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Gene name - inscuteable
Synonyms - not enough muscle Cytological map position - 57B1-4 Function - Links cytoskeleton to spindle-orientation and protein subcellular distribution Kew words - Cytoskeleton, neurogenesis, asymmetric cell division, apical/basal polarity |
Symbol - insc
FlyBase ID: FBgn0013468 Genetic map position - Classification - Novel protein with putative SH3 binding domain, ankyrin repeats and cytoskeletal attachment domain present in the protein 4.1 family, particularly talin Cellular location - cytoplasmic |
The particular dynamics of Inscuteable protein, also termed Not enough muscles, have permitted researchers to gain a deeper understanding of the driving forces behind asymmetric cell division. Before getting into the specifics of Inscuteable, a lengthy aside about asymmetric cell division is in order. The phenomenon has mystified developmental biologists for decades: not only do daughter cells undergo divergent cell fates (often one daughter differentiating and its sister recycling as a stem cell), but asymmetric cell division is often accompanied by an oriented cell division, in which the two daughter cells are oriented to different embryonic locations.
A good example of such asymmetric division is found in Drosophila neurogenesis. In an asymmetric cell division, the neuroblast delaminates from the neuroectoderm and subsequently divides, giving rise to an additional neuroblast and a sister ganglion mother cell (GMC). GMCs are born in a stereotyped position, reflecting a reproducible orientation of the neuroblast division. Neuroblasts (although residing in the CNS) retain contact with the neuroectoderm. Cell divisions have a preferred orientation, ranging from perfectly dorsal (perpendicular to the embryo surface) to nearly lateral (parallel to the embryo surface) (Doe, 1992).
Accompanying the asymmetric cell division process is an asymmetric distribution of two cellular proteins: Prospero and Numb. Both proteins are preferentially distributed to the GMC during the division process, having become asymmetrically associated with the cell cortex during mitosis and passed on to the GMC progeny. The asymmetric cleavage of the cell (giving rise to the GMC and a neuroblast) and the asymmetric localization of cellular proteins are related processes, but what is causal in either phenomenon?
In oriented cell division it is clear that the mitotic apparatus is the causal factor. The plane of orientation of the mitotic apparatus sets the direction of the oriented cell division. But how does the mitotic apparatus become oriented, and does the orientation of the mitotic apparatus direct the asymmetric localization of Prospero and Numb?
At this point, we turn to the role of inscuteable. The Inscuteable protein is expressed in cells that are known to undergo asymmetic cell division, that is, in cells that distribute Numb and Prospero asymmetically. Inscuteable is asymmetically distributed, but to the opposite pole from Numb and Prospero, that is, to cells that remain neuroblasts, whereas Numb and Prospero are distributed to GMCs. In the ectodermal procephalic neurogenic region, where neuroblasts give rise to brain cells, most cells divide perpendicular to the surface; Prospero and Numb segregate into the basal daughter cell (brain precursor). The localization of Prospero and Numb starts in late prophase and persists through metaphase and anaphase. In telophase both proteins segregate into the basal daughter cell. Inscuteable behaves similarly, but becomes delocalized in anaphase, and is not preferentially segregated into one of the daughter cells (Kraut, 1996).
Localization of Inscuteable is dependent on elements of the cytoskeleton. No Inscuteable crescents are detectable upon disruption of the cytoskeleton with cytochalasin D, a drug which disrupts Actin filaments. Although Prospero remains asymmetrically localized, the position of the Prospero crescent is incorrect in many of the scored neuroblasts. There are no defects in Inscuteable localization after destruction of microtubules with colcemid. Therefore, the cytoskeleton and not the mitotic apparatus is linked to the asymmetric localization of Prospero. In inscuteable mutants, both Numb and Prospero are mislocalized. In addition, in mutant neuroblasts, the mitotic spindle is not oriented in the usual direction. Misexpression of inscuteable in the ectodermal cells of abdominal segments causes ectodermal cells to divide perpendicular to the epithelial surface, and not in the usual parallel orientation (Kraut, 1996).
Relevant to this discussion is a structure found oogenesis called the fusome. Fusomes consist of cytoskeletal proteins, alpha-Spectrin, ß-Spectrin, Hu-li tai shao (an adducin-like protein) and Ankyrin. Of particular interest is the association of fusomes with the pole of the mitotic spindle (Lin, 1995). During the first cystoblast (cystoblasts are derived from germ line stem cells) division, fusome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. The association of fusome proteins with the mitotic spindle indicates a direct interaction between cytoskeletal components and only one pole of the mitotic spindle. Surely this must have something to do with the underlying mechanism of asymmetric cell division.
It is concluded that the force that orients spindles operates likewise to give rise to asymmetric distribution of Prospero and Numb, and both phenomena require functional inscuteable. It is suggested that Inscuteable acts to tether the mitotic apparatus to the cytoskeleton, and also functions to connect Prospero and Numb to elements that are oriented with respect to the cytoskeleton (Kraut, 1996)
Transcript size - 4.2 kb
The 459 C-terminal amino acids of INSC have 42% similarity and 24% identity to the C-terminal cytoskeleton attachment domain of talin, but lowered degree of similarity to the other members of the protein 4.1 family, including protein 4.1 itself, ezrin, radixin, and moesin. INSC shares 45% similarity and 23% identity with ankyrin, with a sequence motif that is reminiscent of the ankyrin repeats from other proteins. This motif occurs five times in INSC. Although INSC shares overall 23% identity with ankyrin, the INSC motifs are less similar to any of those 22 repeats found in ankyrin than to those in the yeast mating type switching protein SWI, the product of the human proto-oncogene bcl3, or the IkappaB-related family p100, p105 and Cactus. There is also a short poly-proline stretch that fits the consensus for SH3 target sites, and a perfect nuclear localization repeats. However, there is no indication that INSC is located to the nucleus (Kraut, 1996a). \
The Drosophila Inscuteable protein acts as a key regulator of asymmetric cell division during the development of the nervous system. In neuroblasts, Inscuteable localizes into an apical cortical crescent during late interphase and most of mitosis. During mitosis, Inscuteable is required for the correct apical-basal orientation of the mitotic spindle and for the asymmetric segregation of the proteins Numb, Prospero and Miranda into the basal daughter cell. When Inscuteable is ectopically expressed in epidermal cells, which normally orient their mitotic spindle parallel to the embryo surface, these cells reorient their mitotic spindle and divide perpendicularly to the surface. Like the Inscuteable protein, the Inscuteable mRNA is asymmetrically localized. Inscuteable mRNA localization is not required for Inscuteable protein localization. A central 364 amino acid domain (the Inscuteable asymmetry domain) is necessary and sufficient for Inscuteable localization and function. Within this domain, a separate 100 amino acid region is required for asymmetric localization along the cortex, whereas a 158 amino acid region directs localization to the cell cortex. However, the same 158 amino acid fragment can localize asymmetrically when coexpressed with the full-length protein and can bind to Inscuteable in vitro, suggesting that this domain may be involved in the self-association of Inscuteable in vivo. These results indicate that amino acids 252-615 of Inscuteable are sufficient for directing and orienting asymmetric cell divisions in neuroblasts. This region is therefore named the Inscuteable asymmetry domain. This domain contains five repeats of limited similarity that are characterized by the core motif VRxL/I (in single-letter amino acid code where x represents any amino acid). Further experiments are required to directly test the functional significance of this motif. Amino acids 302-459 of Inscuteable localize to the cell cortex but fail to localize asymmetrically, whereas deletion of amino acids 436-459 of Insuteable affects asymmetric localization but not cortical localization. Thus, Inscuteable localization may be a two-step process, involving cortical localization and asymmetric localization along the cell cortex (Knoblich, 1999).
The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for Band 4.1 family domain signatures.
During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. This study characterizes a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, it has been shown that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. These results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome (Zigman, 2005).
How does mInsc orient mitotic spindles? In Drosophila, Insc is thought to act by polarizing G protein signaling and thereby attracting astral microtubules to the apical cell cortex. In vertebrates, overexpression of heterotrimeric G proteins causes oscillations of the mitotic spindle, suggesting that G protein activity -- as in flies -- regulates the attachment of astral microtubules to the cell cortex. The mammalian Pins homolog LGN is present in the mouse ventricular zone and might activate G proteins. Although the existing LGN antibodies did not allow a determination of its subcellular localization in mouse brain, mInsc might act by recruiting LGN to the apical and lateral cell cortex, resulting in polarized G protein activation. This model predicts that it is the asymmetric distribution of mInsc in vertically dividing progenitors rather than its presence that influences spindle orientation. Consistently, mInsc overexpressed in fibroblasts is without consequence (Zigman, 2005).
In cell polarization of Drosophila neuroblasts, Inscuteable (Insc) functions via tethering Partner of Insc (Pins) to Bazooka, homologous to human cell polarity protein Par3. However, little has been known about mammalian homologues of Insc. Two distinct cDNAs have been cloned from human Insc gene, which is differentially expressed from alternative first exons: one encodes 579 amino acids, whereas the other lacks the N-terminal 47 amino acids. In contrast to human homologues for Pins and Par3, human Insc exhibits a weak homology with the Drosophila counterpart. Nevertheless, human Insc proteins bind to the human Pins homologues LGN and AGS3, and also to human Par3 and its related protein Par3beta. Although LGN by itself is incapable of interacting with Par3, coexpression of human Insc leads to the interaction between LGN and Par3, indicating that human Insc plays an evolutionarily conserved role as an adaptor protein that links Pins to Par3 (Izaki, 2006).
date revised: 15 February 2006
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