Figure 4.
Condensates form ectopic spindle poles during mitosis in TRIM37Δ RPE1 and mulibrey nanism patient cells. (A) Left: Expansion microscopy images of mitotic TRIM37Δ RPE1 cells labeled for acetylated tubulin showing three mitotic configurations. Right: Quantification of mitotic configurations in the indicated cell lines; note that the rarest category, multipolar with a small acetylated tubulin focus, is marked in blue. Scale bars, 20 µm and 2 µm (insets). The experiment was performed three times; quantification was of the pooled data. (B) Images of mitotic cells at different stages stained for microtubules, centrobin, and CEP192; centrosomes (white arrowheads) and condensates (yellow arrows) are marked. Scale bars, 5 µm and 2 µm (insets). (C) Z sections spaced 0.2 µm apart through a metaphase condensate. Scale bar, 2 µm. The experiment in B and C was performed three times. (D) Quantification of condensate-associated ectopic MTOCs in mitotic TRIM37Δ cells. n, number of condensates. (E) Control and mulibrey nanism primary fibroblasts labeled for microtubules and PLK4 (left) or centrobin (right). The condensate (arrows) is marked. Scale bar, 5 µm. Each experiment was performed once. (F) Stills from live imaging analysis of microtubules labeled by expression of mRuby-MAP4-MBD in TRIM37Δ RPE1 cells. Images are of spindles in two different cells. Arrow points to an ectopic spindle pole. Scale bar, 10 µm. The experiment was performed once. (G) Stills from live imaging of TRIM37Δ cells expressing mRuby-MAP4-MBD (pseudocolored green) and WT TRIM37-mNG or Ligmut TRIM37-mNG (pseudocolored magenta). Three observed phenotypes of mitotic TRIM37Δ cells with a Ligmut TRIM37-mNG-labeled condensate (arrows) are shown. Times are in minutes; 0 min is the first time point after nuclear envelope breakdown. Scale bars, 5 µm. (H) Frequency of ectopic spindle poles observed in the indicated conditions analyzed by live imaging of microtubules; cells that exhibited eventual multipolar segregation are indicated. n, number of imaged cells. For G and H, the data from three independent experiments were pooled. (I) Immunostaining of the three configurations observed for mitotic cells with a condensate (arrows) and the normal number of two centrosomes. Scale bar, 5 µm. (J) Relative frequency of the three configurations shown in I. For I and J, the experiment was performed once; data were pooled from seven replicate wells imaged in parallel.

Condensates form ectopic spindle poles during mitosis in TRIM37Δ RPE1 and mulibrey nanism patient cells. (A) Left: Expansion microscopy images of mitotic TRIM37Δ RPE1 cells labeled for acetylated tubulin showing three mitotic configurations. Right: Quantification of mitotic configurations in the indicated cell lines; note that the rarest category, multipolar with a small acetylated tubulin focus, is marked in blue. Scale bars, 20 µm and 2 µm (insets). The experiment was performed three times; quantification was of the pooled data. (B) Images of mitotic cells at different stages stained for microtubules, centrobin, and CEP192; centrosomes (white arrowheads) and condensates (yellow arrows) are marked. Scale bars, 5 µm and 2 µm (insets). (C) Z sections spaced 0.2 µm apart through a metaphase condensate. Scale bar, 2 µm. The experiment in B and C was performed three times. (D) Quantification of condensate-associated ectopic MTOCs in mitotic TRIM37Δ cells. n, number of condensates. (E) Control and mulibrey nanism primary fibroblasts labeled for microtubules and PLK4 (left) or centrobin (right). The condensate (arrows) is marked. Scale bar, 5 µm. Each experiment was performed once. (F) Stills from live imaging analysis of microtubules labeled by expression of mRuby-MAP4-MBD in TRIM37Δ RPE1 cells. Images are of spindles in two different cells. Arrow points to an ectopic spindle pole. Scale bar, 10 µm. The experiment was performed once. (G) Stills from live imaging of TRIM37Δ cells expressing mRuby-MAP4-MBD (pseudocolored green) and WT TRIM37-mNG or Ligmut TRIM37-mNG (pseudocolored magenta). Three observed phenotypes of mitotic TRIM37Δ cells with a Ligmut TRIM37-mNG-labeled condensate (arrows) are shown. Times are in minutes; 0 min is the first time point after nuclear envelope breakdown. Scale bars, 5 µm. (H) Frequency of ectopic spindle poles observed in the indicated conditions analyzed by live imaging of microtubules; cells that exhibited eventual multipolar segregation are indicated. n, number of imaged cells. For G and H, the data from three independent experiments were pooled. (I) Immunostaining of the three configurations observed for mitotic cells with a condensate (arrows) and the normal number of two centrosomes. Scale bar, 5 µm. (J) Relative frequency of the three configurations shown in I. For I and J, the experiment was performed once; data were pooled from seven replicate wells imaged in parallel.

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