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10. The early stages of Drosophila embryogenesis have been a model for mRNA localization and spatial patterning. Four mRNAs in particular are important in the earliest stages of patterning the anterior to posterior axis of the embryo. What are the genes that encode these four mRNAs? Are all four mRNAs localized? For those that are localized, what is the mechanism of localization? For all four of the mRNAs, what is the mechanism for establishing protein gradients along the A-P axis?
- Cytoplasmic polarity Genes: Bicoid, Nanos, Hunchback and Caudal
- Localized genes: BICOID localizes at ANTERIOR of embryo, with NANOS at the Posterior of the embryo.
- Localized mechanism: During oogenesis, the mother's GENOTYPE instructs the activity and localization of these genes. DB…Dynein (- end directing) localizes Bicoid, while Kinesin (+ end directing) localizes Nanos.
- Protein gradient along A-P axis: Established via localizes expression of Bicoid and Nanos, which establish gradient of Hunchback and Caudal to 'tell' the nucleus where it is and what part of the fly it will contribute to.
- BC...Bicoid represses Caudal synthesis
- Nanos represses Hunchback synthesis
11a. Two papers covered in class related to the question of how common mRNA localization is in “everyday” cells, i.e. cells other than eggs or embryos, the Mingle et. al. (2005) and the Mili et. al. (2008) papers. For Mingle et al. (2005), provide a brief summary of the methods, the results and their conclusions. Be sure to include information regarding which specific elements of the cytoskeleton were implicated in the localization of the mRNAs in question.
- Use FISH-TSA to localize mRNA for b-actin and Arp2 and Arp3: All seven Arp2/3 complex mRNAs localize to human foreskin fibroblasts.
- Looked at pattern of dispersion in Poly-A RNAs: shows disperse hybridization pattern
- Looked at localization of Chick Arp2 with and without serum, to see if serum impacted localization: Arp2 with serum showed increased localization over time, and without serum vice versa.
- Looked at WHAT component of the cytoskeleton is necessary for localization, using Cytochalasin D (actin poly inhib) and Colchicine (mt poly inhib): Showed localization of Arp2/3 mRNA dependent on MF and MTs, wherease localization of b-actin mRNA dependent on MFilaments only
- Conclusions: 1. mRNA encoding proteins that function together co-localize together. 2. Localization dependent on intact cytoskeleton, particularly with MF and MTs intact
11b. Two papers covered in class related to the question of how common mRNA localization is in “everyday” cells, i.e. cells other than eggs or embryos, the Mingle et. al. (2005) and the Mili et. al. (2008) papers. For Mili et al. (2008), provide a brief summary of the methods, the results and their conclusions. Be sure to include information regarding which specific elements of the cytoskeleton were implicated in the localization of the mRNAs in question.
- Mouse embryo fibroblast NIH/3T3 cell line placed on filters and induced growth using fibronectin and LPAcid. Pseudopodia grew through membrane, then Cell bodies separated from Pseudopodia and mRNA isolated and stained with Phalloidin. RealtimePCR of RNAs was performed and 50 mRNAs in pseudopodia relative to cell body were found.
- Reporter mRNA constructs (gene/UTR) to analyze mechanisms in localization: looked at Rab13, Pkp4, RhoA and B-globin (2 neg controls).
- Used to determine if the gene (Rab13, Pkp4) alone or the UTR (with 24bp stem loop) was involved in localization
- Distribution of reporter mRNAs determined with MS2-GFP.
- Result: 24bp and 3'UTR are needed for localization
- MS2: binds to 3'UTR and stem loop structures, so has high affinity for mRNA
- To see if microtubules were needed: Nocodazole (MT inhibitor) used and discovered that MT ARE NEEDED
- Looked to see if Rab13 co-localized with APC: Found they DID co-localize at + end of MTs
- Conclusion: Large number of mRNAs that localize throughout the cell, and about 50 localize in the pseudopodia of mouse embryo fibroblasts. 3'UTR and 24bp repeats are REQUIRED for localization. Co-localization also is a factor in the localization of mRNAs
12. What was the major question being addressed in the Katz et. al. (2012) paper and what was the major take home message of the paper? Briefly describe the results that support the take home message.
- Question: Functional significance in the localization of B-actin mRNA to the LEADING edge of migrating cells. Previously shown that B-actin mRNA is localized to the leading edge of migrating FIBROBLASTS. So is B-actin is involved in another process that requires its localization at the leading edge.
- Takehome message: B-actin is localized and is involved with directionality through ZBP1. B-actin protein synthesis is required in these compartments where it is localized. ZBP1 plays a role in regulating localization b-Actin mRNA, allowing it to dwell, where it can play a functional role, forming connections between adhesions and newly synthesized actin filaments. Also shown that it is translating when it is dwelling.
- 1. ZBP1 directionality and 3'UTR: Zipcode binding protein 1 (ZBP1) is important for both localization and directionality of fibroblasts. Experiments shown that with interruption of the 3’ UTR of b-actin mRNA or with loss of c-terminal mRNA binding domains in ZBP1, there was a DECREASE in directionality of fibroblasts.
- These experiments done with mouse embryo fibroblasts that were homozygous for ZBP-/-MBS+/+, which were then infected with MS2 and immortalized in cell culture.
- MBS = b-actin-MS2-binding sites (tagged with MS2 stem loop in 3'UTR)
- 2. Dwelling: ZBP1 specifically localizes B-actin mRNA to the adhesion compartment, where it DWELLS for minutes. The focal adhesion compartment environment is therefore responsible for gathering the b-actin mRNA. These dwelling mRNAs were also translating.
- These experiments done by labeling the mRNA with GFP bound to the MS2 stem loops in the 3’ UTR. Any mRNAs that persisted in the location for more than 1minute were considered ‘dwelling’ mRNAs. To test translation, puromycin (antibiotic causing premature translation termination) was added and the percent of dwelling mRNAs was significantly reduced.
- 3. Focal adhesion STABILITY: related to ZBP1 expression in these Mouse embryonic fibroblasts.
- Using paxillin-mCherry staining to label adhesion sites, ZBP1 knockouts showed lack of directionality whereas wildtypes showed clear directionality with waves of adhesions
- 4. b-Actin mRNA tethering to the focal adhesions: INCREASED lifetimes and adhesion sizes, and local translation of B-actin mRNA increased adhesion stability.
- Tethered cells were introduced to cyclohexamide (inhibitor for translation elongation) and showed reduced adhesion stability.
d13. What are the reasons that have been suggested for localizing mRNAs rather than simply localizing the proteins? Are there known examples to support each of these suggestions and what are they? Be as specific as possible.
- 1. functional efficiency: Proteins that function together need to be co-localized to improve functional efficiency. (Mili and Rab13 / APC).
- 2. Energy efficiency: Transport of individual proteins could be inefficient or “costly” in regards to energy requirements, whereas transport of single mRNA can provide source of many copies of the protein. (Polyribosomes)
- 3. Loss in transit: In situ translation prevents the possibility of inappropriate interactions during transport. (no examples)
- 4. High conc: In situ translation allows generation of high local concentrations of specific proteins. (Katz and β-actin, Crofts, plant storage protein complexes).
- 5. Fast responses: In situ translation allows synthesis in response to localized signals (rapid response mechanism).