Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes - PubMed (original) (raw)
Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes
James R LaFountain Jr et al. Mol Biol Cell. 2014 May.
Abstract
We use liquid crystal polarized light imaging to record the life histories of single kinetochore (K-) fibers in living crane-fly spermatocytes, from their origins as nascent K-fibers in early prometaphase to their fully matured form at metaphase, just before anaphase onset. Increased image brightness due to increased retardance reveals where microtubules are added during K-fiber formation. Analysis of experimentally generated bipolar spindles with only one centrosome, as well as of regular, bicentrosomal spindles, reveals that microtubule addition occurs at the kinetochore-proximal ends of K-fibers, and added polymer expands poleward, giving rise to the robust K-fibers of metaphase cells. These results are not compatible with a model for K-fiber formation in which microtubules are added to nascent fibers solely by repetitive "search and capture" of centrosomal microtubule plus ends. Our interpretation is that capture of centrosomal microtubules-when deployed-is limited to early stages in establishment of nascent K-fibers, which then mature through kinetochore-driven outgrowth. When kinetochore capture of centrosomal microtubules is not used, the polar ends of K-fibers grow outward from their kinetochores and usually converge to make a centrosome-free pole.
Figures
FIGURE 1:
LC-PolScope images of flattened spermatocytes, colorized by ImageJ lookup table “green fire blue” (see the text). (A) Wide-field view of two spermatocytes at diakinesis. Top, two centrosomes properly positioned on opposite poles of the nucleus; bottom, one displaced centrosome and one with a normal position next to the nucleus. (B–E) Selected time-lapse images: (B) just after NEB, (C) when nascent K-fibers (arrows) are evident in the centrosome-free half-spindle at angles similar to centrosomal microtubules from the opposite half-spindle, (D) at metaphase showing the disparity in K-fiber retardance in the centrosome-free and centrosomal half-spindles, and (E) mid anaphase A. For time-lapse movie, see Supplemental Movie S1. Time, hours:minutes. Bar, 10 μm.
FIGURE 2:
LC-PolScope images of a flattened spermatocyte presented as black and white retardance images. (A) Prometaphase when nascent K-fibers are first evident in the centrosome-free half-spindle; red arrow locates the displaced centrosome. (B) Retardance area of nascent K-fiber at the plane pointed out with white arrow is equivalent to 19 microtubules. (C) LC-PolScope image at metaphase; retardance area measured at the plane of the white arrow. (D) Plot profile of plane in C is equivalent to 52 microtubules. For time-lapse movie, see Supplemental Movie S2. Time, hours:minutes. Bar, 10 μm.
FIGURE 3:
K-fiber maturation in a centrosome-free half-spindle. (A–D) LC-PolScope images at different times during maturation with nascent fiber on top and mature metaphase fiber on the bottom. (A) Nascent K-fiber at early prometaphase. (B, C) Elongation of nascent fiber during progression from prometaphase to metaphase. (D) Mature K-fiber at metaphase. Profiles of retardance magnitude along the length of K-fiber were obtained by positioning a 5 × 150 pixel ROI over the fiber of interest and then using the Image J
command to generate a curve of retardance magnitude as a function of distance. Red arrows are positioned at the approximate left and right margins of the ROI. (E) Retardance magnitude plots; the black arrow locates the retardance peaks at kinetochores (presumed to be due to edge birefringence and not directly related to microtubules). (a) Profile from A; (b) profile from B; (c) profile from C; (d) profile from D. Maximal retardance poleward away from kinetochores increases with time, as does the magnitude of retardance along the length of a fiber, both interpreted to be a consequence of kinetochore-driven outgrowth of microtubules from the kinetochore. Time, hours:minutes. Bar, 10 μm.
FIGURE 4:
Chart of microtubules in pairs of K-fibers from a bivalent to the two poles. Dark bars: from a bivalent to the centrosomal pole; light bars: to the centrosome-free pole. Percentage disparity calculated as in Materials and Methods.
FIGURE 5:
K-fibers gain microtubules during maturation. (A) At 18 min after NEB; white arrow locates the plane of a nascent K-fiber that produced the retardance area plot in the inset, an area equivalent to 14 microtubules. (B) At 1 h, 5 min later than A and 3 min before anaphase onset; the retardance of the fiber at the same plane (white arrow) indicates significantly more microtubules, and retardance area (inset) is equivalent to 49 microtubules. Time, hours:minutes. Bar, 10 μm.
FIGURE 6:
Microtubules are added to K-fibers at kinetochore ends in normal spindles. (A–D) LC-PolScope images at different times during maturation; images were rotated to position the K-fiber of interest horizontally. (A) Nascent fiber: red arrows locate the left and right margins of the 5- by 125-pixel ROI that was positioned over each image to generate plots of retardance as a function of distance along the fiber; the right margin of all plots is the plane of cohesion between homologues. (B) Prometaphase nascent fiber gains retardance at kinetochore end. (C) Retardance increases at kinetochore end and spreads poleward. (D) Metaphase showing maximal retardance along the length of the K-fiber and then dropping as fiber approaches the pole. (E) Profiles of retardance magnitude along the length of the K-fiber from each of the images. (a) Profile from A; (b) profile from B; and so on. Black arrow locates positions of kinetochores; retardance increases are at the kinetochore end, and they progress poleward with time along the length of a fiber. Images from Supplemental Movie S3 (left cell). Time, hours:minutes. Bar, 10 μm.
FIGURE 7:
Divergent K-fiber growth and attraction of its minus end to a distant centrosome. LC-PolScope images from diakinesis to metaphase I. (A) At diakinesis, the bivalent of interest is encircled at the nuclear periphery, near the nuclear envelope; nuclear space around bivalents appears isotropic. (B) After NEB, centrosomal microtubules invade nuclear space; the region located with the arrow remains isotropic as it was before NEB; there is no evidence of microtubules in this region; bivalent of interest appears to be shielded from the distal centrosome by other bivalents. (C) At ∼29 min after NEB, a nascent K-fiber (arrow) is detectable as it grows out with a divergent trajectory, not aimed at the distal centrosome. (D) Further growth and increase in retardance of the divergent K-fiber (arrow) is evident. (E) The trajectory of the divergent K-fiber (arrow) changes as its minus end is attracted to the distal centrosome. (F) Metaphase after the previously divergent K-fiber (arrow) has established normal alignment with respect to the centrosome. Images from Supplemental Movie S4 (upper left cell). Time, hours:minutes. Bar, 10 μm.
FIGURE 8:
(A–F) LC-PolScope evidence for kinetochore capture after NEB. (A) Diakinesis; the bivalent of interest is in the circle. (B) At 2 min after NEB; centrosomal microtubules invade the nuclear space; arrow points to microtubule-free nuclear space between the centrosomal array and the bivalent, which is not yet attached. (C) At 3 min after NEB; the bivalent attaches to the centrosomal bundle and moves toward the pole; nascent K-fiber (arrow) is formed. (D) At 12 min after NEB; the nascent K-fiber is well defined (arrow), as is its partner K-fiber to the opposite pole. (E) Prometaphase: 27 min after NEB; K-fiber maturation is underway, and birefringence increases at the kinetochore end of the K-fiber. (F) Approaching metaphase: 43 min after NEB; further maturation. (G) Metaphase: 61 min after NEB; maximal retardance of K-fiber along its length. (H) Distance (ordinate) vs. time (abscissa) plot after NEB for the captured kinetochore in A–D. Initial movement (before time of magenta arrow) is away from the pole, followed by poleward movement (ending at the gold arrow, as in C). (I) Retardance profiles from A–G. (a) Profile of A in plane indicated by red arrows in A with left margin at the center of the centrosome (location of polar basal bodies) and right margin at the edge of the bivalent of interest (blue arrowhead); black arrowhead locates nuclear envelope. (b) Profile of plane between red arrows in B; kinetochore (magenta arrowhead) at the right margin is not yet attached; note that the retardance magnitude between the chromosome and the plus end of the centrosomal bundle is similar to that of microtubule-free, isotropic nuclear space. (c) Profile of plane between red arrows in C; the kinetochore (gold arrowhead) is attached and has moved poleward (see distance vs. time plot in I). (d) Profile of plane between red arrows in D; the attached kinetochore (green arrowhead) of nascent fiber starts to congress, moving toward the equator. (e) Profile of plane between red arrows in E; magnitude of birefringence at the kinetochore end of the K-fiber increases (blue arrowhead). (f) Profile of plane between red arrows in F; magnitude of birefringence at the kinetochore end of the K-fiber increases further (fuchsia arrowhead). (g) Profile of plane between red arrows in G; magnitude of birefringence is maximal along the length of the K-fiber (gold arrowhead). Images from Supplemental Movie S5 (bottom right cell). Time, hours:minutes. Bar, 10 μm.
References
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