Figure 2.
Figure 2. Localization of type 1 nodes across the cell cycle. Images are time series of maximum intensity projections of confocal fluorescence micrographs with time in minutes from SPB separation except in G. (A) Reverse contrast and merged images early in mitosis of a cell expressing Cdr2p-mEGFP, Blt1p-mCherry, and Sad1p-mRFP. Blt1p incorporated into the contractile ring as type 1 nodes moved from the equator and dispersed Cdr2p into the cytoplasm. Projections of four z sections taken at 400-nm steps. (B) Demonstration of the asymmetric movements of the nodes from the midline by a false-color kymograph of the time course of Cdr2p-mEGFP fluorescence along the length of the cell in Fig. 2 A. At each time point, the fluorescence intensity across the cell width was summed to obtain a fluorescence profile and colored according to intensity per area. (C) Images of a cell expressing Cdr2p-mEGFP and Cdc7p-mCherry showing that Cdr2p nodes moved in greater numbers and to a larger extent (arrowhead) in the direction of the active SPB marked with Cdc7p-mCherry. The same correlation was present in every cell we observed (n > 13). (green images) 10 z sections taken at 360-nm steps comprising half the cell. (red images) 20 z sections taken at 360-nm steps comprising the entire cell. (D) Time series of confocal maximum intensity projection images of a cell expressing Cdr2p-mEGFP, Blt1p-mCherry, and Sad1p-mRFP showing Cdr2p nodes reappearing in a cortical band of type 1 nodes around the daughter nuclei ∼10 min before the cell separated at time 80 min. Same conditions as A. (E) Outcome plots of the time course of the localization of Cdr2p-mEGFP to nodes in wild-type (n = 21; closed circles) and temperature-sensitive sid2-250 cells (n = 28; open circles). Cells were imaged at a permissive temperature (23–25°C). In wild-type cells, Cdr2p disappeared from nodes into the cytoplasm for the 40-min eclipse period. Most sid2-250 cells never dispersed Cdr2p from their nodes; in 18% of cells, Cdr2p disappeared from nodes into the cytoplasm for an abbreviated 1–3-min eclipse period. (F) Time-lapse images of temperature-sensitive sid2-250 cells expressing Cdr2p-mEGFP and Sad1p-mCherry at a permissive temperature (23–25°C) showing incomplete dispersal of type 1 nodes marked with Cdr2p-mEGFP. Projections of 20 z sections taken at 360-nm steps. (G) Movement of type 1 nodes from the equator of cells treated with 131 µM MBC to depolymerize microtubules. Time series of images at 3-min intervals of cells expressing Cdr2p-mEGFP and Sad1p-mCherry. The arrowhead points to Cdr2 nodes after movement from the equator. The arrow points to divided SPBs. Images from a representative experiment repeated twice. (H) Plot of mean squared displacement (MSD) versus time step for a sample of type 1 nodes imaged at 1-s intervals and marked with Cdr2p-mEGFP during interphase (n = 23; dark green) and mitosis (n = 6; light green). Bars, 2 µm.

Localization of type 1 nodes across the cell cycle. Images are time series of maximum intensity projections of confocal fluorescence micrographs with time in minutes from SPB separation except in G. (A) Reverse contrast and merged images early in mitosis of a cell expressing Cdr2p-mEGFP, Blt1p-mCherry, and Sad1p-mRFP. Blt1p incorporated into the contractile ring as type 1 nodes moved from the equator and dispersed Cdr2p into the cytoplasm. Projections of four z sections taken at 400-nm steps. (B) Demonstration of the asymmetric movements of the nodes from the midline by a false-color kymograph of the time course of Cdr2p-mEGFP fluorescence along the length of the cell in Fig. 2 A. At each time point, the fluorescence intensity across the cell width was summed to obtain a fluorescence profile and colored according to intensity per area. (C) Images of a cell expressing Cdr2p-mEGFP and Cdc7p-mCherry showing that Cdr2p nodes moved in greater numbers and to a larger extent (arrowhead) in the direction of the active SPB marked with Cdc7p-mCherry. The same correlation was present in every cell we observed (n > 13). (green images) 10 z sections taken at 360-nm steps comprising half the cell. (red images) 20 z sections taken at 360-nm steps comprising the entire cell. (D) Time series of confocal maximum intensity projection images of a cell expressing Cdr2p-mEGFP, Blt1p-mCherry, and Sad1p-mRFP showing Cdr2p nodes reappearing in a cortical band of type 1 nodes around the daughter nuclei ∼10 min before the cell separated at time 80 min. Same conditions as A. (E) Outcome plots of the time course of the localization of Cdr2p-mEGFP to nodes in wild-type (n = 21; closed circles) and temperature-sensitive sid2-250 cells (n = 28; open circles). Cells were imaged at a permissive temperature (23–25°C). In wild-type cells, Cdr2p disappeared from nodes into the cytoplasm for the 40-min eclipse period. Most sid2-250 cells never dispersed Cdr2p from their nodes; in 18% of cells, Cdr2p disappeared from nodes into the cytoplasm for an abbreviated 1–3-min eclipse period. (F) Time-lapse images of temperature-sensitive sid2-250 cells expressing Cdr2p-mEGFP and Sad1p-mCherry at a permissive temperature (23–25°C) showing incomplete dispersal of type 1 nodes marked with Cdr2p-mEGFP. Projections of 20 z sections taken at 360-nm steps. (G) Movement of type 1 nodes from the equator of cells treated with 131 µM MBC to depolymerize microtubules. Time series of images at 3-min intervals of cells expressing Cdr2p-mEGFP and Sad1p-mCherry. The arrowhead points to Cdr2 nodes after movement from the equator. The arrow points to divided SPBs. Images from a representative experiment repeated twice. (H) Plot of mean squared displacement (MSD) versus time step for a sample of type 1 nodes imaged at 1-s intervals and marked with Cdr2p-mEGFP during interphase (n = 23; dark green) and mitosis (n = 6; light green). Bars, 2 µm.

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