Mitotic spindles orientation

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). 

In Pins - embryos the initiation steps of apical complex formation occur normally. However, this complex cannot be maintained in mitotic neuroblasts. Hence, the importance of the maintenance of this complex for asymmetric cell division can be ascertained by assessing how Pins- neural progenitors divide. Pins- embryos exhibit all of the defects seen in insc mutants. Mitotic spindle orientation is defective. In the cells of mitotic domain 9 the 90° reorientation, which normally occurs in wild-type resulting in the orientation of the spindle along the apical—basal axis (Fig. 3A), fails to occur in the mutant (Fig. 3B). Mitotic spindle orientation of neuroblasts in the segmented CNS, deduced from DNA staining, also often fails to... 

Fig. 2.3 The development of polarity and asymmetric division in Saccharomyces cerevisiae. The diagram is reproduced in a slightly simplified form from the work of Lew Reed (1995) with the permission of Current Opinion in Genetics and Development, (a) The F-actin cytoskeleton strands = actin cables ( ) cortical actin patches, (b) The polarity of growth is indicated by the direction of the arrows (arrows in many directions signifies isotropic growth), (c) 10-nm filaments which are assembled to form a ring at the neck between mother and bud. (d) Construction of the cap at the pre-bud site. Notice that the proteins of the cap become dispersed at the apical/isotropic switch, first over the whole surface of the bud, then more widely. Finally, secretion becomes refocussed at the neck in time for cytokinesis, (e) The status and distribution of the nucleus and microtubules of the spindle. Notice how the spindle pole body ( ) plays an important part in orientation of the mitotic spindle.

The centromere (Fig. 24-9) is a sequence of DNA that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle. This attachment is essential for the equal and orderly distribution of chromosome sets to daughter cells. The centromeres of Saccharomyces cere-visiae have been isolated and studied. The sequences essential to centromere function are about 130 bp long and are very rich in A=T pairs. The centromeric sequences of higher eukaryotes are much longer and, unlike those of yeast, generally contain simple-sequence DNA, which consists of thousands of tandem copies of one or a few short sequences of 5 to 10 bp, in the same orientation. The precise role of simple-sequence DNA in centromere function is not yet understood. 

A FIGURE 22-20 General features of asymmetric cell division. Various mechanisms can lead to asymmetric distribution of cytoplasmic components, such as particular proteins or mRNAs (red dots) to form a polarized parental cell. Division of a polarized cell will be asymmetric if the mitotic spindle is oriented so that the localized cytoplasmic components are distributed unequally to the two daughter cells, as shown here. However, if the spindle Is positioned differently relative to the localized cytoplasmic components, division of a polarized cell may produce equivalent daughter cells. 

Orientation of the Mitotic Spindle Is Linked to Cytoplasmic Cell-Asymmetry Factors... 

For localized protein complexes to be differentially incorporated into two daughter cells requires that the plane of cell division be appropriately oriented. In dividing fly neuroblasts, the mitotic spindle first aligns perpendicular to the apical-basal axis and then turns 90 degrees to align with it at the same time that the basal complexes become localized to... 

Asymmetry factors exert their influence at least in part by controlling the orientation of the mitotic spindle, so that asymmetrically localized proteins and structures are differentially incorporated into the two daughter cells. Myosin proteins bind to proteins that control asymmetry factors of cells to control spindle orientation. 

Petritsch, C., et al. 2003. The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts. Devel. Ceil 4 273-281. 

A class of motile systems completely different from and unrelated to the actin-myosin contractile systems is used in cellular structures as diverse as the mitotic spindle, protozoan and sperm flagella, and nerve axons. These systems are constructed from microtubules, very long, tubular structures built from a helical wrapping of the protein tubulin (Figure 8.19). There are two kinds of tubulin subunits, oi and each of molecular weight 55,000. They are present in equimolar quantities in the microtubule, which can be considered a helical array of ot-/i dimers. Alternatively, we can view the microtubule as consisting of 13 rows, or protofilaments, of alternating ot and subunits. Because the oi and b units are asymmetrical proteins, with a defined and reproducible orientation in the fiber, the microtubule has a definite sense of direction. 

Stebbins, G. L., Shah, S. S., Jamin, D., and Jura, P. (1967). Changed orientation of the mitotic spindle of stomatal guard cell divisions in Hordeum vulgare. Am. J. Botany 54, 71-80. 

Ostergren s (1951) simple explanation for reduction of chromosome number in meiosis is clearly supported by evidence for preferential orientation, and recourse to unspecified physiological differences between meiotic and mitotic cells (Lima-de-Faria, 1958) as the immediate cause is not necessary at present (Nicklas, 1967). Moreover there is direct evidence favoring factors intrinsic to the chromosomes rather than physiological differences. First, merely the observation that both amphi- or synoriented chromosomes may occur on the same spindle suggests this (Schwartz, personal communication). Notable examples are found in meiosis when normally paired chromosomes are present as unpaired half-bivalents (reviewed by John and Lewis, 1965, pp. 52 ff. related... 

Only some unpaired half-bivalents amphiorient, and this variation may well seem puzzling. Briefly, a probable explanation is that on Ostergren s (1951) hypothesis, metiotic versus mitotic orientation depends upon the same factors involved in initial orientation the position of kinetochores relative to each other and preferential orientation. As has been seen, initial orientation is not errorless, because chromosomes may by chance lie in positions making appropriate orientation of their kinetochores more or less unlikely. Hence, amphiorientation of ha -bivalents may be viewed as an enor, especially likely when univalency induces frequent reorientation (see p. 272) and the half-bivalent is exposed repeatedly to the hazard of amphiorientation which may be possible whenever the half-bivalent s kinetochores happen to lie perpendicular to the spindle axis during reorientation. 

Fig. 7. Low-power electron photomicrograph of isolated mitotic apparatus at metaphase (X3400). After isolation, mitotic apparatus was treated with neutral 0s04, embedded in methacrylate, and sectioned. Chromosomal fibers apparent. Spindle and asters appear to be gels with oriented regions corresponding to fillers seen with light microscope. Electron microscopy carried out by Miss P. F. Harris.)...

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