Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry.
TL;DR: It is proposed that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.
Abstract: Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.
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TL;DR: The centrosome is a unique organelle: the semi-conservative nature of its duplication generates an inherent asymmetry between'mother' and 'daughter' centrosomes, which differ in their age as discussed by the authors.
Abstract: The centrosome is a unique organelle: the semi-conservative nature of its duplication generates an inherent asymmetry between 'mother' and 'daughter' centrosomes, which differ in their age. This asymmetry has captivated many cell biologists, but its meaning has remained enigmatic. In the last two decades, many stem cell types have been shown to display stereotypical inheritance of either the mother or daughter centrosome. These observations have led to speculation that the mother and daughter centrosomes bear distinct information, contributing to differential cell fates during asymmetric cell divisions. This review summarizes recent progress and discusses how centrosome asymmetry may promote asymmetric fates during stem cell divisions.
17 citations
TL;DR: The role of PLK-1 in cell polarization and asymmetric division of C. elegans embryos has been investigated in this article, showing that it is essential for the entry into and progression through mitosis.
Abstract: PLK1 is a conserved mitotic kinase that is essential for the entry into and progression through mitosis. In addition to its canonical mitotic functions, recent studies have characterized a critical role for PLK-1 in regulating the polarization and asymmetric division of the one-cell C. elegans embryo. Prior to cell division, PLK-1 regulates both the polarization of the PAR proteins at the cell cortex and the segregation of cell fate determinants in the cytoplasm. Following cell division, PLK-1 is preferentially inherited to one daughter cell where it acts to regulate the timing of centrosome separation and cell division. PLK1 also regulates cell polarity in asymmetrically dividing Drosophila neuroblasts and during mammalian planar cell polarity, suggesting it may act broadly to connect cell polarity and cell cycle mechanisms.
5 citations
TL;DR: In this article, the authors highlight the recent use and future potential of optogenetic approaches to study homeostasis, and apply them to Drosophila melanogaster.
Abstract: Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.
4 citations
TL;DR: The latest advances in this field are reviewed with special emphasis on the complex structure-function relationship of centrosomes with regards to ACD and on mechanistic insight derived from cell types that divide symmetrically but is likely to be relevant in ACD.
4 citations
11 Aug 2021
TL;DR: Most cells in the body go through a cycle of life in which their genetic information is retained, fixed, and passed down to daughter cells through a highly coordinated and regulated process, but during this cell cycle, there are many situations where mistakes are made by the cycle or by a regulating system that causes the cell to proliferate uncontrollably, leading to cancer.
Abstract: Most cells in the body go through a cycle of life in which their genetic information is retained, fixed, and passed down to daughter cells through a highly coordinated and regulated process. However, during this cell cycle, there are many situations where mistakes are made by the cycle or by a regulating system that causes the cell to proliferate uncontrollably, leading to cancer. Somatic cells are the best know for this cell cycle and go through 2 main phases called interphase and mitosis.During interphase, some subdivisions are important to cell division and maintenance of the genetic material. The subdivisions consist of G1, S, and G2. The cells may also enter into a 4th phase known as G0 when the cell is destined to die and no longer divide.
3 citations
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TL;DR: A bacterial CRISPR RNA/Cas9 system is adapted to precisely engineer the Drosophila genome and it is reported that Cas9-mediated genomic modifications are efficiently transmitted through the germline.
Abstract: We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
1,067 citations
"Dynamic centriolar localization of ..." refers methods in this paper
...were then cloned into pU6-BbsI-ChiRNA [50] between BbsI sites....
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TL;DR: Recent advances in understanding of the function and biogenesis of centriole organelles are reviewed, and their connection to human disease is emphasized.
822 citations
TL;DR: Immunofluorescence studies with an antibody that recognizes an antigen associated with the centrosome indicate that the organization of this organelle is disrupted in the mutant embryos, and these defects appear to lead to the production of polyploid cells.
Abstract: Neuroblast cells in larvae homozygous for mutant alleles of the locus polo show a high frequency of metaphases in which the chromosomes have a circular arrangement, and anaphase figures in which chromosomes appear to be randomly oriented with respect to at least one of the spindle poles. These defects appear to lead to the production of polyploid cells. Sex chromosome disjunction is affected in male meiosis, primarily in the second division, and the meiotic spindles of living cells are abnormal. One allele is a larval lethal, whereas another is semi-lethal with about 7% of homozygotes surviving as adults. Embryos from homozygous polo females have aberrant mitotic spindles that are highly branched and have broad poles. Immunofluorescence studies with an antibody that recognizes an antigen associated with the centrosome indicate that the organization of this organelle is disrupted in the mutant embryos.
577 citations
Additional excerpts
...RFP-}ZH-2A, w(1118), DNAlig4(169) (BDSC #58492), worGal4, UAS-mCherry::Jupiter [16], cnb e00267 [19], Df(3L) ED4284 (cnb deficiency; BDSC #8056), polo(1) [48], polo(16-1) [49], pUASp-YFP::Cnb::PACT [19]....
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TL;DR: A simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila, which demonstrates rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described.
Abstract: We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.
567 citations
TL;DR: Using transposon mutagenesis in Drosophila, a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, is constructed to facilitate gene expression mapping at single-cell resolution and finds that 600–900 different genes are trapped in the collection.
Abstract: Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.
567 citations
"Dynamic centriolar localization of ..." refers background or methods in this paper
...Fly strains, trans-genes and fluorescent markers The following fly strains were used: Cnb RNAi (VDRC, 28651GD), wdr62Δ 3–9 allele [20], Df (2L)Exel8005 (a deficiency removing the entire wdr62 locus and adjacent genes; BDSC #7779), worniu-Gal4 [45], pUbq-DSas-6::GFP [46], Cnn::GFP, Polo::GFPCC01326 (protein trap line) [34], GFP::Polo (genomic rescue construct using Polo’s endogenous enhancer) [40], pUbq-Asl::GFP [47], pUbq-YFP::Cnb [8], YFP::CnbT4A,T9A,S82A [8], nos-Cas9/Cyo (BDSC #78781), y1, w67c23, P {y[+mDint2] = Crey}1b; D/TM3, Sb1 (BDSC #851), y1,M{Act5C-Cas9....
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...Representative 3D-SIM images of (A) apical or (B) basal third instar larval neuroblast centrioles, expressing Polo::GFP (protein trap line [34]; middle row; green in merge)....
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...Taken together, we conclude that Polo-dependent phosphorylation of Cnb is necessary for the establishment of molecularly distinct centrioles during mitosis, impacting subsequent molecular interphase asymmetry. https://doi.org/10.1371/journal.pbio.3000762.g003 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000762 August 6, 2020 8 / 29 Polo becomes enriched on the daughter centriole whereas Plp remains localized on the mother centriole Having implicated Polo in Cnb’s localization dynamics, we then analyzed the localization of Polo (Polo::GFP) and Plp (Plp::EGFP; generated by CRISPR/Cas9; see Methods)....
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...Fisher’s exact test: Polo::GFP [34] control versus cnb RNAi: p = 1....
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...Representative interpolated images of apical interphase or early prophase and late metaphase or early anaphase centrosomes, expressing (D) Polo::GFP (protein trap line [34]; green in merge) or (E) YFP::Cnb (green in merge) and stained for Plp (magenta in merge)....
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