Citation
Characterization of Filamin's Involvement in Response to Mechanical Stimuli

Material Information

Title:
Characterization of Filamin's Involvement in Response to Mechanical Stimuli
Creator:
Sarah Buckler
Yulia Artemenko
Publication Date:

Notes

Abstract:
Directed migration of cells is vital for a number of processes, including uncoordinated migration of cancer cells. The social amoeba Dictyostelium discoideum is very similar to mammalian cells, making it an excellent model organism for studying migration. Cells can migrate directionally in response to mechanical stimuli, such as shear flow, although how cells sense this type of stimulus is unclear. Previous studies in our lab showed that filamin, which is a protein that cross-links actin cytoskeleton in cells, is involved in the ability of cells to respond to mechanical cues. We have now shown that filamin does not affect response to chemical stimuli, suggesting that filamin’s role is specific to responsiveness to mechanical stimuli. Filamin requires the actin-binding domain (ABD) and the dimerization domain (DD) to work together to ensure proper binding and cross-linking of the actin cytoskeleton. To further understand filamin’s role in sensing physical stimuli, we generated an expression construct for filamin with no ABD. Future studies will test the ability of filamin without ABD to rescue the reduced response of filamin-null cells in a mechanical assay.
Acquisition:
Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Sarah Buckler.

Record Information

Source Institution:
SUNY Oswego Institutional Repository
Holding Location:
SUNY Oswego Institution
Rights Management:
All applicable rights reserved by the source institution and holding location.

Downloads

This item is only available as the following downloads:


Full Text

PAGE 1

Vector and Insert Preparation In order to confirm if PCR worked, gel electrophoresis is used. It is a technique used to separate DNA fragments according to their size. The PCR product is verified based on its size (Figure 1). This technique is used to isolate a certain fragment of DNA from an agarose gel. The bands of interest are cut out of the gel, and the DNA is purified. Gel electrophoresis is done to verify size of DNA. Sarah Buckler, Yulia Artemenko Department of Biological Sciences, SUNY Oswego, Oswego, NY Abstract Directed migration of cells is vital for a number of processes, including uncoordinated migration of cancer cells. The social amoeba Dictyostelium discoideum is very similar to mammalian cells, making it an excellent model organism for studying migration. Cells can migrate directionally in response to mechanical stimuli, such as shear flow, although how cells sense this type of stimulus is unclear. Previous studies in our lab showed that filamin, which is a protein that cross links actin cytoskeleton in cells, is involved in the ability of cells to respond to mechanical cues. We have now shown that filamin does not responsiveness to mechanical stimuli. Filamin requires the actin binding domain (ABD) and the dimerization domain (DD) to work together to ensure proper binding and crosslinking of the actin cytoskeleton. To further understand for filamin with no ABD. Future studies will test the ability of filamin without ABD to rescue the reduced response of filamin null cells in a mechanical assay. Social Amoeba Dictyostelium discoideum D. discoideum has an unusual life cycle, including periods of both unicellular and multicellular stages. D. discoideum is a valuable model in human disease analysis, as well as studying cell cell communication and roles in cell shape changes. 1 Filamin Figure A: Actin cytoskeleton with filamin crosslinks. ht tps://study.com/academy/lesson/actin filaments function structure quiz.html Figure C: pDRH plasmid. Filamin/no ABD gene will be inserted between Sal1 and Not1 restriction sites. mCherry fluorescent tag is not shown. Figure B: D. discoideum filamin. ABD is located at the N terminus (yellow) and DD is located at rod 6. Results Measure response of filamin null cells expressing mCherry filamin or vector to a stimuli. to mechanical stimulation Generate an expression construct for filamin without ABD by inserting the appropriate filamin fragment amplified by PCR into the Sal1 and Not1 restriction sites of the pDRH mCherry vector (Figure C). PCR Gel electrophoresis Figure 1. Response of filamin null cells expressing pDRH vector or mCherry filamin, as well as GFP tagged Ras Binding Domain (RBD) to stimulation with folic acid. Cells grown on a bacterial lawn were collected, washed, plated in 8 well chambers, and imaged with epifluorescence microscopy every 3 seconds for 1 min. 100 µM folic acid was added after frame 5 (time 0). Response was measured as an inverse of a drop in cytosolic intensity of RBD GFP. Data shown are mean+/ SE; n=29, collected from several wells in one experiment. Gel extraction Digestion Ligation Transformation and Plasmid DNA Purification Results 1 2 3 4 5 6 1 2 1 2 Figure 2 : Large scale PCR for filamin lacking ABD (1 6). The top bands (2238 bp) were cut out for DNA extraction. Note the DNA ladder on the left. Figure 3 : DNA extraction test for filamin lacking ABD. Lanes 1 and 2 show that the product of the expected size (2238 bp) was successfully purified. Figure 4 : Digestion of the pDRH mCherry filamin vector (1 3) and filamin/no ABD insert (4 6) with SalI and NotI. The pDRH mCherry fragment of the vector at 10 kb and insert at 2.2 kb were cut out for gel extraction. The bottom band of the vector is the full length filamin gene. Response of filamin null cells to stimulation with a chemoattractant Moving Forward Since filamin null cells responded to stimulation with a chemoattractant similarly in the presence or absence of filamin, it app ears that filamin is not involved in response to chemical stimuli. Thus, its role is likely specific to mechanical stimuli. To further understand the role of filamin in sensing chemical and mechanical stimuli, an expression product with no ABD was suc cessfully prepared and will be tested for its ability to rescue the phenotype of filamin null cells in a mechanical stimulation assay. Cloning of filamin with no DD is in progress. References Acknowledgements 1 Mondal S, Burgute B, Rieger D, Müller R, Rivero F, et al. (2010) Regulation of the Actin Cytoskeleton by an Interaction of IQ GAP Related Protein GAPA with Filamin and Cortexillin I. PLoS ONE 5(11) 2 Sunderland, Mary E., "Dictyostelium discoideum". Embryo Project Encyclopedia (2009 06 10). ISSN: 1940 5030 3 Su, W., Mruk, D. D., & Cheng, C. Y. (2012). Filamin A: A regulator of blood testis barrier assembly during post natal developmen t. Spermatogenesis , 2 (2), 73 78. assisting and supporting this research. This work is supported by NSF RUI grant no. 1817378 (to Y.A.). Figure 5 : DNA extraction test after digestion. Vector (2) and insert (1) show correct sizes. Figure 6 : Diagnostic digestion of plasmid DNA isolated from bacteria transformed with the ligated pDRH mCherry filamin/no ABD. Plasmids were digested with SalI and NotI. Clones in lanes 1,3,5,7,9,11 gave the expected sizes. Corresponding uncut plasmids are shown in lanes 2,4,6,8,10,12. 1 2 3 4 5 6 ABD DD DD ABD 1 2 3 4 5 6 7 8 9 10 11 12 Filamins are large actin binding proteins that stabilize three dimensional actin webs and link them to cellular membranes (Figure A). 2 filamin is composed of actin binding domain (ABD) located at the N terminus, followed by 6 rod domains. Dimerization is mediated through rod 6 (Figure B). 3 Filamin can regulate multiple cellular functions including its involvement as the mechanotransduction element of the cytoskeleton. 3 The focus of this study is to test whether filamin requires its ABD to allow the cell to sense and respond to mechanical stimuli. Approach and Methods Cloning of filamin lacking ABD Several clones were verified by sequencing and large amounts of plasmid were prepared. Vector and Insert Preparation