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Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential

¹ Department of Biology, Duke University, Durham, NC 27708-1000, USA
² Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA
³ Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA

View larger version (26K): [in a new window]   Fig. 1. Three states of kinetochore attachment to the spindle. Chromosomes in blue, kinetochore microtubules, spindle outlines and poles in gold. (A) Mitosis in vertebrate cells. Top to bottom: a chromosome with both kinetochores (blue ovals) unattached (u), a chromosome with both kinetochores strongly attached (s), under tension from oppositely directed poleward forces (green arrows), and a misattached chromosome with one kinetochore unattached (u) and one weakly attached (w); tension from antagonistic antipoleward forces (red arrowheads) and poleward forces (green arrow) is present. (B) First division of meiosis in grasshopper spermatocytes. As for A, except the misattached chromosome at the bottom has both kinetochores weakly attached (w) to the same pole and tension is absent – only poleward forces are present.

View larger version (85K): [in a new window]   Fig. 2. Mad2 antibodies recognize a protein of approximately 24 kDa on an immunoblot of grasshopper testes. Coomassie blue-stained SDS-PAGE of molecular weight markers (MW), bacterially expressed Xenopus Mad2 protein (XMad2), and homogenized grasshopper testes (GH) is shown on the left. A blot of homogenized grasshopper testes labeled with Mad2 antibodies is shown on the right. The blot lane was run on the same gel shown stained on the left (see Materials and Methods).

View larger version (130K): [in a new window]   Fig. 3. Mad2 and 3F3/2 phosphoprotein double immunolabeling of an early grasshopper spermatocyte. (A) Mad2 immunofluorescence (green) superimposed on a phase contrast image. (B) 3F3/2 immunofluorescence (red) superimposed on the phase contrast image. In this early cell, one chromosome (asterisk) is improperly attached – both kinetochores (yellow arrows) are attached to the same (upper) pole and other chromosomes have only recently attached as shown by high 3F3/2 brightness of some kinetochores (blue arrow). The upper spindle pole (p) is labeled with 3F3/2 and the approximate position of the lower pole is indicated (p). Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 4. Detaching kinetochores from microtubules leads to Mad2 binding, and tension from a microneedle leads to Mad2 loss. (A,B) Phase contrast images of the cell in life. The chromosomes labeled with blue and yellow asterisks were detached from the spindle with a micromanipulation needle, moved to the cytoplasm, and kept detached for 10 minutes. The chromosome labeled with the yellow asterisk was then moved so that both kinetochores pointed to the lower pole. After 3 minutes, its kinetochores had attached and the chromosome was pulled away from the pole (red arrow), imposing tension; tension was greater on the left kinetochore than the other one (note that the left arm of the chromosome is more greatly stretched and is thinner). The chromosome was kept under tension for 7 minutes and then the cell was fixed and immunostained. (C) Mad2 immunostaining (green) superimposed on a phase contrast image. The kinetochores of the detached chromosome are brightly labeled (blue arrows), but the kinetochores of the chromosome under tension from the microneedle (yellow arrows) are unlabeled (left) or very lightly labeled (right). The kinetochores of unmanipulated chromosomes, which had been under tension from mitotic forces, are unlabeled (yellow arrowheads). The approximate positions of the spindle poles are labeled ‘p’. Bar, 10 µm.

View larger version (107K): [in a new window]   Fig. 5. Weak attachment greatly reduces kinetochore microtubule accumulation. (A-C) Phase contrast images of the cell in life. Arrows in A, B and ‘w’ and ‘u’ in C, kinetochores of the manipulated chromosome; arrowheads, some control kinetochores. (A) Before the operation. (B) Both kinetochores (arrows in A and B) of one chromosome were detached from spindle microtubules and the chromosome was moved to the cytoplasm and kept from reattaching for 10 minutes. (C) One kinetochore (‘w’) was then allowed to attach to spindle microtubules but the other one was not (‘u’), so that the attached kinetochore was not under tension and was weakly attached. After 10 minutes the cell was fixed for anti-tubulin immunofluorescence. (D,E) Superimposed phase contrast and immunofluorescence images (D) and immunofluorescence alone (E). The kinetochore that was not under tension (‘w’ in D) had a very weakly fluorescent kinetochore microtubule bundle (arrow) compared with the bundles of the controls (arrowheads) that had been under tension. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 6. Weak attachment can inhibit kinetochore phosphorylation and Mad2 binding. (A,B) Phase contrast images of the cell in life. One kinetochore (upper arrow in A, ‘u’ thereafter) of a metaphase chromosome was detached from the spindle and kept detached for 10 minutes, while the other kinetochore (lower arrow in A and ‘w’ thereafter) remained weakly attached in the absence of tension. (C,D) The cell after fixation and immunostaining for 3F3/2 (C, red) and Mad2 (D, green). In both C and D the unattached kinetochore (u) is bright – it is highly phosphorylated and loaded with Mad2. The weakly attached kinetochore (w) is dim, as are the kinetochores of unmanipulated chromosomes (yellow arrowheads), which had been strongly attached. Bar, 10 µm.

View larger version (97K): [in a new window]   Fig. 7. Weak attachment sometimes, but not always, inhibits dephosphorylation and Mad2 loss. (A-C) Phase contrast images of a cell in life. A chromosome (kinetochores identified by arrows in A and B, ‘w’ and ‘u’ thereafter) was completely detached from the spindle, moved to the cytoplasm (B) and kept detached for 10 minutes, allowing rephosphorylation and Mad2 binding. One kinetochore (‘w’) was then allowed to attach to the spindle, while the other (‘u’) was kept unattached. (D) After 3F3/2 immunostaining (red; superimposed on a phase contrast image). The unattached kinetochore (‘u’) is bright but the weakly attached kinetochore (‘w’) is only a third as bright (from fluorescence measurements) as the unattached kinetochore and not much brighter than the kinetochores (yellow arrowheads) of unmanipulated chromosomes, which had been under tension from mitotic forces and were strongly attached. (E,F). Mad2 immunofluorescence (green; superimposed on a phase contrast image) in two additional cells after the same experimental procedure in A-D. In both E and F the unattached kinetochore (‘u’) is bright; it contained lots of Mad2 and the kinetochores (yellow arrowheads) of unmanipulated chromosomes are not detectably labeled. In one cell, Mad2 was undetectable at the weakly attached kinetochore (E, ‘w’), while in the other (F, ‘w’) it was almost as bright as the unattached kinetochore. Bar, 10 µm.