The centrosome is a dynamic structure that ejects PCM flares

Abstract

The Drosophila Centrosomin (Cnn) protein is an essential core component of centrosomes in the early embryo. We have expressed a Cnn-GFP fusion construct in cleavage stage embryos, which rescues the maternal effect lethality of cnn mutant animals. The localization patterns seen with GFP-Cnn are identical to the patterns we see by immunofluorescent staining with anti-Cnn antibodies. Live imaging of centrosomes with Cnn-GFP reveals surprisingly dynamic features of the centrosome. Extracentrosomal particles of Cnn move radially from the centrosome and frequently change their direction. D-TACC colocalized with Cnn at these particles. We have named these extrusions`flares'. Flares are dependent on microtubules, since disruption of the microtubule array severs the movement of these particles. Movement of flare particles is cleavage-cycle-dependent and appears to be attributed mostly to their association with dynamic astral microtubules. Flare activity decreases at metaphase, then increases at telophase and remains at this higher level of activity until the next metaphase. Flares appear to be similar to vertebrate PCM-1-containing `centriolar satellites' in their behavior. By injecting rhodamine-actin, we observed that flares extend no farther than the actin cage. Additionally, disruption of the microfilament array increased the extent of flare movement. These observations indicate that centrosomes eject particles of Cnn-containing pericentriolar material that move on dynamic astral microtubules at a rate that varies with the cell cycle. We propose that flare particles play a role in organizing the actin cytoskeleton during syncytial cleavage.

Centrosomin represses dendrite branching by orienting microtubule nucleation

“…GFP::Cnn reproduces Cnn localization 28 and rescues cnn loss of function phenotypes 28 ( Supplementary Fig. 8).…”

Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

“…To simultaneously observe the behavior of MTs and spindle poles and see if anti-KLP61F has any detectable effect on pole organization, we microinjected anti-KLP61F with rhodamine-labeled tubulin into embryos expressing GFP-centrosomin (CNN), an integral component of the mitotic centrosome (Megraw et al, 2002; Figure 2E; Supplemental Video 3). During collapse, intact spindle poles steadily moved toward one another at a rate of ϳ0.06 m/s, and the appearance of focused, radial sets of fluorescent MTs projecting from discrete centrosomes persisted throughout the collapse ( Figure 2E).…”

The mob as tumor suppressor Gene Is Essential for Early Development and Regulates Tissue Growth in Drosophila

“…Then PLP was replaced by methanol, and the samples were vigorously shaken to remove vitelline membrane. Embryos were then rehydrated with balanced salt solution (BSS) Triton 0.3% (Ashburner 1989) and stained with anti-Cnn antibodies (a gift of Thomas Kaufman; Megraw et al 2002) ; ey-Gal4/SM6ÁTM6B to generate flies for comparison. Flies carrying loss-of-function alleles of cyclin E, diap1, cyclin A, and cyclin B were also crossed with w 1118 flies to determine their heterozygous phenotypes.…”