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First published online 31 May 2005
doi: 10.1242/jcs.02410

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Klp67A destabilises pre-anaphase microtubules but subsequently is required to stabilise the central spindle

Melanie K. Gatt^1,*, Matthew S. Savoian^1,*,, Maria G. Riparbelli², Chiara Massarelli², Giuliano Callaini² and David M. Glover¹

¹ Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
² Department of Evolutionary Biology, University of Siena, Siena, Via Aldo Moro, 2-53100, Italy

View larger version (91K): [in a new window]   Fig. 1. klp67A mutant primary spermatocytes lack robust central spindles and midbodies. (A) Late anaphase cells isolated from klp67A mutants or wild-type (inset) testes. Microtubules are in green, DNA in blue and the centriole marker Centrin in red. During late anaphase control cells form a symmetrical central spindle that spans the cell's equator. In klp67A mutants the central spindles are diminished and asymmetrical with microtubule bundles positioned on a single side of the cell (white arrowhead) or concentrating in the interior but lacking at the periphery (white arrow). Some centrosome-associated MTs can be abnormally long and run along the periphery (black arrowhead). In other cells, the spindles become bent or S-shaped (black arrow). (B) In control cells constriction of the contractile ring results in the formation of a dense midbody connecting the two daughter cells (inset). This structure was rarely observed in the mutant cells, which although partially constricted lacked discreet microtubule bundles at this later stage. Note how most cells homogenously contain individual, disorganised microtubules but that in some instances few microtubules are visible at the equator (arrowhead). Bar, 10 µm.

View larger version (152K): [in a new window]   Fig. 2. Anaphase B kinetics are similar between klp67A mutants and wild-type cells. Selected frames from time-lapse series comparing anaphase B in a wild-type (A) and klp67A mutant primary spermatocyte (B) expressing ß-tubulin-EGFP. The maximum intensity projection of the six optical sections and the corresponding single, centre-most DIC image for each time point is shown here and a similar format is used for subsequent figures. (A) In wild-type cells anaphase B occurs after the dyads disjoin (0 minutes). Once initiated, the spindle continually elongates, moving the centrosomes in opposite directions until a new steady-state length is reached (10-18 minutes) (B). (B) Before anaphase onset (0 minutes) klp67A mutants formed dense spindles surrounded by long astral microtubules. Unlike in control cells, the centrosomes in the mutants did not undergo significant separation during anaphase B. As the spindle elongated (5-13 minutes) it buckled out of this focal plane before also assuming a new steady-state length (18 minutes). Note how the mutant lacks a discreet central spindle. See main text for details. (C) Kinetic plot of the elongating spindles depicted in (A) and (B). In both cases there is a lag before anaphase B initiates, after which time the spindles extend at the indicated velocities. See Materials and Methods for details of measurements. Bar, 10 µm. Times are in minutes relative to anaphase onset.

View larger version (50K): [in a new window]   Fig. 3. klp67A mutants mislocalise the central spindle-dependent proteins Pav-KLP, KLP3A and Asp. Microtubules are in green and DNA is shown in blue. (A) In control cells (inset) pavarotti-kinesin like protein (Pav-KLP; red) assumes a band-like distribution on the overlapping and interdigitating plus ends of central spindle microtubules. Loss of klp67A prevents the formation of this homogeneous band, but Pav-KLP still associates with the variable numbers of central spindle microtubule bundles that form, which in severe cases is very few, where it appears as aggregates (arrow). (B) Like Pav-KLP, KLP3A (red) localises to the midzone of wild-type central spindles and midbodies (inset). In klp67A mutants the motor protein is detected as ragged streaks that correspond to the poorly formed central spindle microtubule bundles. (C) Asp (red) associates with microtubule minus ends. In wild-type cells (inset) Asp localises to the centrosomes as well as the ends of the microtubules that have been released from the centrosomes and spindle poles to form the central spindle. Similarly, in mutants, Asp associates with the centrosomes and at the termini of the central spindle microtubules (arrowheads). As indicated by the arrows, irregularly spaced Asp signals are found near the equators of klp67A mutant cells, indicative of variable microtubule lengths or positions. Bars, 10 µm.

View larger version (163K): [in a new window]   Fig. 4. klp67A mutants form diminished and unstable central spindles. Selected frames from time-lapse sequences showing central spindle formation in wild-type (A) and (B) klp67A mutant cells. At anaphase onset in wild-type cells (A; 0 minutes) the spindle is linear and has well defined asters that cap each spindle pole. As anaphase ensues the spindle remodels itself into an equatorially positioned central spindle composed of bundles of cytoplasmic, peripheral astral microtubules and interior microtubules within the spindle envelope. This structure initiates and promotes the advancement of the cleavage furrow (20 minutes) before being compacted into a midbody. (B) Central spindle formation is aberrant in klp67A mutants. Before anaphase onset (0 minutes) the cell contained increased numbers of abnormally long astral microtubules that encircled the robust spindle. As the cell advances into anaphase these peripheral astral microtubules collect in the upper portion of the cell, where they initiate a unilateral cleavage furrow that advances but fails to fully cleave the cell (10-29 minutes; arrowheads). Concomitantly, the interior central spindle fails to form ostensible bundles as seen in the wild-type and rapidly degrades (compare arrows at 6 and 29 minutes). The peripheral microtubules are also unstable and as a result this cell lacks a recognisable central spindle. See main text for details. Time is in minutes relative to anaphase onset. Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 5. Ectopic furrows in klp67A mutants generate cytoplasts. Selected frames from a time-lapse sequence of a klp67A mutant during cytokinesis. At anaphase onset (0 minutes) the spindle was abnormally long and surrounded by a symmetrical cage of astral microtubules. These translocate during spindle elongation and coalesce at the right hand side and lower portion of the cell, where they initiate (12 minutes) the primary (arrows) and ectopic (16 minutes; arrowheads) cleavage furrows, respectively. Ingression of the two unilateral furrows leads to the formation of an anuclear cytoplast (26 minutes). Time is in minutes after anaphase onset. Bar, 10 µm.

View larger version (159K): [in a new window]   Fig. 6. Interior central spindles can initiate cleavage furrows in primary spermatocytes. Time-lapse sequence of a klp67A mutant during cytokinesis. 0 minutes: Image of the cell at anaphase onset. Note the characteristic rings of astral microtubules encircling the dense spindle. After dyad disjunction the interior central spindle (9 minutes; white arrowhead) undergoes anaphase B elongation causing it to buckle outwards. During this time the peripheral astral microtubules concentrate and contact the cell boundary at the equator where they initiate a primary furrow (arrows). The interior central spindle further protrudes and approaches the cell cortex in a region of diminished peripheral microtubule density (9-16 minutes; white arrowheads). As the primary furrow continues to advance (16-23 minutes; arrows), an ectopic furrow (19 minutes; black arrowhead) initiates where the interior central spindle apposes the cell membrane (white arrowhead). This ectopic furrow then ingresses towards the cell centre (21-23 minutes; black arrowheads). Time is in minutes relative to anaphase onset. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 7. klp67A mutants fail to form continuous actin and anillin rings. Wild-type (insets) and klp67A mutants were fixed and stained for actin (A; red) or anillin (B; red). Microtubules are shown in green and DNA in blue. In contrast to wild-type cells, which form continuous rings of actin at their equators, actin staining is often disorganised or in streaks in the mutants and in some instances is found primarily as cortically associated foci. Similarly, anillin staining is aberrant in mutants (B) and is patchy or discontinuous. The extent to which either actin or anillin form homogenous bands correlates with the integrity of the central spindle, with the proteins being more symmetrically distributed when robust microtubule bundles are present. Bars, 10 µm.