cytokinesis during the indicated time period in a single BJ fibroblast treated with FAK inhibitor (PF). The arrows mark the uncut intercellular bridge between two daughter cells.
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