JCS052514 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -
• Supplemental Tables S2 -
• Supplemental Table S3 -
• Supplemental Table S4 -
• Supplemental Figure S1 -

  Fig. S1. The severity of Baz mislocalization at the extended germ band stage differs substantially in germ-line clone embryos mutant for class I and class II aPKC alleles. (A-G) Embryos of the indicated genotypes at the extended germ band stage (stage 10-11) were stained for Baz (green). The top panel shows an overview of the embryo and the bottom panel a superficial optical section of the same embryo at the plane of the ZA. Note that in wild type (A) and in the class II aPKC mutants (D,E) Baz forms contiguous belts at the ZA, whereas in class I aPKC mutants (B,C) and in aPKC^k06403 (F) and par-6Δ²²⁶ (G) null mutants only scattered dots of Baz staining are visible. Scale bar for top panels: 100 µm. Scale bar for bottom panels: 10 µm. Anterior is to the left and dorsal up.

• Supplemental Figure S2 -

  Fig. S2. aPKC mutant embryos show a strong increase in apoptotic cell death. Apoptotic cells were labeled by TUNEL staining (green) and antibody staining for the active caspase Drice (red) in wild-type (A) and aPKC^psu69 mutant (B) embryos. Scale bar: 100 µm (A,B). (C) In wild-type embryos, apoptotic bodies (red arrowheads) are engulfed by macrophages without interfering with the integrity of the ectodermal epithelium. (D) In aPKC^psu69 (class I) mutant embryos, apoptotic bodies remain on the surface of the embryo and are not internalized by macrophages. Scale bars: 1 µm (C,D). In A and B anterior is to the left and dorsal up. In C and D, apical is up. The embryos shown in A-D are at stage 11.

• Supplemental Figure S3 -

  Fig. S3. Spindle orientation defects in neuroblasts of aPKC mutants. (A) In a wild-type neuroblast the mitotic spindle (dark yellow) is orientated perpendicular to the crescents of apical proteins (red), including Bazooka, aPKC and PAR-6 and basal proteins (blue), including Miranda, Prospero and Numb. We define the orientation of the spindle with respect to these proteins as the intrinsic orientation. In addition, the spindle is also perpendicular to the plane of the overlying ectodermal epithelium. We define this latter orientation as the extrinsic orientation. (B) To quantify extrinsic spindle orientation defects, we have defined two categories, which were determined by measuring the angle of the metaphase plate (green) with respect to the plane of the overlying ectodermal epithelium. In category 1, which we define as correct spindle orientation, the angle of the metaphase plate deviates less than 20° from the plane of the epithelium. In category 2, which we define as extrinsic misorientation, the metaphase plate deviates more than 20° from the plane of the epithelium. In both categories 1 and 2, the spindle is oriented perpendicular to the crescent of Baz, so the intrinsic orientation is correct. The orientation of the spindle with respect to the localization of Miranda could not be scored, because in the majority of cases Miranda was mislocalized all around the neuroblast cortex. In category 3 we scored all neuroblasts in which the spindle was not properly aligned with the crescent of Baz. Apical is up.

• Supplemental Figure S4 -

  Fig. S4. Four new mutant alleles of aPKC change single highly conserved amino acid residues. The sequence alignment of the environment of the mutated amino acid residues was performed with Megalign (DNAStar) software. The numbers on the left correspond to the first amino acid shown in the respective line.

• Supplemental Figure S5 -

  Fig. S5. Two dimensional principal component analysis of the modeled protein. Shown are the final structures taken from ten simulated annealing simulations of the modeled protein, projected on their first two common eigenvectors (grey dots). The circle shows two independently sampled structures, converged to a common point in the essential space. This structure has therefore been chosen for the subsequent simulations.

• Supplemental Figure S6 -

  Fig. S6. Root mean square deviations (RMSD) of the simulated systems. (A) Backbone RMSDs of the modeled wild-type (WT) and the respective mutant structures. (B) Backbone (bb, solid line) and Mg²⁺-ATP (dashed line) RMSDs of the wild-type and the respective mutant structures after Mg²⁺-ATP insertion.