Redistribution of actin during assembly and reassembly of the contractile ring in grasshopper spermatocytes

Abstract

Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end-directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules.

Figure 1. Mechanically induced reorganization of microtubules mediated actin redistribution into ectopic contractile rings.

(A, a–d) The diagram shows how an anaphase spindle can be mechanically collapsed by pushing the spindle poles together, using a micromanipulation needle. The procedure results in ectopic cleavage in two locations, as shown in (B). Microtubules, green; chromosomes, blue; kinetochores, magenta; centrosomes, orange; cortical actin, red; needle, gray. (B) Polarization microscopy sequence of ectopic cleavage, with time shown in minutes. Following anaphase onset (0 min, arrows show direction of impending collapse; m, spindle-associated mitochondria; also see Video S1), the spindle was collapsed with a microneedle, resulting in lateral growth of microtubules that dislodged the mitochondria (5 min). On opposite sides of the cell, these microtubules reorganized into two lateral spindles, while the displaced mitochondria appeared to rebundle with the microtubules (18 min). Cleavage furrows initiated simultaneously around both of the continuously reorganizing lateral spindles (45 min, arrows). Ultimately, each furrow ingressed around the approximate midpoint of its spindle (48 min), producing two anucleate membrane pockets (64 min). (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at furrow initiation (a–c) and ingression (d–f) showed that each lateral spindle was in fact an independent bipolar spindle complete with a midzone (a and d, arrows). Bands of actin filaments (b and e), presumably contractile rings, encircled the midzone regions, while another subset of actin filaments (marked by asterisks) appeared to colocalize with spindle microtubules. Remnants of contractile rings from previous cell cleavages, i.e., “cell division scars”, were also present (b and e; small circles or ovals at cortex). Bars, 10 µm.

Figure 2. Spindle folding generated a concentrated source of actin for building a new contractile ring.

(A) The central spindle can be folded using a microneedle, before furrow formation. Color scheme, as in Fig. 1A. (B) Polarization microscopy sequence (Video S2). After folding (0 min), the mechanically generated monopolar spindle reorganized into a bipolar spindle. Microtubules from the pair of spindle poles radiated toward the cell cortex (11 min, arrows) where a furrow initiated (23 min, arrows). The furrow ultimately ingressed at the equator of the spindle (37–52 min), after realignment of the spindle midzone. (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at stages similar to those in (B). Upon folding of the spindle by micromanipulation, the deformed midzone (a, arrow) and remnants of the contractile ring (e and i) remained relatively organized. As central spindle microtubules reorganized, the original midzone disappeared (b). Concurrently, actin filaments dispersed from the disintegrating ring, oriented in apparent alignment with central spindle microtubules (f and j). Meanwhile, a few very short microtubule bundles (b, arrowheads) emerged at the spindle tip and initiated formation of a new midzone, transverse to the new spindle axis (b, arrow). Around the time of furrow initiation, reorganization of the elongated bundles created a new “pole” (c), thereby establishing a bipolar spindle with a broad midzone (c, arrows). Actin filaments appeared to disassociate from spindle microtubules as they entered the new contractile ring (g and k). The furrow ingressed as microtubules at the distal pole elongated (c–d, green), shifting the midzone (c–d, arrows) and the contractile ring (g–h and k–l) toward the spindle equator. Bars, 10 µm.

Figure 3. Incorporation of actin filaments into the contractile ring during furrow induction and ingression.

(A) Surface view of induction. Actin filaments were labeled by microinjection with Alexa 488-phalloidin and imaged every 10 seconds. The large panels show overall actin redistribution over time. The small panels show the dynamics of a single actin filament (arrows), as seen in selected time-lapse images within the boxed region of interest (Video S3). Immediately prior to furrow initiation (0 sec image), some cortical actin filaments began to move toward the spindle equator where they coalesced into nascent bundles of the emerging contractile ring (arrowhead). The arrows mark the end of a filament of interest, discernible at this focal plane. The filament moved toward the equator, underwent a sharp change in trajectory, and disappeared into the emerging contractile ring. The unlabeled spindle was flanked by microtubule-associated, autofluorescent mitochondria (also present in (B)) that hampered visualization of actin filaments in the vicinity. Time in seconds. Bars, 10 µm. (B) Midplane view of ingression. Actin filaments were labeled and observed as described in (A) except imaged at the midplane of the cell to follow furrow ingression. In the large panels, both labeled actin filaments and autofluorescent mitochondria could be seen. During furrow ingression (arrowheads), actin filaments continued to move toward and incorporate into the contractile ring, presumably along spindle microtubules. The small panels (selected from Video S4; within the boxed region) show the dynamics of a single actin filament (arrows) moving into the ingressing contractile ring. Time in seconds. Bars, 10 µm.

Figure 4. Redistribution of actin filaments labeled by microinjection of Qdot 655-Phalloidin.

At the onset of cytokinesis (10), Qdot-decorated actin filaments formed a pattern vaguely mirroring that of the spindle (10–13), reminiscent of the distribution of central spindle microtubules (as in Video S5). The Qdots gradually cleared from the non-equatorial regions and accumulated at the cleavage furrow (18 onward) as cytokinesis proceeded. Time in minutes. Bar, 10 µm.

Figure 5. Redistribution of actin filaments labeled by Alexa 488-Phalloidin and Qdot 655-Phalloidin.

(A–B) Actin filaments (green) speckled with Qdots (red) continuously merged into the contractile ring during furrow induction (A; Video S6) and furrow ingression (B; Video S7). Occasionally, Qdot-decorated actin filaments could be seen moving away from the contractile ring (B, 3–9). The unlabeled spindles were flanked by a pair of green bundles that contained microtubule-associated, autofluorescent mitochondria. Time in minutes. Bar, 10 µm.

Figure 6. Tracking individual actin filaments labeled by Alexa 488-Phalloidin and speckled with Qdot 655-Phalloidin.

As shown in the kymographs (Videos S8, S9, S10), actin filaments could be seen moving into the cleavage furrow (A, B) and moving away from the furrow (C). Arrows depict Qdot-marked reference points on the actin filaments, and a white vertical line marks the location of each cleavage furrow. Time in seconds. Bar, 10 µm.

Figure 7. The movement of an actin filament along sparsely distributed spindle microtubules during furrow ingression.

For fluorescent speckle microscopy, microtubules (false-colored green) and actin filaments (false-colored red) were labeled by microinjection with Rhodamine-tubulin and a minute amount (∼0.2 µM) of Alexa 488-phalloidin, respectively. (A) A selected image from a time lapse series showing a dividing cell in which a single, clearly visible microtubule (boxed) happened to be isolated from the rest of the central spindle. Bar, 10 µm. (B) Sequential images of the boxed region, containing the cell cortex and the microtubule of interest, were enlarged and reoriented, with the microtubule horizontal and the cell cortex toward the right. A speckled actin filament (or bundle) appeared to move along a single microtubule toward the cortex. Bar, 5 µm.

Figure 8. Cortical flow and de novo synthesis of actin filaments in grasshopper spermatocytes.

(A) Time-lapse sequence of actin filaments undergoing cortical flow (from Video S11). Flow of actin was induced by collapsing the spindle and repositioning it near one side of the cortex. The final location of the spindle is marked by “sp”. Actin filaments were labeled by microinjection with Alexa 488 phalloidin. Note the clearing over time of the region closest to the spindle, as the actin flows to the cortex on the opposite side of the cell. Spindle-associated mitochondria were seen as a pair of large bright patches (labeled “m”) that autofluoresced in the FITC channel. Because mitochondria were localized to the cytoplasm, their displacement was not related to cortical flow, but rather was driven by the elongation of dynamic microtubules with which they were associated. Time in minutes. (B) A confocal micrograph showing actin aggregates (red, marked by arrows) localized to the tips of bundled microtubules (green) at the spindle midzone. These aggregates were strikingly similar in their location, timing of appearance and morphology to aggregates found in silkworm spermatocytes , which have been shown to be assembled de novo at the midzone. Actin was labeled by microinjection of rhodamine phalloidin during anaphase, and microtubules were labeled with paclitaxel green. A cell division scar (red circle, lower edge of cell) was visible. Bars, 10 µm.

Figure 9. Cell division did not require the presence of spindle-associated mitochondria.

A time-lapse sequence of DIC images. During anaphase (0 min), both groups of spindle-associated mitochondria (m) that flanked the central spindle (sp) were mechanically repositioned distal to opposing sets of segregating chromosomes (c; 12 min). Although the equatorial region was thus cleared of mitochondria, cleavage at the spindle equator was not inhibited (12 min onward). Arrows show the locations of the cleavage furrows: at the equator, and in two ectopic locations induced by the relocated, mitochondria-associated bundles of microtubules. The furrows produced two daughter cells (d), each associated with an anucleate membrane pocket (p). Time in minutes. Bar, 10 µm.