Phase transition of tensin-1 during the focal adhesion disassembly and cell division

Abstract

Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

Keywords: TNS1; biomolecular condensate; cell division; focal adhesion; tensin.

Fig. 1.

GFP-tagged TNS1 proteins form condensates in cells. (A) Schematic diagram and IUPred3 identification of the disordered region of the human TNS1 protein. A score >0.5 indicates disordered. ABD, actin-binding domain; PTPh, protein tyrosine phosphatase homology; SH2, Src homology 2; PTB, phosphotyrosine-binding; FAB, focal adhesion binding. (B) Equal amounts of protein lysates from MDCK wild-type, TNS1-KO, and two KO-GFP-TNS1 cell lines were immunoblotted against TNS1 and GAPDH antibodies. (C) Live-cell imaging showing two TNS1 condensates were fused into one. (D) FRAP studies. The top TNS1 condensate (arrow) larger than the laser beam was bleached at 0’’, and its fluorescence recovery was recorded; the bottom punctum was an unbleached control. (E) The average fitting curve (blue) of the FRAP experiments suggested the half-time (t1/2) and percentage of recovery were 30 ± 18 s and 55 ± 11% (n = 25), respectively.

Fig. 2.

Dynamics of cellular TNS1 condensates. (A) TNS1 condensates (arrows) were derived from and displaced the rear end of GFP-TNS1-positive focal adhesions. (B) Intensity quantification of developing condensates [as framed in the yellow square in (A)] revealed four phases: precondensate formation (phase 1), condensate developing (phase 2), departing (phase 3), and departed (phase 4). The intensity values were measured and normalized based on the highest intensity during each time course as 100. The averages (connected dots) and SDs (shaded area) were plotted (n = 20 condensates from five cells). The intensities at phase 4 were significantly lower than phase 1 (two-tailed Student’s t test, P < 0.0001), suggesting that condensate formation/departure has displaced a portion of FA. (C) A kymograph showed the condensate development in the tracking area (yellow line). (D) Live-cell imaging of GFP-TNS1 condensates during mitosis (h:min): prophase (00:00, defined by the nuclear envelope breakdown), metaphase [00:15, the GFP-excluded area (arrow) indicated chromosome alignment at the metaphase plane], cytokinesis (00:25, the formation of two daughter cells). No condensates were detected from 00:00 to 00:25. (E) Condensate counts in each cell (left vertical axis) and the relative fluorescence intensity in cytoplasm (right vertical axis, the highest intensity was set as 100). Averages were connected by lines, and shaded regions denote SDs (n = 6 dividing cells); (F) Area/size of condensates (n = 15 to 100 each time point).

Fig. 3.

Phase separation of GFP-TNS1 recruits selected proteins to condensates in KO-GFP-TNS1 cells, and endogenously tagged TNS1 proteins also form condensates in TNS1-GFP-KI cells. Cells were immunofluorescence-stained with indicated antibodies. Representative results show TNS1 condensates colocalized with (A) ILK, but not FAK, and (B) pT308Akt, not pS473Akt. (C) Immunoblots of wild-type and TNS1-GFP-KI cell lysates against indicated antibodies. (D) TNS1 condensates were derived from disassembling focal adhesions in TNS1-GFP-KI cells. (E) pT308Akt localized to condensates in TNS1-GFP-KI cells.