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14. What are the two major mechanisms involved in mRNA localization that were covered in class and what is the evidence for each?
- 1a. mRNA need INTACT cytoskeleton: Mingle showed localize by utilizing both microfilaments and Microtubules. Mingle showed by using Cytochalasin D (MF inhibitor) and Colchicne (MT inhib) Arp2/3 mRNAs did not localize to fibroblast protrusions, and B-actin could not localize without MF. Mili Et al. showed with NOCODAZOLE (MT inhib) the localization of the reporter mRNAs were DECREASED
- 1b. mRNA need myosin motors: Using myosin inhibitors on chicken embryo fibroblasts, Arp2/3mRNA localization was inhibited.
- Sum: MTs & kinesins get particles to intracellular locations, but MFs needed to get to cell periphery.
- 2a. mRNA needs zip code: Katz et al. showed zip codes in the 3UTR that forms a stem loop structure NECESSARY in mouse embryonic fibroblasts, where it dwells for minutes in focal adhesion compartment. This ZBP1 is important for focal adhesion stability and the mRNA tethered to focal adhesion specifically INCEASED lifetime and size, so FUNCTIONal importance.
- 2b. mRNA need cellular activity: Mingle showed staining in the absence of serum, minimal localization occurred with Arp2/3 mRNA in fibroblast protrusions. Localization is cELLULAR RESPONSE to Extracellular stimuli
mRNAs are associated with various proteins
15. What are the SUN and KASH proteins? Where are they located in the cell? What is their proposed function?
- SUN and KASH: New classes of proteins form BRIDGES between nuclear matrix and cytoskeleton, spanning the PNS. (perinuclear space).
- SUN proteins: embedded in INM (inner nuclear membrane). They have at least 1 transmembrane domain, with the C-terminal domain extending into PNS.
- KASH proteins: span the outer nuclear membrane (ONM).
- SUN function: SUNs C-terminal domain extends 35aas from the C-terminus. The C-terminus is in the PNS, and recruits KASH proteins and LOCALIZES them to the ONM. N-terminal of SUN interacts with the chromosome binding proteins, connecting the CHROMOSOME/chromatin to the INM
- KASH function: N-terminus of KASH on the cytoplasmic side of the ONM, so aids in binding of cytoplasmic elements. Connecting the ONM to actin and INTERMEDIATE filaments. Also migration of nuclei (both attached to and without centrosome), and ANCHORING nuclei
16. What was the major question addressed in the Luxton et. al. (2010) paper? What were the primary methods they used to address the question? What were their conclusions?
- Question: What controls the positioning, movement and reorientation of the NUCLEUS in cells? What is the mechanism for transmitting FORCE BTW the cytoskeleton and the nucleus?
- Methods: Used wound healing in an epithelial cell layer. Grew cells in culture producing a monolayer that was contact inhibited. They created a wound by scraping some of the cells off the plate. Had two wound cultures, one with a serum-free medium and the other with growth factor. Then observed the behavior of the cells. Expressed different constructs in cells adjacent to the wound edge and monitored effects of the expressed construct on reorientation on the nucleus. Constructs: RFP-SR-KASH and RFP-KASH (poison proteins, disrupt LINC and displace nespris from INM, RFP-KASH?L (Lacks SUN, no effect on LINC or nesprins), various siRNAs, GFP-mini-N2G-nesprin (no actin binding domain).
- Results: RFP-KASH expression BLOCKS centrosome/nucleus REORIENTATION and nuclear MIGRATION.
- Nuclear movement depends on Nesprin 2G (N2G): N2G deletion results in blocked nuclear migration (MT dependent) but has NO effect on actin cable migration.
- TAN lines form: Expressed GFP-mini-N2G co-localizes with actin cables and SUN2 complexes in N2G depleted cells. TAN lines coupled with DORSAL ACTIN cables during nuclear MOVEMENT
- SUN2 depletion also inhibited nuclear positioning and centrosome orientation
- Nuclear positioning and orientation is not random and in migrating cells: depends on both the ACTIN and MT components of the cytoskeleton. Recently discovered SUN and KASH proteins for bridges (LINC complexes) that interconnect the two layers of the nuclear membrane and anchor the NM to the lamina on the inner surface and the cytoskeleton on the cytoplasmic surface.
- LINC complexes provide a mechanism: for transmitting force (generated by cytoskeletal motors) TO the NUCLEUS.
- Not clear if this is true communication or whether the nucleus is passively dragged to the appropriate position.