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- Investigation of The Role of KrsB in Rap1-Mediated Adhesion of Dictyostelium discoideum
- Kelsey Roberts
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- Dictyostelium discoideum is a model organism commonly used to study cell migration because of its similarities to other amoeboid cells, such as neutrophils and metastatic cancer cells. Rap1 is a small GTPase that increases cell adhesion, which is necessary for proper migration, by regulating myosin II and talin. Kinase responsive to stress B (KrsB) is a kinase that negatively regulates cell adhesion, although the molecular mechanism of its action is unknown. Preliminary evidence from our laboratory suggested that while Rap1 does not require KrsB for its effects on adhesion, KrsB might be a negative regulator of Rap1. The goal of this study was to examine the behavior of cells expressing Rap1 in the presence or absence of KrsB to determine if KrsB indeed negatively regulates Rap1. It was predicted that cells expressing Rap1 in the absence of KrsB would show increased spreading and, consequently, decreased migration, while cells expressing both Rap1 and KrsB would show decreased spreading and increased migration. Two cell lines were generated from KrsB-null cells, one with RFP-tagged Rap1 G12V (constitutively active Rap1) and one with RFP-tagged pDM318 (empty vector). In addition, both cell lines were also transformed with GFP-tagged KrsB under an inducible promoter. Each cell line was either induced with doxycycline to express KrsB-GFP, or not induced (KrsB-null). Random migration and spreading were imaged using brightfield illumination, and RFP-Rap1 and KrsB-GFP expression was confirmed using epifluorescence. Under the conditions tested, spreading appeared to be equal between all four cell lines, whereas migration increased in the presence of KrsB both in Rap1 G12V and vector expressing cells. These findings were unexpected given the known effect of Rap1 G12V on cell spreading. Future studies will examine behavior of the cell lines under conditions that are more optimal for cell spreading and will also select cells that have high Rap1 G12V expression for analysis.
- Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Kelsey Roberts.
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Investigation of The Role of KrsB in Rap1 Mediated Adhesion of Dictyostelium discoideum Introduction Dictyostelium discoideum is a soil dwelling amoeba. D . discoideum is a good model organism for cell migration studies because its movement is like other amoeboid cells, such as neutrophils and metastatic cancer cells. Cell adhesion to substrate is an essential component of migration, yet little is known about its mechanisms in D . discoideum. Rap1 is a small GTPase. Its expression has been previously shown to increase cell adhesion, possibly by regulating Talin and Myosin II. 1,2 Kinase responsive to stress B ( KrsB ) is a negative regulator of cell adhesion, so activation of KrsB leads to decreased cell adhesion. This could be because it is negatively regulating Rap1. Previous studies have shown that expression of KrsB is not required for Rap1 to function, as its absence does not appear to change effect of Rap1 on adhesion. Research Question and Hypothesis Methods Methods Results Results Discussion and Conclusions Kelsey Roberts and Dr. Yulia Artemenko Question: Does the expression of KrsB in D. discoideum reduce cell adhesion by negatively regulating Rap1? Hypothesis: KrsB is a negative regulator of Rap1. If this hypothesis is true, the overexpression of KrsB should reduce the ability of Rap1 to increase spreading. Quantification: Cell velocity was measured using Tracking tool pro. Area, perimeter, and GFP brightness were measured using FIJI (Image J). For KrsB induced cells, only cells with high KrsB expression (measured by GFP brightness values) were quantified. Statistical Analysis: A one way ANOVA was conducted to test for a relationship between KrsB expression/ mutant type and cell velocity. Acknowledgements Department of Biological Sciences, SUNY Oswego I would like to thank Dr. Yulia Artemenko department for the opportunity to conduct this study. This work was supported by National Science Foundation Research in Undergraduate Institutions (NSF RUI) grant no. 1817378 (to Y.A.). References Results of the random migration show that KrsB expression in KrsB null cells with or without constitutively active Rap1 improves migration. Interestingly, this is not accompanied by changes in cell spreading. Since Rap1 G12V did not show the expected increase in spreading, the results of this study are currently inconclusive. Problems and set backs: Rap1 expression was not taken into account during quantification, meaning that some of the cells that were measured may have lacked Rap1. Transformed Rap1 G12V cells tended to lose KrsB expression as they aged, at a much faster rate compared to the other cell lines. Another problem faced during this study was contamination of the mutant plates, leading to less data being collected. Future directions: Data will be checked to ensure that all cells have Rap1. To avoid other similar problems in the future, new plates of cells expressing Rap1 G12V can be made more frequently, since they appear to lose the plasmid quickly. To avoid contamination in the future, a clean and sterile environment must be ensured while always handling the cells. This includes always using pipette tips that are sterile, and sterilizing the hood regularly using UV light. It is also possible that the DB used during data collection influenced migration and spreading. Future experiments will try using a different media . Future directions of this study aim to apply these results to chemotaxis. No significant differences in cell spreading (measured by average area) were found between cell lines. Image of D . discoideum cells in brightfield view Random migration and cell spreading was imaged at 400X magnification on an LSM700 Zeiss confocal microscope. Three different positions were imaged per well in brightfield. Images were acquired every 2 0 sec for 45 frames. Images from the last frame were used in measuring cell spreading. After random migration, GFP and RFP images were taken for each ending position using epifluorescence. Brightfield images were acquired every 2 0 sec for 15 min and quantified to determine velocity. The last frame, which was used to quantify cell spreading, is shown. RFP and GFP images were captured for each position immediately after the last frame. One representative field is shown for each cell line. Testing expression of RFP Rap1 G12V or RFP alone (vector) and KrsB GFP following doxycycline induction in KrsB null cells 1 Artemenko et al. Assessment of development and chemotaxis in Dictyostelium discoideum mutants. Methods Mol Biol . 2011;769:287 309. doi:10.1007/978 1 61779 207 6_20 2 Artemenko Y, Devreotes PN. Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation. J Vis Exp . 2017;(129):56411. Published 2017 Nov 9. doi:10.3791/56411 Data shown as mean +/ SD n=32 n=49 n=27 n=29 Data shown as mean +/ SD; ***P<0.0001 n=32 n=49 n=27 n=29 *** ***