A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome
- Stanford University, United States
Figures
Figure 1 with 2 supplements
C. elegans PCM is organized into layered spheres that disassemble using different behaviors, see also Figure 1—figure supplement 1, Figure 1—figure supplement 2, Videos 1–4.
(A) Left: Cartoon representing the C. elegans 4-cell stage embryo with ABp in red. Right: 7.5 µm z-projection from a live pie-1p::GFP::TBB-1/β-tubulin (green); tagRFP::SPD-5 (red) expressing embryo …
Figure 1—figure supplement 1
Methods for quantifying PCM width.
(A) The width of each PCM protein was determined using the same image analysis pipeline. Image stacks of about 30 images separated by 0.5 µm z-steps (15 µm total) were acquired at NEBD using the …
Figure 1—figure supplement 2
Time-lapse analysis of TAC-1, ZYG-9 and TPXL-1 during disassembly compared to SPD-2 and SPD-5.
(A) Time-lapse analysis of the disassembly of SPD-2 (first row), SPD-5 (second row), TAC-1 (third row), ZYG-9 (fourth row) and TPXL-1 (fifth row) starting at NEBD (t = 0 min) and imaged every minute …
Figure 2
The PCM fragments into SPD-5 and GIP-1 containing packets that localize dynamic microtubules.
(A–C) Analysis of colocalization of SPD-5 packets (red) with GIP-1 (A, green), or microtubules (B, TBA-1/α-tubulin, green), and TBG-1 (red) with AIR-1(C, green) in early packets (left panels) or …
Figure 3 with 1 supplement
Dissolution of SPD-2 and MZT-1 precedes rupture and packet formation, see also Figure 3—figure supplement 1 and Video 5.
(A–C) Comparison of tagRFP::SPD-5 (red) to SPD-2::GFP (A, green), GFP::MZT-1 (B, green), or GFP::GIP-1 (C, green) disassembly. ‘Dissolution’ (light grey arrow) begins as SPD-2 (t = 2 min. post-NEBD) …
Figure 3—figure supplement 1
Dynamics of PLK-1, TAC-1, AIR-1 and TPXL-1 during disassembly.
(A) Average pixel intensity at the centrosome of PCM proteins during disassembly starting at NEBD (t = 0 min): SPD-2::GFP (green, n = 11), GFP::TAC-1 (yellow, n = 7) and PLK-1::GFP (black, n = 7). …
Figure 4 with 2 supplements
Cortical forces rupture the PCM into packets, see also Figure 4—figure supplement 1 and Figure 4—figure supplement 2.
(A) Time-lapse analysis starting at NEBD (t = 0 min) of the disassembly of endogenous tagRFP::SPD-5 (red) and SPD-2::GFP (green) treated with lacZ(RNAi) (control, top panels, grey (A–E)), gpr-1/2(RNA…
Figure 4—figure supplement 1
Cortical forces regulate SPD-5, but not SPD-2, intensity and regrowth in the next cell cycle.
Total SPD-5 (A) or SPD-2 (B) intensity at the centrosome during disassembly starting at NEBD (t = 0 min) in embryos treated with lacZ(RNAi) (control, grey), gpr-1/2(RNAi) (blue), or csnk-1(RNAi) …
Figure 4—figure supplement 2
Localization of astral microtubules and cortical force generating proteins during PCM disassembly.
Time lapse analysis from embryos expressing pie-1::TBB-2/ß-tubulin::GFP and endogenous tagRFP::SPD-5 (A), LIN-5::mNG (B), DNC-1::mNG (C), or DHC-1::mNG (D, E). Times relative to nuclear envelope …
Figure 5
PP2A phosphatases regulate PCM disassembly.
(A) Time-lapse analysis of embryos expressing pie-1p::mCherry::TBA-1/α-tubulin (red) and endogenous GFP::GIP-1 (green) and treated at anaphase (t = 0 min) with DMSO (left panels), 30 µM okadaic acid …
Figure 6 with 1 supplement
Kinases and phosphatases shape the PCM throughout mitosis, see also Figure 6—figure supplement 1.
(A) Time-lapse analysis of the disassembly of endogenous LET-92 starting at NEBD (t = 0 min) and imaged every minute for 9 min. (B) Time-lapse analysis of embryos expressing endogenous …
Figure 6—figure supplement 1
Microtubule behavior following cell fusion.
(A) Time-lapse analysis of an ABp – P2 fusion experiment in embryos expressing pie-1::TBB-2/ß-tubulin::GFP (green), histone::mCherry (red) and PLC∂::mCherry (red). Fusion site is marked by the …
Figure 7
A two-step model of PCM disassembly.
The centrosome is assembled through the activity of mitotic kinases that phosphorylate PCM proteins to be incorporated into an inner (magenta) and outer (blue) sphere. Phosphatases oppose this …
Videos
Video 1
Centrosome disassembly in the ABp cell in a 4-cell embryo expressing endogenous SPD-2::GFP.
Scale bar, 5 µm.
Video 2
Centrosome disassembly in the ABp cell in a 4-cell embryo expressing endogenous GFP::MZT-1.
Scale bar, 5 µm.
Video 3
Centrosome disassembly in the ABp cell in a 4-cell embryo expressing endogenous GFP::SPD-5.
Yellow arrowhead and ‘c’ mark the centrioles. White arrowhead and ‘p’ mark the packets. Scale bar, 5 µm.
Video 4
Centrosome disassembly in the ABp cell in a 4-cell embryo expressing endogenous GFP::GIP-1.
Yellow arrowhead and ‘c’ mark the centrioles. White arrowhead and ‘p’ mark the packets. Scale bars, 5 µm.
Video 5
Centrosome disassembly in the ABp cell in a 4-cell embryo expressing endogenous tagRFP-T::SPD-5; GFP::GIP-1.
Yellow arrowhead and ‘c’ mark the centrioles. White arrowhead and ‘p’ mark the packets. Scale bars, 5 µm.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.47867.021
