BIOLOGICAL OVERVIEW

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)

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). This study investigated the mechanism of spindle orientation by the Insc-Pins complex. Previously, two Pins spindle orientation pathways were defined: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. The current study examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. It was found that the Insc-Pins complex requires Galphai for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. These results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud) (Mauser, 2012).

Spindle positioning is important in many physiological contexts. At a fundamental level, spindle orientation determines the placement of the resulting daughter cells in the developing tissue, which is important for correct morphogenesis and tissue organization. In other contexts, such as asymmetric cell division, spindle position ensures proper segregation of fate determinants and subsequent differentiation of daughter cells. This study examined the function of a protein thought to provide a 'passive' mark on the cortex for subsequent recruitment of the spindle orientation machinery. During neuroblast asymmetric cell division, Insc has been thought to mark the cortex based on the location of the Par polarity complex (Mauser, 2012).

Ectopic expression of Insc in cells that normally do not express the protein has revealed that it is sufficient to induce cell divisions oriented perpendicular to the tissue layer, reminiscent of neuroblast divisions. Expression of the mammalian ortholog of Inscuteable, mInsc, in epidermal progenitors has shown that this phenotype is not completely penetrant over time. Expression of mInsc leads to a transient re-orientation of mitotic spindles, in which mInsc and NuMA initially co-localize at the apical cortex. After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another (Mauser, 2012).

This study examined the effect of Insc-Pins complex formation both in an induced polarity spindle orientation assay and in in vitro binding assays. The results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. The Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding (Mauser, 2012).

Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila (this work) and mammalian proteins (LGN-NuMA) suggests that Insc regulation of Mud-binding is a highly conserved behavior (Mauser, 2012).

This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins (Yu, 2000b). This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system (Mauser, 2012).

The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/Dynactin generates force to draw the centrosome towards the center of the cortical crescent. Fusion of the Pins TPR motifs, which recruit Mud, to Echinoid does not lead to spindle alignment, indicating that the Mud pathway is not sufficient for spindle alignment. The PinsLINKER domain does have partial activity on its own, however, and when placed in cis with the TPRs leads to full alignment. In this framework, the function of Insc may be temporal control, ensuring that microtubule attachment by the Dlg pathway occurs before the force generation pathway is activated (Mauser, 2012).

In the temporal model of Insc function, what might cause the transition from the Insc-Pins-Dlg complex, which mediates astral microtubule attachment, to the Mud-Pins-Dlg complex, which generates spindle pulling forces? By early prophase, Inscuteable recruits Pins and Gαi to the apical cortex. During this phase of the cell cycle, Mud is localized to the nucleus in high concentration. Apically-localized Pins binds Dlg, creating an apical target for astral microtubules. During early phases of mitosis, Inscuteable would serve to inhibit binding of low concentrations of cytoplasmic Mud to the Pins TPRs to prevent spurious activation of microtubule shortening pathways. After nuclear envelope breakdown, Mud enters the cytoplasm in greater concentrations and could then act to compete with Insc for binding to Pins, allowing Pins output to be directed into microtubule-shortening pathways (see Proposed model for Inscuteable regulation of spindle orientation). Future work will be directed towards testing additional aspects of this model (Mauser, 2012).

GENE STRUCTURE

Transcript size - 4.2 kb

PROTEIN STRUCTURE

Amino Acids - 859

Structural Domains

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).

mInscuteable regulates meiotic spindle organization during mouse oocyte meiotic maturation

Establishment of cellular polarity is one of the key events during oocyte maturation. Inscuteable (Insc) has been identified as a key regulator of cell polarity during asymmetric division in Drosophila. However, the function of its evolutionarily conserved mammalian homologue, mInscuteable (mInsc), in mouse meiotic maturation is not clear. This study investigated the roles of mInsc in mouse oocyte maturation. mInsc was detected at all stages of oocyte maturation. The protein level of mInsc was slightly higher at the germinal vesicle breakdown (GVBD) stage and remained constant during mouse oocyte maturation. The subcellular localization of mInsc overlapped with spindle microtubules. Disruption of microtubules and microfilaments caused changes in the localization of mInsc. Depletion or overexpression of mInsc significantly decreased the maturation rates of mouse oocytes. Depletion of mInsc significantly affected asymmetric division, spindle assembly, alignments of chromosomes and actin cap formation. Taken together, these results demonstrated that mInsc regulates meiotic spindle organization during mouse meiotic maturation (Xiao, 2019).

date revised:  25 April 2024

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.