Figure 8.
Figure 8. Architecture of spindle machinery in mitotic cells without cohesion. (A) Immunofluorescence image of metaphase-arrested cells (Cdc20 depleted) in which tension is absent because the cohesin subunit Scc1 is cleaved by TEV protease (Scc1-TEV268 cells). Blue: DNA. Magenta: MTs. Inset: schematic showing the loss of cohesion. (B) Serial cryo-ET model of one such cell. The nuclear envelope is colored blue and the spindle MTs colored magenta. The few Dam1C/DASH rings that were found are colored green. Note that the discontinuities in the nuclear envelope model are from the interfaces between adjacent cryosections, which could not be accurately modeled. (C) Enlargement of the spindle modeled in B, rotated to a view perpendicular to the spindle’s axis. (D) Cryotomographic slices (6 nm) of front views of complete (left) and partial (right) Dam1C/DASH rings around kMTs. (E) Box-and-whisker plots and raw values (colored circles) of the distances between kMT plus ends and Dam1C/DASH ring centers of mass. Two Dam1C/DASH rings were located in front of kMT plus ends, which gave rise to negative distance values. Meta: cells arrested in metaphase (complete: n = 12; incomplete: n = 8). TEV: cells arrested in metaphase and with Scc1 cleaved (complete: n = 4; incomplete: n = 5). Noc: cells treated with nocodazole (n = 3). ns: not significant, Student’s t test P > 0.05. Asterisk: F-test of equal variance P < 0.01. (F) A model of a portion of a metaphase spindle. MTs are magenta and the nuclear envelope membranes are blue. (G) Models of two metaphase spindles with Scc1 cleaved. These models came from single cryosections that were cut almost exactly transverse to the spindle axis. In metaphase cells without tension (Scc1 cleaved), the spindle MTs are arranged in isolated bundles surrounding a MT-free core. The spindle on the right (boxed) includes Dam1C/DASH rings (green) and is enlarged 3.5-fold on the right inset. Note that Dam1C/DASH rings within the cluster (green brackets) were spread out along the spindle axis (coming out of the image) and were not in contact.

Architecture of spindle machinery in mitotic cells without cohesion. (A) Immunofluorescence image of metaphase-arrested cells (Cdc20 depleted) in which tension is absent because the cohesin subunit Scc1 is cleaved by TEV protease (Scc1-TEV268 cells). Blue: DNA. Magenta: MTs. Inset: schematic showing the loss of cohesion. (B) Serial cryo-ET model of one such cell. The nuclear envelope is colored blue and the spindle MTs colored magenta. The few Dam1C/DASH rings that were found are colored green. Note that the discontinuities in the nuclear envelope model are from the interfaces between adjacent cryosections, which could not be accurately modeled. (C) Enlargement of the spindle modeled in B, rotated to a view perpendicular to the spindle’s axis. (D) Cryotomographic slices (6 nm) of front views of complete (left) and partial (right) Dam1C/DASH rings around kMTs. (E) Box-and-whisker plots and raw values (colored circles) of the distances between kMT plus ends and Dam1C/DASH ring centers of mass. Two Dam1C/DASH rings were located in front of kMT plus ends, which gave rise to negative distance values. Meta: cells arrested in metaphase (complete: n = 12; incomplete: n = 8). TEV: cells arrested in metaphase and with Scc1 cleaved (complete: n = 4; incomplete: n = 5). Noc: cells treated with nocodazole (n = 3). ns: not significant, Student’s t test P > 0.05. Asterisk: F-test of equal variance P < 0.01. (F) A model of a portion of a metaphase spindle. MTs are magenta and the nuclear envelope membranes are blue. (G) Models of two metaphase spindles with Scc1 cleaved. These models came from single cryosections that were cut almost exactly transverse to the spindle axis. In metaphase cells without tension (Scc1 cleaved), the spindle MTs are arranged in isolated bundles surrounding a MT-free core. The spindle on the right (boxed) includes Dam1C/DASH rings (green) and is enlarged 3.5-fold on the right inset. Note that Dam1C/DASH rings within the cluster (green brackets) were spread out along the spindle axis (coming out of the image) and were not in contact.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal