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The Cell Cycle and Development - Novartis Foundation

Novartis Foundation The Cell Cycle and Development - John Wiley & Sons, 2001. - 268 p.
ISBN 0-471-49662-6
Download (direct link): thecellcycleanddevelopment2001.pdf
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A schematic summary of the localization of the key components of the asymmetric division process is given in Fig. 1.
FIG. 1. Localization of key proteins involved in the neuroblast asymmetric cell division. During late interphase a complex of proteins including Insc, Baz (and Pins) are localized to the apical cell cortex. This complex acts to mediate the basal cortical localization of the cell fate determinants Numb (and its partner Pon), Pros (and its partner Miranda) and pros RNA (and its partner Staufen) during mitosis. During interphase, Numb is cytoplasmic and Pros is localized to the apical cortex.
Results and discussion
Pins interacts and co-locali^es with Insc
We have previously shown that a 209 amino acid region (aa288-497, asymmetric localization domain) of Insc is necessary and sufficient for apical cortical localization and for mitotic spindle orientation along the apical-basal axis (Tio et al 1999). In a yeast two-hybrid screen we identified Partner of Inscuteable (Pins), a novel 658aa protein with multiple repeats of the Tetratricopeptide (TPR) motif. Affinity purification experiments using embryonic extracts demonstrate that Pins complexes with Insc in vivo. In vitro protein interaction assays demonstrates that Pins interacts with the Insc asymmetric localization domain (see Yu et al 2000).
Antibodies were raised against Pins fusion proteins which specifically recognize Pins. In neuroblasts Pins is localized as a crescent to the apical cortex starting at interphase following delamination. More intensely labelled Pins apical crescents can be seen during mitosis from prophase to anaphase. Double labellings with anti-Insc indicate that Pins and Insc are largely co-localized during the neuroblast cell cycle. The exception being in delaminating neuroblasts where high levels of Insc staining can be seen on the apical stalk which extends from the neuroblast towards the surface of the neuroectoderm; in contrast, high levels of apical Pins are detected only following neuroblast delamination. Hence although Insc and Pins are co-localized in fully delaminated neuroblasts, the initial localization of Insc to the apical stalk during delamination (interphase) may precede that of the Pins apical localization.
Apical cortical localisation of Pins, Insc and Bas in mitotic neuroblasts is codependent
In order to assess the function of pins, we generated several small deletions which removed all or part of the pins coding region. Two alleles which are antigen negative were viable and allowed us to obtain Pins- embryos which lacked both the maternal and zygotic components of pins. The availability of embryos lacking bas, insc or pins enabled us to assess the localization of each of the three apical components in embryos lacking either of the other two functions. In wild-type mitotic neuroblasts, Baz, Insc and Pins co-localize as apical crescents. However, in mitotic neuroblasts lacking either bas or insc, Pins becomes distributed throughout the cortex. In bas or pins mutant embryos, Insc is localized to the cytoplasm of mitotic neuroblasts. Similarly, in mitotic neuroblasts lacking either pins or insc, Baz apical crescents are either undetectable or its levels are drastically reduced. Hence in neuroblasts which have completed delamination or are undergoing mitosis, the asymmetric localization/stability of Baz/Insc/Pins are
FIG. 2. The formation of the apical protein complex involves two distinct steps. Bazooka is localized apically in the epithelium from which neuroblasts are derived. In the interphase (G2), delaminating neuroblast formation ofthe apical complex is initiated. It is thought that Baz acts to allow neuroblasts to retain the apical/basal polarity inherent in the epithelium. Baz recruits Insc to the neuroblast apical stalk during delamination before Pins becomes part of the complex. During this initiation step Baz, Insc and Pins are part of a linear hierarchy. However following delamination and during mitosis, the maintenance of the apical localization of each of these proteins requires all three proteins.
mutually dependent. Loss of any single component will result in defective localization or instability of the remaining two components (see Fig. 2).
bainsc andpins share a hierarchical relationship during
the initiation of apical complexformation in interphase neuroblasts
However, in interphase delaminating neuroblasts, which are known to have completed S-phase and are at the G2 stage of the cell cycle, this codependence of Baz/Insc/Pins seen in mitotic neuroblasts does not apply. Delaminating neuroblasts possess an apical membrane stalk which retains contact with the epithelial surface and this is where apical cortical localization of Insc is initially seen (see Fig. 2). This initial localization of Insc to the apical stalk occurs
normally in Pins- embryos. In contrast, it is not seen in delaminating neuroblasts of bas mutants. Moreover, the localization of Baz to the stalk does not depend on either insc or pins. These results indicate that the initial localization of Baz to the stalk of delaminating neuroblasts requires neither insc nor pins; the initiation of Insc apical localization to the stalk requires bas but not pins; however, the maintenance of apical Baz/Pins/Insc following delamination and in mitotic neuroblasts is mutually dependent, requiring all three components. Hence during the initiation of apical complex formation, Baz/Insc/Pins appear to have a hierarchical relationship with Baz being the most upstream and Pins being the most downstream component. However for the maintenance of this apical complex during mitosis, they are codependent. A schematic summary of these conclusions is shown in Fig. 2.
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