Asymmetric Cell Division in the Drosophila Neuroblast Asymmetric cell division is the process by which certain cells divide, producing unevenness between the two daughter cells. This unevenness can take the form of a difference in size, a difference in the distribution of cellular components, or a combination of both. Examples of this type of division are seen throughout nature. Budding yeast divide asymmetrically by the creation of daughter buds that pinch off the mother and are noticeably smaller. It can be seen in plant cells, whereby a mother epithelial cell divides asymmetrically to reproduce itself and a smaller guard mother cell which in turn gives rise to two guard cells lining the stomata of the leaves. The nematode C. elegans uses the process of asymmetric cell division as a basis for mapping out its body plan in the very first division of the embryo. However, the most interesting model organism where asymmetric cell division has been studied is in Drosophila melanogaster. In the Drosophila embryo, there are a number of symmetrical divisions which give rise to an epithelial layer. Within this epithelium, some cells are given cues to assume a neural fate and delaminate from the epithelial layer. These cells are the progenitor cells of the neurons which will eventually become the nervous system of the larval Drosophila. These neuroblast cells then divide asymmetrically to produce another neuroblast cell as well as a much smaller ganglionic mother cell (GMC). Finally, the GMC then divides asymmetrically to produce two neurons with different allocations of cellular components, but of similar size. The pathway involved in the process of division from neuroblast mother cell to neuroblast and GMC daughters to neurons is a complex process involving many different protein pathways. Not only are the two daughter cells of different size, but the proteins contained within them are drastically different as well. The discovery and understanding of how these pathways work to produce this asymmetric division has been a long process and its entirety still continues to elude the scientific community. Starting from the discovery of what are called fate determinants, to understanding the process of spindle orientation, to the recent realization that cell cycle regulators play an additional role in affecting asymmetric cell division, the veil cast over a beautiful and intricate mechanism has been lifted and only now are we beginning to see how everything fits together. To begin this journey of understanding how the asymmetric division of the neuroblast is achieved, first there must be a general overview of what happens in this process. As stated before, some epithelial cells in the drosophila embryo are given cues governed by gene signalling to assume a neuronal fate (White and Kankel 1978). These cells then lose their adherens junctions and delaminate from the epithelium and are then called neuroblasts. Neuroblasts then undergo a process by which certain proteins, called fate determinants, are segregated to opposite sides of the cell. While this is occurring the spindle, which is responsible for separating the duplicated chromosomes, rotates 90 degrees (Kaltschmidt et al. 2000). This rotation causes the cell to divide in along the plane which separates the apical region from the basal region. This is perpendicular to the epithelial cells from which it delaminated, that divide in a posterior/anterior manner. The spindle midbody then moves basally where the cleavage furrow is forming, resulting in a daughter cell which retains its neuroblast identity and a smaller daughter cell which takes of the identity of a ganglionic mother cell (Bingwei et al. 1998). This identity change of the GMC is due to certain proteins which were preferentially allocated to the GMC daughter during the division of the neuroblast mother cell. These fate determinants govern the process by which the GMC loses its self-renewing properties and divides only once more to produce two neurons (Doe et al 1991). The first fate determinant was called Numb, which was originally discovered in sensory organ precursor (SOP) cells, and was shown there to confer different fates to the daughter cells of the SOP (Rhyu et. al 1994). Numb is a signalling protein containing an N-terminal phosphotyrosine binding domain which has been found to interact with a signal transduction pathway known as the Notch pathway. Specifically numb acts as a linking protein between an α-adaptin protein and the Notch protein which facilitates the endocytosis and repression of Notch (Knoblich et al 1997). This pathway in the GMC is thought to be responsible for the determination of the position of the axon in the developing neuron cells. It has been seen that since the Numb protein maintains its position in the apical cortex of the GMC, it becomes preferentially segregated to one of the neuron daughter cells. This means that only one neuron will have a repression of the Notch pathway, and that repression is thought to direct the axon growth on the posterior side of the cell. Opposite this, the other cell will have a fully functioning Notch pathway and the axon growth will occur on the anterior side (Bingwei et al. 1998). The second fate determinant is named Prospero. This protein is a transcription factor, and after segregation to the basal cortex of the neuroblast mother cell, and subsequent transfer into the GMC, it enters the nucleus of the GMC and is responsible for activating and repressing certain genes which specify the fate of the GMC (Doe et al. 1991). Prospero contains a homeodomain and binds to the DNA upstream of approximately 700 genes and is known to repress genes that are involved in maintaining self renewal capabilities as well as upregulate genes involved in halting the cell cycle and directing division into the two neuron daughter cells. This homeodomain consists of two regions, a highly divergent homeodomain adjacent to a conserved novel Prospero domain. The Prospero domain region is involved directly with the binding to DNA (Ryter et al. 2002). Interestingly, not only is Prospero preferentially segregated into the GMC, but an RNA transcript of Prospero is segregated as well. This transcript is brought to the GMC from the neuroblast by a protein named Staufen (Ferrandon et al. 1994). Staufen is a double stranded RNA binding protein that was discovered in the Drosophila oocyte, where it plays a role in the localization of RNA transcripts to both the anterior and posterior poles of the oocyte (Li et al. 1997). The double-stranded binding domain found in Staufen is a fairly common one consisting of approximate 65 amino acids and is conserved from bacteria to humans. Staufen contains 5 copies of this motif and it has been shown that the 3rd copy binds to double-stranded RNA in vitro (Bycroft et al. 1995). In the neuroblast, Staufen binds ProsperoRNA and localizes it to the basal cortex. After division Staufen is known to release ProsperoRNA into the cytoplasm of the GMC. The apparent reason for this transfer of an RNA transcript into the GMC is because the GMC is not known to transcribe Prospero itself. This is in addition to the fact that the Prospero mRNA transcript is a very long transcription unit that takes approximately 20 minutes to transcribe from the DNA. The current view is that the mRNA transcript was delivered to the GMC at cell division in order to avoid delays in the next cell division by having to wait until more Prospero RNA was transcribed (Li et al. 1997). The third and latest fate determinant to be discovered goes by the name of Brain Tumour (Brat). Brat is a member of a family of tumour suppressor proteins that has been evolutionarily conserved (Betschinger et al. 2006). This protein consists of a 1037 amino acid chain containing two Bbox zinc finger domains, a coiled coil domain and a C-terminal beta-propeller domain, all of which are known to mediate protein-protein interactions (Slack and Ruvkun 1998). Although currently the exact mechanism by which Brat operates is unknown, the fact that it plays a role in tumour suppression (Betschinger et al. 2006) suggests that Brat works in conjunction with Prospero to inhibit cell growth, cause the loss of self-renewal function in the GMC, and induce the GMC to undergo terminal differentiation resulting in two neurons. These fate determinant proteins do not localize themselves to the basal cortex and segregate into the GMC at cell division. There are two proteins that act as scaffolds to bind and move the determinants to the basal pole of the neuroblast and ensure they end up in the GMC. The first of these proteins to be discovered was Miranda, and consequently much is known about this protein. Miranda is an adaptor protein that contains a coiled coil region known to bind the proteins Prospero, Staufen, and Brat (Shen et al. 1997, Ikeshima-Kataoka et al. 1997, Lee et al. 2006). In the neuroblast cell, Miranda initially is localized at the apical cortex then moves to the basal cortex where it tethers its cargo to the cortex and awaits cell division (Matsuzaki et al. 1998). Once division occurs, Miranda then dissociates and releases its protein cargo into the cytoplasm (Schuldt et al. 1998). Miranda first accumulates in the cytoplasm during interphase, then it is recruited to the apical cortex by a protein named Inscuteable, which will be discussed later, where it forms an apical crescent (Kraut et al. 1996). At this point a number of things occur which causes Miranda to be excluded from the apical cortex and redistributed into the cytoplasm. First, atypical Protein Kinase C (aPKC) phsophorylates a protein named Lethal (2) giant larvae (Lgl) (Lee et. al 2006). This phosphorylation causes the inactivation of Lgl, which in turn allows for the activation of Myosin II. Myosin II is responsible for the exclusion of Miranda from the apical cortex (Erben et al. 2008). Once Miranda is localized in the cytoplasm, another protein, Myosin VI, interacts with Miranda and helps transport it to the basal cortex. It is not known whether Myosin VI then acts as an anchor to tether Miranda to the cortex or if there is another anchor protein involved (Erben et al. 2008). After cell division, Miranda is now tethered to the apical region of the GMC. It has been discovered that the Miranda protein contains a number of consensus sequences that allow it to be targeted for destruction (Fuerstenburg et al. 1998). These destruction boxes show similarity to the destruction boxes that exist in the cell regulators cyclins A and B. The theory is that Miranda is degraded by ubiquitination, causing the release of Staufen, Prospero and Brat into the cytoplasm of the GMC (Schuldt et al. 1998). The fate determinant Numb is localized and tethered to the cortex by a different adaptor protein by the name of PON (Partner of Numb). PON is also a coiled-coil protein which binds only Numb, and although it helps in localizing Numb to the basal cortex, it has been shown that it is not strictly required for the localization (Lu et al. 1998). Experiments have uncovered that the mechanisms responsible for the asymmetrical localization acts on Numb as well as PON (Wang et al. 2007), although recently it was discovered that Myosin II plays a major role in localizing PON, and therefore Numb to the basal cortex (Erben et al. 2008). It should be noted that after cell division, PON doesn’t release Numb from the cell cortex. Numb remains there and it is there it acts to repress the Notch pathway. This tethering to the cell cortex of the GMC is also responsible for its preferential segregation into only one of the neuron daughter cells (Lu et al. 1998). In the neuroblast it is of grave importance that polarity between the apical and basal sides of the cell be set up and maintained. The proteins responsible for this were originally discovered in C. Elegans, and their homologues were then found in Drosophila (Knoblich 2008). These proteins are named Bazooka, Par-6 and aPKC, mentioned earlier. Bazooka is a large protein containing three PDZ domains, required for anchoring proteins to the cytoskeleton of the cell (Schober et al. 1999). Bazooka protein is found in the apical cortex of all epithelial cells in the drosophila embryo, where it helps maintain apical/basal polarity. Because epithelial cells divide laterally, Bazooka is distributed equally between the two epithelial daughter cells. This apical localization of Bazooka is maintained when some cells assume a neuronal fate and delaminate to become neuroblasts. In the neuroblast, Bazooka is responsible for recruiting the protein Inscuteable, which has ties to other protein localization as well as spindle orientation and will be described later (Wodarz et al. 1999). Par-6 is a smaller protein containing only one PDZ domain and shares a similar role as Bazooka, although it is not responsible for recruiting Inscuteable. Par-6 does however interact with the cell cycle regulator cdc42, which is responsible for its apical localization to the cell cortex (Atwood et al. 2007). Par-6 also interacts with aPKC causing it to be localized to the apical cortex as well. These three proteins are the main players in providing localization information for the processes involved in asymmetric cell division. Although it has be said that how cell fate determinants are localized asymmetrically remains the greatest mystery of asymmetric cell division, recent studies have begun to uncover the mechanisms that lay behind this (Kaltschmidt et al. 2000). Previously it has been shown how Myosin II and VI interact with Miranda and Numb to transport the proteins to the basal cortex of the neuroblast, although this seems only to be the tip of the iceberg. The protein Lgl, as well as aPKC has been shown to play a part in this localization process by interacting with each other in a negative way (Lee et al. 2006). Because Lgl is normally found throughout the cytoplasm and is directly inactivated by aPKC which phosphorylates three serines on Lgl thereby preventing it from binding to the actin cytoskeleton (Betschinger et al. 2005), Lgl is only found in an active form at the basal cortex. This dance between Lgl activation/inactivation by aPKC was thought to be directly involved in the asymmetric localization of fate determinants until it was found the Lgl does not bind to any of these proteins (Lee et al. 2006). The current theory is that Lgl and aPKC indirectly localize fate determinants by interacting with the cortical binding sites of the determinants with aPKC inhibiting the sites, or Lgl activating them (Chia et al. 2008). The final process necessary for a neuroblast to undergo asymmetric cell division is the coordination of the orientation of the spindle with the asymmetric localization of the fate determinants during mitosis. In neuroblast cells, as previously stated, the spindle rotates 90 degrees, and the midbody moves toward the basal cortex of the cell so that when division occurs, not only does the cell divide perpendicular to the epithelial cells, but the division results in a neuroblast daughter cell that is 75% larger than the GMC daughter cell on average (Morgan 2007). The main protein responsible for the correct orientation of the spindle is named Inscuteable. Inscuteable is a novel protein, 859 amino acids in length containing a 252 amino acid domain named the asymmetry domain and is partly responsible for directing the orientation of the spindle (Kraut et al. 1996, Schober et al. 1999). Inscuteable is recruited to the apical cortex by Bazooka through an interaction in a 100 amino acid coiled coil domain, where it forms a complex with a number of other proteins, namely Pins (Partner of Inscuteable), Discs large (Dlg), Mushroom body defect (Mud), Canoe, and a heterotrimeric G protein subunit, Gαi (Schaefer et al. 2000, Cai et al. 2003, Speicher et al. 2008). Inscuteable recruits Pins to the apical cortex where it binds the protein. Pins then binds Gαi by way of three GoLoco domains that exist of the C-terminal domain of Pins (Nipper et al. 2007). This binding not only tethers Pins to the cortical membrane, but also causes Pins to undergo a conformational change and allows it to bind the protein Mud. Mud is a microtubule binding protein that is thought to act as a dock for astral microtubules, which makes it partly responsible for the orientation of the spindle poles (Siller et al. 2006). Recently it was found that the protein Canoe, which is known to be a scaffolding protein containing kinesin-like and myosin V-like domains, also forms in a complex with Inscuteable-Pins- Gαi, and that it acts upstream of Mud, helping with the orientation of the spindle (Speicher et al. 2008). Although the precise mechanisms for the orientation of the spindle are unclear, it is known that the mechanisms are dependent on where the centrosome is located in the cell (Kaltschmidt et al. 2000). Before replication, the location of the centrosome dictates the direction of the orientation of the spindle. If the duplication occurs in the apical region of the cell, the posterior centrosome moves basally in a counter-clockwise manner. However, if the duplication occurs in the basal region, the anterior centrosome moves apically in a clockwise fashion (Kaltschmidt et al. 2000). This method of spindle orientation and movement is unique to the drosophila neuroblast. Normally in asymmetric cell division, the spindle poles are formed and one pole basically maintains its position, moving slightly back and forth, whereas the other pole moves away from the middle of the cell towards the outer cortex (Morgan 2007). This causes the midbody to move from the center of the cell and the creation of a cleavage furrow is formed at the location of the midbody leading to an asymmetrical division. This type of division can be seen in the C. Elegans embryo and results in one daughter cell being two thirds larger than the other. In drosophila, it is interesting to see how the spindle is reoriented and how the astral microtubules, which attach to the chromosomes, undergo differences in growth and reduction. The apical microtubules lengthen dramatically, while the basal ones shorten. This results in the movement of the spindle midbody towards the basal region of the cell (Morgan 2007). In most cases of cellular division, it has been shown that the spindle midbody dictates the position of the cleavage furrow and subsequent cytokinesis (Cai et al. 2003). In the neuroblast however, a very unique event occurs. As the midbody moves towards the basal region of the cell, the cleavage furrow has already started to invaginate (Kaltschmidt et al. 2000). This discovery has only been seen in the case of the neuroblast and goes contrary to the popular belief that the midbody dictates the site of cleavage. Apart from the unique proteins that facilitate asymmetric division, the direction a lot of research has turned to recently is the involvement of cell cycle regulators and kinases and their role in this process. It is known that the kinase Aurora A functions in the process of centrosome maturation and spindle formation when activated by TPX2 (Morgan 2007), however it was discovered that Aurora A has an inhibitory function on the cell’s ability to self renew (Berdnik and Knoblich 2002). This was conjoined with the discovery that Aurora A also functions in the localization of Numb to the basal cell cortex, although the mechanism by which it does this is not certain (Barros et al. 2005). Another cell cycle regulator, Polo, has also been shown to play a role in the localization of Numb. The experiments that determined these separate functions of the cell cycle regulators were brought about by monitoring cells with phenotypes that involve a mutation in the Aurora A, and Polo transcribing genes (Wang et al. 2007). As the neuroblast cell attempted to divide asymmetrically, it was seen that Numb was either distributed throughout the cytoplasm, or that a crescent of the protein was formed, just not at the basal cortex where it normally would be found. The G1 cell cycle regulator CyclinE was found to play a key role in the determination of neuroblast cell fate this year. Previously it had been discovered that Prospero represses the expression of CyclinE (Choksi et al. 2006), but this discovery brought to light that CyclinE also inhibits the function of Prospero. This negative feedback loop has been deemed essential in the decision of the neuroblast to continue to divide, or enter into differentiation into the GMC, and subsequent neuronal cells. CyclinE was found to inhibit Prospero function by affecting its localization to the cell cortex by potentially phosphorylating the protein and causing a disruption in its ability to bind to Miranda (Berger et al. 2010). This function of CyclinE was also found to be independent of its role in cell cycle regulation. The act of asymmetrical cell division has turned out to be a very complex and intricate cascade of proteins that interact with each other in order to facilitate the neuroblast cell cycle. Starting from the delamination of the neuroblast from the cortical epithelia of the neuroectoderm, the protein Bazooka interacts with aPKC and recruits the protein Inscuteable and PAR6 to form a complex that not only acts in directing the localization of the cell fate determinants Miranda and PON and maintaining the overall polarity of the cell, but also interacts with the complex formed by Pins, Gαi, Dlg, Canoe, and Mud in directing the proper spindle orientation and facilitate the lengthening/shortening of the astral microtubules of the spindle. Miranda acts in the localization of the cell fate determinants Prospero and Brat by acting as a transporter protein to tether the fate determinants to the basal cortex of the cell. Miranda also binds Staufen, which in turn binds the Prospero mRNA transcript as well. Numb is localized to the basal cell cortex by the transport protein PON, which along with Miranda utilize the proteins Myosin II and VI to transport their respective protein cargo to the cortex for segregation into the GMC daughter cell. In addition to this, it has recently been discovered that the regular cell cycle kinases Polo and Aurora A assist in the localization of Numb to the basal cortex in addition to their regular known function. As well, the cell cycle regulator, CyclinE is suggested to inhibit the function of Prospero which allows for the neuroblast cell to maintain its self-renewal capabilities. 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