Anomalous centriole configurations are detected in Drosophila wing disc cells upon Cdk1 inactivation

Abstract

The centriole, organizer of the centrosome, duplicates by assembling a unique daughter identical to itself in overall organization and length. The centriole is a cylindrical structure composed of nine sets of microtubules and is thus predicted to have nine-fold symmetry. During duplication, a daughter lacking discrete microtubular organization first appears off the wall of the mother centriole. It increases in length perpendicularly away from the mother and terminates growth when it matches the length of the mother. How a unique daughter of the correct length and overall organization is assembled is unknown. Here, we describe three types of unusual centriole configurations observed in wing imaginal discs of Drosophila following inactivation of Cdk1. First, we observed centriole triplets consisting of one mother and two daughters, which suggested that centrioles have more than one potential site for the assembly of daughters. Second, we observed centriole triplets comprising a grandmother, mother and daughter, which suggested that subsequent centriole duplication cycles do not require separation of mother and daughter centrioles. Finally, we observed centriole pairs in which the daughter is longer than its mother. These findings suggest that regulatory events rather than rigid structural constraints dictate features of the stereotyped duplication program of centrioles.

Fig. 1

Wing discs from sevelen (wild-type) larvae or Cdk1^ts larvae. Eggs were collected and aged to the late second instar larval stage at 18°C. Larvae were then shifted to 30°C for 2 days, after which they were fixed. Discs were dissected and stained for DNA with the dye, bis-benzidine (Hoechst). Wing discs in which Cdk1 has been inactivated are, typically, smaller in size compared with similarly treated sevelen discs and have fewer cells that are bigger than normal. Bar, 25 μm.

Fig. 2

Centriole length is misregulated upon inactivation of Cdk1. In all cases, the mother centriole is on the left and the daughter on the right. Furthermore, we have oriented images so that mothers in longitudinal section have their long axes running top-bottom and daughters in longitudinal section have their long axes facing left-right. Since the section is thinner than the centriole, a longitudinal section through a centriole appears as a pair of parallel lines (representing the walls of the centriole). A centriole in cross-section appears as a circle (Vidwans et al., 1999). The pairs of pictures in A, D and E are adjacent sections from a series. (A,B) Control centrioles from sevelen wing discs incubated at 30°C for 2 days. (C,D,E) Centrioles from Cdk1-inactivated wing disc cells. (A) Both the mother and the daughter in this centriole pair are in longitudinal section. Note that the daughter is shorter than its mother (compare the mother in the left section to daughter in the right). (B) Both the mother and the daughter centrioles are in longitudinal section, with the daughter shorter than its mother. (C) A longitudinal mother-daughter centriole pair with a long mother. Compare the length of this mother to those in A and B. (D) A mother in cross-section with a long daughter in longitudinal section. Compare the length of this mother to that in A and B. (E) A longitudinal centriole pair with long mother and daughter. The daughter is longer than its mother. Bar, 100 nm.

Fig. 3

Distribution of lengths of mother and daughter centrioles from sevelen and Cdk1^ts wing discs incubated at 30°C for 2 days. The x-axis is centriole length (measured in nm) and the y-axis is the number of centrioles at any given length. Note that the distribution of daughter centrioles from wild-type discs varies between 46 and 93 nm, while the corresponding distribution from ts discs varies between 46 and 162 nm. The wider distribution of daughter centriole lengths in ts discs is reflected in the higher average: 114 nm compared with 70 nm in wild-type. The distribution of mother centrioles from ts discs is also broader than wild-type (105 to 184 nm in ts discs compared with 93 to 139 nm in wild-type). The average mother centriole length in ts discs is 134 nm compared with 115 nm in wild-type.

Fig. 4

Mother centrioles with two daughters. Three examples of mother centrioles with two daughters. Each triplet is shown in two consecutive serial sections with a schematic to orient the reader. (A) A mother centriole (in the center) that is slightly angled with two longitudinal daughters, one to the left and the other to the right. (B) A mother in cross-section with two daughters, both in longitudinal section, one to the left and one (seen only in the section at the right) at the top. (C) A mother in cross-section with two longitudinal daughters, one to the right and the other to the left. Note that one of the daughters in each triplet is always longer than the other, suggesting that the two daughters were assembled at different times. Furthermore, note that the differences in the angle between the two daughter centrioles suggest that the initiation of a second daughter can occur at a variety of positions with respect to the first daughter. Bar, 100 nm.

Fig. 5

Examples of mother, daughter and granddaughter triplet centrioles. Each triplet is shown in two consecutive serial sections with a schematic on the right to orient the reader. All the centriole triplets are oriented with the mother on the left, the daughter to the right and the granddaughter to the top or bottom. To accomplish this orientation, adjustments were made in Adobe PhotoShop, some of which appear as discontinuities within the images (D,E). Note that mother and daughter in B are not quite orthogonal, while the arrangement in D and E appears to be orthogonal. Note that if mother daughter orientation is fixed, the granddaughter can emerge from different faces of the daughter, suggesting that the asymmetry is not programmed by the preceding generation of centriole. Bar, 100 nm.