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17. Briefly describe the centriole/centrosome cycle in animal cells. Be sure your answer includes explanation of the distinction between the mother and daughter centrioles.
- Centrioles: pair of these is found at the center of the centrosome in animal cells.
- Centrosome: composed of both a mother and daughter centriole, with the mother centriole containing distal and sub-distal appendages containing ninein. The daughter centriole lacks the appendages.
- PCM: space surrounding centrioles
the site of centrioles maturation. Gamma-TuRC is a major component of the PCM, which aids in assembly of MTs by acting as a template for assembly (gamma TuRC includes many proteins as well as ?-tubulin) as cell enters mitosis.
- During S phase: Both the mother and daughter centrioles undergo replication. The mother centriole becomes the old mother and the daughter centriole becomes the new mother. With each mother centriole and new daughter centriole forms. Cdk2-cyclin and tubulins are involved here to aid in this replication.
- During G2 phase: Mother and daughter fully replicated. The centrioles now move to opposite poles of the cell during prophase, and the original daughter of new mother centrosome gains the APPENDAGES.
- OLD mother centriole distinguished from the centrosome with the NEW mother centriole: staining with ODF2/cenexin. The centrosome with the OLD mother centriole will stain BRIGHTER vs. the NEW.
18. What is asymmetric cell division and where, that is in what cell types, does it most commonly occur? Why is it important?
- Asymmetric cell division is an unequal division of committed stem cells. The asymmetry can be either functional (in that the cells do different things) or structural (size, shape, orientation of cell components). It is seen most often in committed stem cell lines were one cell of the division will remain a stem cell and the other cell will begin to differentiate.
- In neural stem cells: one cell remains a stem cell and the other enters a neuronal developmental pathway. Asymmetric division also is used in the establishment of tissue organization: by allowing for the MAINTENANCE of the stem cell line while also producing DIFFERENTIATING cells that will differentiate into specialized tissue cells.
19. The papers covered in the lecture on asymmetric cell division followed the behavior of mother versus daughter centrosomes in various cell types. For the following proteins indicate which are specifically associated with the mother centrosome/centriole and which associate only/primarily with the daughter. If some associate with both equally indicate that as well. Where known, also indicate the function(s) of the proteins from this list.
- a. a. NuMA (Nuclear mitotic apparatus protein): Fxn in interphase in nucleus, mitosis in spindle poles and at cortex, especially in late metaphase-telophase. (In ACD, the daughter centrosome with NEW mother centriole is inherited to the daughter cell with NuMA preferentially localized to the cell cortex, whereas the daughter centrosome with the NEW mother centriole migrates to the other daughter cell. Izumi and Kaneko et.al. During SCD, the daughter centrosome with the old mother centriole migrates to the daughter cell with NuMA localized to the cell cortex.)
- b. ?-tubulin: primary component of PCM, and is centrosome specific. Aids in helping to form template of MTs, along with gamma-TuRC.
- c. ODF2/cenexin : OLD MOTHER (mostly) centrosome marker, protein acquired by new mother during maturation (leading up to the G2/M transition), but amount of staining GREATER for the old mother relative to the new mother centriole.
- d. Centrobin: DAUGHTER cell marker. Necessary for daughter centrioles to keep PCM and MTOC activity during Interphase in NB cells. Phosphorylation required of CNB to work. Also controls mother-daughter centriole Asymmetry in Drosophila neuroblasts (Januschke et al. 2013)
- e. Ninein: MOTHER centiole marker. Fxn in positioning and anchoring microtubules minus ends in epithelial cells. It concentrates at the appendages surrounding the mother centrioles and the microtubule minus ends.
- f. Centrin 1: MOTHER and DAUGHTER associated, each centriole contains Centrin protein. Required for DUPLICATION of centrioles. This component of centriole, found in centrosome of eukaryotes
20. What is/was the significance of amplification of the MYCN gene discussed in the Izumi & Kaneko paper? What cell/tissue type did they study in this paper? Did amplification of MYCN affect cell division? If yes, in what way did it affect cell division?
- MYCN is known to play role in self-renewal of normal neural stem cells and precursor cells. When neural stem cells divide asymmetrically (ACD) one cell retains the stem cell identity and the second enters a neuronal developmental pathway. Neural stem cells can also divide symmetrically (SCD) to produce two stem cells
- Neuroblastomas (aggressive forms): correlate with amplification of the MYCN oncogene (segmental amplification).
- Note: amplification of MYCN does not appear to cause neuroblastoma..
- Paper focus: does Neuroblastoma patterns of SCD and ACD correlate with MYCN gene AMPLIFICATION?
- Cells studied: human neuroblastoma cells, control used were HeLa
- MYCN results: ACD DOES occur in human neuroblastoma cells with a normal MYCN copy, but NO ACD with MYCN AMPLIFICATION.
- MYCN protein knockout: in cells with MYCN amplification, ACD restored.
- MYCN protein overexpression: REPRESSES ACD
- Frequency of ACD (normal cell division) vs. SCD (stem cell expansion) varies between NB cell lines and correlates with amplification of the MYCN gene and levels of MYCN protein.
- Pattern of inheritance affected: In ACD cells, opposite site inheritance of ODF2 and NuMA observed.
- Conclusion: MYCN protein regulates genes that affect symmetry of SPINDLE and of cell division, but that gene unknown.
21. What was the primary question being addressed in the Habib et. al. (2013) paper, what methods did they use and what were their primary conclusions?
- Primary question: Consequences of local Wnt signaling on pluripotency gene expression, using various ES reporter cells.
- Wnts: Developmental signals which are often presented to cells in an oriented manner.
- Methods: Both Wnt3 and Wnt5 proteins were chemically immobilized to beads and their biological activity was confirmed. Then, beads were added to the Embryonic Stem Cells in culture.
- Wnt3: Involved in stem cell renewal, acts through ß-catenin pathway.
- Wnt5: (neg control) not associated with stem cells and does not act via ß-catenin.
- Cells with single bead attached were observed by live microscopy and antibody staining as they divided for redistribution of:
- ?-catenin: via labeling APC (component of B-catenin destruction) and Lrp6 (Wnt receptor)
- Inheritance of old (mother) vs. new (daughter) centrosomes: labeled Ninein and Centrin 1- centriole components (to find old mother centriole)
- Orientation of the spindle apparatus
- Wnt3a setting up for cells to differentiate: using Claudin 6 and H3K27me3 as markers of epiblast stem cell fate.
- Wnt3a beads induce asymmetric distribution of components of the Wnt/?-catenin pathway before, during and after cell division. The asymmetric distribution of these Wnt components was maintained after the cells divided. The daughter cell in proximity to the Wnt3a bead preserved high amounts of LRP6 and APC, in contrast to the lower amounts in the distal cell.
- Wnt pathway components can interact with astral microtubules and other components of the mitotic spindle, including centrosomes.
- Investigation of the effect of Wnt beads on the asymmetric inheritance of the centrosomes: By the end of division, centrosomes in 78% of cells (n=18) that were attached to Wnt3a beads had a high abundance of Ninein, whereas the segregation of Ninein was almost random in the presence of the Wnt5a beads (54%, n=15).
- High expression levels of pluripotency genes are observed in the Wnt3a proximal cells, but not in control cells.
- Distal cells express markers of differentiation towards Epiblast stem cell fate (express EpiSCs markers Claudin 6 and H3K27me3)
- Some component of the centriole/centrosome causes the mother centrosome to orient towards a localized source of Wnt3a, setting up ACD in embryonic stem (ES) cells.
- By orienting cell division, the Wnt signal positions the distal daughter cell out of its signaling range, leading to differentiation in that cell.
- Habib et al. developed a system, using the single cells exposed to Wnt signal, that allows for precise real time examination of processes involved in asymmetric cell divisions.
22. Compare and contrast the inheritance of the mother vs. daughter centrosomes during asymmetric cell divisions in Drosophila NBs and in mammalian embryonic stems cells (ESCs).
- Drosophila Neuroblast cells (NBs): the mother-daughter centriole asymmetry is dictated by Centrobin (CNB). CNB is responsible for daughter centrioles maintaining the PCM and MTOC mitotic activity during INTERPHASE. Centrobin was also used as a marker the denote the daughter/new mother centriole.
- Centrioles replicate AFTER segregation.
- During division, the OLD mother centrosome migrates to and is inherited by the distal cell [the ganglion mother cell (GMC)]. Distal daughter cells migrate during ACD where it undergoes differentiation while the proximal daughter cells remains as a stem cell.
- Embryonic stem cells (ESCs), asymmetric distribution is induced by Wnt3a, for the centrosome containing old mother.
- Ninein used as a marker to determine the location of the centrosome containing the OLD mother centriole. When using ninein after cell division, centrosomes in 78% of the cells attached to the Wnt3a beads had a high abundance of Ninein showing in the cell with the mother centriole.