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When the time is right: regulation of cell division in Drosophila

  • Author(s): Brose, Lotti Alicia
  • Advisor(s): Sullivan, William
  • et al.
Abstract

Accurate cell division is paramount to growth, development, and repair in all multi- cellular organisms. Unsurprisingly, cell division is highly regulated, with checkpoints in place following the major growth phase, DNA duplication, and spindle assembly. However, no checkpoint exists to determine whether or not the resulting daughters physically divide. It is on this process, cytokinesis, which serves as the focal point of my thesis.

Cytokinesis results in the physical separation of two daughter cells following mitosis. In animal cells, this process relies heavily on the contraction of the actomyosin ring at the ingressing furrow. Along with this mechanical pinching, cells rely on the addition of membrane components to the furrow to increase the membrane surface area of both daughters. New membrane is delivered via vesicles derived from both the secretory and endocytic pathways. Cytokinesis failure results in binucleate cells, which can lead to aneuploidy, a hallmark of cancer, or cell death in the following divisions.

Vesicle delivery must be highly regulated during cytokinesis. Because these vesicles function in other processes prior to mitosis, redirection and timing are everything. Our lab specifically focuses on the functional transition of the recycling pathway from interphase to mitosis in the early Drosophila embryo. During mitosis, the recycling endosome (RE) is positioned at the centrosomes and sends vesicles to the ingressing furrow. These vesicles contribute membrane, actin, and actin remodelers to the furrow, and mutants in major parts of this pathway result in incomplete ingression, aneuploidy, and failure of the embryo to develop. The primary focus of my thesis is the cell cycle regulation of RE-derived vesicle delivery to the ingressing furrow. Specifically, I examine the role of the conserved

Drosophila protein, Nuf, in regulating vesicle delivery during the early embryonic divisions and during the asymmetric stem cell divisions of the larval neuroblast.

Rab11 is a small RE-associated GTPase required for vesicle-mediated membrane delivery to the ingressing furrow. Nuf is a Rab11 effector that binds Rab11 GTP at the RE and is required to activate vesicle delivery. In the early embryo, without Nuf vesicle delivery is not initiated, leading to cytokinesis failure and embryonic lethality. However, Nuf localization to the centrosome must be regulated to occur only during mitosis. Indeed, we observe that Nuf localization is cell cycle-regulated, with its highest concentration at the centrosome occurring at prophase followed by loss of Nuf from the centrosome. Stimulation of the embryo by cyclin-B injection to jumpstart mitosis similarly displaces Nuf from the centrosome. We find that this localization correlates with high phosphorylation levels of Nuf shortly following its cytoplasmic dispersal. This is achieved through phosphorylation of Ser225 and Thr227 specifically by the cell cycle kinase Polo. In vivo, inhibition of Polo activity results in a loss of phosphorylated Nuf as well as Nuf maintenance at the centrosome during metaphase, when it should have already dispersed to the cytoplasm. Constitutive activation of Polo showed an opposing effect, with less Nuf at the centrosome even during prophase, though these results were not significant.

As we probed further into the importance of Nuf phosphorylation, we generated non- phosphorylatable and phosphomimetic Nuf mutants to determine whether these two residues alone are responsible for the localization defects observed in the different polo backgrounds. It is important to parse these differences, as Polo plays several roles during the cell cycle that may directly influence Nuf localization. While non-phospho Nuf did not have an observable effect on Nuf localization, phosphomimetic Nuf was not maintained at the centrosome at any phase of mitosis. Further, both mutants exhibited furrowing defects in the form of actin gaps and ectopic actin. These results strongly suggest phosphorylation of these residues specifically plays a key role in Nuf localization and activity during cytokinesis.

Nuf localization to the centrosome is driven by the minus-end directed motor, Dynein/Dynactin and dependent on microtubules. Indeed inhibition of microtubule polymerization or Dynein heavy chain expression prevents Nuf accumulation at the

centrosome regardless of mitotic phase. Cytoplasmic Nuf binds Dynein/Dynactin and is transported along the microtubules to the centrosome-associated RE. Nuf is continually dispersing from the RE into the cytoplasm, requiring constant recruitment via Dynein/Dynactin until metaphase. We hypothesize that phosphorylation of Nuf at these residues disrupts the Nuf/Dynein binding site. These residues lie outside of the Rab11- binding domain, suggesting another mechanism for Nuf loss from the centrosome when it is phosphorylated. Nuf has been shown to bind both Dynein heavy chain as well as a component of the dynactin complex, Arp1. In a dosage-sensitive interaction between nuf and Dynein/Dynactin components, flies heterozygous for nuf and dynactin 2 are semi-sterile, suggesting a strong interaction. Further work is required to determine whether these mutant residues are capable of binding Dynein/Dynactin directly.

In addition to its role in cytokinesis regulation in the early embryo, we examined a potential role for Nuf in regulating division asymmetry later in development. Division asymmetry is controlled by many factors intrinsic and extrinsic to the cell. Many early studies demonstrate the importance of proper segregation of small RNA and protein determinants to specific regions of the cell cortex. Additionally, the centrosome has been shown to influence division asymmetry in several cell types and organism. Much like the cortical determinants, inheritance of the mother or daughter centrosome is specified to the daughter stem cell faithfully in each division. Recently, we learned that exogenous formation of an active RE in Drosophila sensory organ precursor (SOP) cells increases cell fate transformation. Using our developed tools for RE examination and manipulation, we sought to determine how Nuf expression affects the asymmetric divisions of the Drosophila larval neuroblast (NB) and pupal SOP cells.

The Drosophila larval neuroblast is a neural stem cell that produces a population of differentiated neurons that comprise much of the adult central nervous system. It divides asymmetrically into a self-renewing daughter neuroblast and a differentiating ganglion mother cell (GMC). In this division, the daughter centrosome is preferentially segregated to the self-renewing daughter NB. We find that Nuf is also preferentially segregated to the daughter NB following division. Expression of our Nuf phospho-mutants maintains this preference, in which both non-phospho and phosphomimetic Nuf are more frequently inherited by the daughter NB. Interestingly, when Nuf is symmetrically distributed following division, non-phospho Nuf is frequently lost from both daughters following division, while phosphomimetic Nuf is maintained in both daughters. We hypothesize that phosphorylation of Nuf may protect it from degradation during division. Polo, a known kinase of Nuf, is repressed in the daughter GMC, while its activity is enhanced in the daughter NB. This provides a model in which Polo phosphorylates Nuf in the daughter NB, while any Nuf remaining in the daughter GMC is targeted for degradation.

Drosophila SOP cells divide asymmetrically to expand the peripheral nervous system including the bristles, socket cells, sensory neurons, and sheath cells. Following the first division, only one of the daughter cells (pIIb) forms an active RE. Overexpression of both Rab11 and Nuf result in RE formation in both pIIb and pIIa daughters, and this leads to cell fate transformations toward the epithelial fate that manifest as bristle duplications. We find that expression of phosphomimetic Nuf, but not non-phospho nor GFP Nuf, in SOP cells results in statistically significant bristle duplications. We hypothesize that phosphorylation of Nuf stabilizes the RE in SOP cells, resulting in RE formation in pIIb and pIIa, driving a preference for epithelial fate.

Altogether, this thesis explores the role of the RE-associated protein Nuf in the regulation of RE throughout development in Drosophila. Nuf influences and directly regulates cytokinesis in the early embryo via RE-derived vesicle delivery to the ingressing furrow. Nuf phosphorylation influences its subcellular localization and stabilization in the embryo, larval neuroblast, and sensory organ precursor cells.

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