Figure 1.
Figure 1. Two types of interphase nodes observed by confocal fluorescence microscopy of asynchronous cells expressing fluorescent fusion proteins. (A and B) Localization of kinase Cdr2p-mEGFP, node protein Blt1p-mCherry, and SPB protein Sad1p-mRFP (black arrowheads in the reverse contrast image of the red channel). (A) Maximum intensity projections of five z sections in 400-nm steps including 2 µm (about half the thickness) of the cells. Cdr2p and Blt1p colocalize in nodes near the equator of some cells (arrow) but in separate nodes in other cells (paired white arrowheads). In cells with Blt1p concentrated in the contractile ring (single white arrowhead), Cdr2p is dispersed in the cytoplasm. (B) Cells such as in A were classified into interphase stages by length and other criteria (Materials and methods). (C and D) Localization of proteins in two types of nodes across the cell cycle. Reverse contrast, confocal fluorescence, and maximum intensity projections of 21 z sections taken at 260-nm steps and arranged in montages according to cell length. We adjusted the brightness and contrast of each image to account for differences in illumination intensity and exposure time. (bottom) Drawings of the distributions of the two types of nodes across the cell cycle. (C) Type 1 nodes located in broad bands around the equator (green boxes in one column) contain Cdr2p-mYFP and Cdr1p-3GFP. Mid1p-mEGFP is present in these nodes and transiently in the contractile ring (red box). (D) Type 2 nodes containing Blt1p-mYFP, mECitrine-Gef2p, and Klp8p-mYFP appear at the division site, redistribute to the equator during interphase (red boxes), and incorporate into the contractile ring. (E) Histogram of the distribution of nodes along the length of asynchronous interphase cells such as in A and B. Nodes were defined as colocalized if their intensity centers of mass in the red and green channels were less than one point spread function (3 pixels) apart. n = 188 nodes from 12 cells in two separate experiments. Dotted ovals outline cells. Bars, 2 µm.

Two types of interphase nodes observed by confocal fluorescence microscopy of asynchronous cells expressing fluorescent fusion proteins. (A and B) Localization of kinase Cdr2p-mEGFP, node protein Blt1p-mCherry, and SPB protein Sad1p-mRFP (black arrowheads in the reverse contrast image of the red channel). (A) Maximum intensity projections of five z sections in 400-nm steps including 2 µm (about half the thickness) of the cells. Cdr2p and Blt1p colocalize in nodes near the equator of some cells (arrow) but in separate nodes in other cells (paired white arrowheads). In cells with Blt1p concentrated in the contractile ring (single white arrowhead), Cdr2p is dispersed in the cytoplasm. (B) Cells such as in A were classified into interphase stages by length and other criteria (Materials and methods). (C and D) Localization of proteins in two types of nodes across the cell cycle. Reverse contrast, confocal fluorescence, and maximum intensity projections of 21 z sections taken at 260-nm steps and arranged in montages according to cell length. We adjusted the brightness and contrast of each image to account for differences in illumination intensity and exposure time. (bottom) Drawings of the distributions of the two types of nodes across the cell cycle. (C) Type 1 nodes located in broad bands around the equator (green boxes in one column) contain Cdr2p-mYFP and Cdr1p-3GFP. Mid1p-mEGFP is present in these nodes and transiently in the contractile ring (red box). (D) Type 2 nodes containing Blt1p-mYFP, mECitrine-Gef2p, and Klp8p-mYFP appear at the division site, redistribute to the equator during interphase (red boxes), and incorporate into the contractile ring. (E) Histogram of the distribution of nodes along the length of asynchronous interphase cells such as in A and B. Nodes were defined as colocalized if their intensity centers of mass in the red and green channels were less than one point spread function (3 pixels) apart. n = 188 nodes from 12 cells in two separate experiments. Dotted ovals outline cells. Bars, 2 µm.

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