Senior Thesis-Analyzing the interactions between MYOGEF and a component of erythrocyte cytoskeleton SPTA1
- 1. Fordham University Analyzing the interactions between MYOGEF and a component of erythrocyte cytoskeleton SPTA1 Dougan McGrath Department of Biological Sciences Dr. Qize Wei May 5, 2015
- 2. 2 Abstract Erythroid spectrin, a major component of the membrane cytoskeleton of erythrocytes, plays a central role in maintaining the shape and elasticity of red blood cells, thus conferring to erythrocytes the capability of travelling through the circulatory system while withstanding shear and stress in the process of circulation. The basic molecular organization of erythroid spectrin is a heterodimer consisting of an alpha-subunit (SPTA1) and a beta-subunit (SPTB1). Mutations in the SPTA1 or SPTB1 gene have been associated with hereditary elliptocytosis. Importantly, it has also been shown that RhoA, a key component of Rho GTPase signaling, is localized to the erythrocyte membrane and implicated in the regulation of erythrocyte cytoskeleton. However, it is essentially unknown whether and how Rho GTPase signaling is implicated in regulating the shape and elasticity of red blood cells. A previous screening in Dr. Wei’s laboratory has identified SPTA1 as an interacting partner of MYOGEF, a guanine nucleotide exchange factor that is capable of activating RhoA and is involved in the regulation of cell division and cell migration. In this study, we constructed various cDNA fragments encoding different regions of MYOGEF or SPTA1 and examined their interactions using yeast two-hybrid and in vitro pull- down assays. Our results showed that the C-terminal region of MYOGEF interacted with the EF- hand motifs that are situated at the C-terminal region of SPTA1. We propose that the MYOGEF- SPTA1 interaction may lead to MYOGEF-mediated RhoA activation at the erythrocyte membrane, thus contributing to the regulation of membrane cytoskeleton in erythrocytes.
- 3. 3 Introduction Erythroid spectrin, a major component of the membrane cytoskeleton of erythrocytes, plays a central role in maintaining the shape and elasticity of red blood cells, thus conferring to erythrocytes the capability of travelling through the circulatory system while withstanding shear and stress in the process of circulation. The basic molecular organization of erythroid spectrin is a heterodimer consisting of an alpha-subunit and a beta-subunit encoded by genes SPTA1 and SPTA2, respectively. The lateral association between the alpha- and beta-chains forms a spectrin dimer that in turn joins head-to-head to form a spectrin tetramer (see Figure 1). There is an actin-binding domain at both ends of the spectrin tetramer. Therefore, both ends of the spectrin tetramer bind to a cytoskeleton complex consisting of short actin filaments, protein 4.1, and actin-binding proteins dematin, adducin, tropomyosin, and tropomodulin (see Figure 1). Such interactions form a spectrin-actin network that is in turn linked to the cell membrane via ankyrin, which binds to both spectrin and the transmembrane protein band 3 (Figure 1). In addition, protein 4.1 binds not only to the ends of the spectrin tetramers, but also to the cytoplasmic domain of the transmembrane protein glycophorin (see Figure 1). Therefore, protein 4.1 also plays a critical role in linking the spectrin-actin network to the cell membrane (Machnicka et al, 2011).
- 4. 4 Figure 1: A simplified cross-section of the red blood cell (erythrocyte) membrane. Taken from Lancet 372:1411-26, 2008. Defects in the alpha-spectrin have been shown to have loss of membrane surface area as well as shear-induced deformation of the cell (Salomao et al, 2006). Illnesses that can arise from this defect, including hereditary spherocytosis, which refers to a group of heterogeneous inherited anemias that have spherical-shaped erythrocytes, and hereditary elliptocytosis, which is a heterogeneous group of diseases that cause elongation of the erythrocyte (Perotta et al, 2008; Vaya et al, 2014). Erythrocyte integral membrane components also include many GTP binding proteins. These proteins have been shown to bind to Rho GTPases. Rho GTPases belong to the Ras superfamily of small G proteins that function as molecular switches in actin cytoskeleton, cell polarity, cell motility, and cell cycle (Kalfa et al, 2014). The main subfamilies of Rho GTPases are Rho, Rac, and Cdc42. Rho GTPases are important for assembly of cell-cell junctions in
- 5. 5 epithelial cells and tumor invasion. The three main types of Rho are RhoA, RhoB, and RhoC (Kalfa et al, 2014). RhoA stimulates assembly of contractile actomyosin filaments and is critical for tumor invasion in breast carcinoma. Previous studies also show how cytosolic RhoA binds to the cytoplasmic side of erythrocyte membranes, and this interaction regulates actin filaments and actomyosin complexes of the contractile ring (Boukharov et al, 1998). This interaction of RhoA with the erythrocyte membrane has an effect on the erythroid spectrin on the RBCs. Many signaling proteins and exchanges factors can mediate this interaction; in particular, myosin- interacting guanine exchange factor (MyoGEF) activates RhoA and other Rho GTPases. Decreased levels of MyoGEF decrease the activation of RhoA and RhoC (Wu et al, 2009). MyoGEF also plays an important role in regulating cytokinesis and cell migration. Like other GEFs, MyoGEF contains a Dbl homology (DH) domain and pleckstrin homology (PH) domain. The DH domain is involved in catalyzing the exchange of GDP/GTP, while the PH domain is involved in intracellular pathways such as ß-adrenergic and Phospholipase C (Wu et al, 2014: Rebecchi et al, 1998). This important signaling molecule is highly expressed in invasive breast cancer cells. Previous studies Dr. Wei’s lab has already MyoGEF interacting with the C-terminal of Spta1, as shown in Figure 2. Figure 2: A map of Spta1, consisting of 2415 amino acid residues and its specific domains, and its interaction with MyoGEF. Previous studies determine MyoGEF interacting with the C
- 6. 6 terminal half of Spta1, specifically, between amino acid residues 2040-2415. The aim of this study is to characterize the interaction between Spta1 and MyoGEF, previously identified through yeast 2-hybrid and in vitro pulldown assays. The data collected indicates the C-terminal of Spta1, specifically the EF-hand, is important for the interaction with the C- terminal half of MyoGEF. Experimental Procedures High-Fidelity Polymerase Chain Reaction (Hi-Fi PCR) System The Hi-Fi PCR system was used to amplify cDNA fragments corresponding to the C- terminal half of mSpta1, amino acid residues 2040-2256, as well as amino acid residues 2040- 2340. The following reaction mixture was used to amplify the desired fragments: 0.5µL template DNA, 10µL 5x PCR HiFi buffer, 1µL appropriate primer, 1µL appropriate reverse primer, 2µL 10mM dNTP mix, 0.8µL HiFi enzyme, and 34.7µL ddH2O. The samples were then placed in PCR system at the following conditions: denatured at 94˚C for 30 seconds, annealed at 48˚C for 30 seconds, and elongated at 68˚C for 1 minute. This process continued for 30 cycles. The samples were then purified via PCR purification protocol (Qiagen). TOPO Cloning Reaction for PCR Products and Transformation into Competent Cell Each purified PCR product was then cloned into pCR8-GW-TOPO entry vector. The following reaction mixture was used: 3µL of PCR product, 1µL salt solution, 1µL ddH2O, and 1µL pCR8-GW-TOPO vector. This reaction mixture incubated for 20 minutes at room temperature and then placed on ice. 2µL of reaction mixture was added to 25µL of TOPO One shot competent cells, and incubated on ice for 30 min. The samples were then heat shocked at 42˚C for 40sec, and then placed on ice for 2 min. 250µL of SOC medium was added to each
- 7. 7 sample, and then placed in shaker at 225rpm for 1 hour at 37˚C. The samples were then plated on Luria-Bertani (LB)-spectinomycin agar plates. These plates were incubated overnight at 37˚C. The cultures were then harvested and grown in 4mL of LB broth with 4µL of spectinomycin. These liquid bacteria cultures shook overnight 250rpm at 37˚C. Plasmids were then extracted by QIAprep Miniprep protocol (Qiagen). The fragments’ length were confirmed by EcoRI enzyme digestion and then the fragments’ orientations were confirmed by EcoRV enzyme digestion. The fragments were also sequenced to confirm length and orientation. LR Recombination Reaction Once the plasmids were confirmed, they were then cloned into the destination vector pACT-2.2-gtwy. The following reaction mixture was used: 3.5µL plasmid, 0.5µL pACT-2.2- gtwy vector, and 1µL clonase II mix. The reaction mix was then incubated at room temperature for 2 hours. Then 0.5µL of proteinase k was added and subsequently incubated at 37˚C for 10min and then placed on ice. Reaction mixes were then transformed into competent cells as described previously. The only difference is that the destination vector is ampicillin-resistant. Positive cloning was confirmed through EcoRI and XhoI enzyme digestions. Yeast 2-Hybrid- Preparation of Competent AH109 Yeast Cells A yeast 2-hybrid protocol (Clontech) was used to determine if the clones purified from the PCR system interact with C-terminal half MyoGEF. The yeast strain AH109 was used in this protocol. After AH109 was streaked on a YPDA agar plate and incubated until colonies appeared, one colony was inoculated into 3mL of YPDA medium. This was incubated in the shaker overnight at 30˚C. 10µL of the culture was transferred to 100mL of YPDA and incubated at 30˚C shaking at 230rpm for 16-20hr. The culture was then centrifuged for 5min and the cell pellet was resuspended in 200mL of YPDA. The culture was incubated at 30˚C until the optical
- 8. 8 density (OD) was 0.4-0.5. Then 50mL of the cells were transferred to a separate tube, then centrifuged and resuspended in 1.5mL of 1.1X TE/LiAc. The solution was then centrifuged for 15sec and the cell pellet was resuspended in 300µL of 1.1X TE/LiAc. Yeast 2-Hybrid- Transformation of Competent AH109 Yeast Cells The transformation mix was then set up with the following mixture: 5µL of bait plasmid, 5µL of prey plasmid, 5µL Herring Testes Carrier DNA, 50µL Competent Cells, 0.5mL PEG/LiAc. The mixtures were mixed by vortexing and then incubated at 30˚C for 30min; the cells were mixed every 10min. 20µL of DMSO was added and then placed in 42˚C water bath for 15min; vortexed every 5min. The mixtures were centrifuged for 15sec to pellet the yeast cells. The supernatant was removed and pellet was resuspended in 1mL of YPD. The solution was then centrifuged for 15sec, the supernatant was discarded, and was then resuspended in 400µL of 1x TE buffer. Yeast 2-Hybrid: Plating the Transformation Mixture 200µL of the transformation mixture was plated on each of the SD selected medium: (SD/-Trp-Leu) and (SD/-Trp-Leu/-Ade/-His). The plates were incubated at 30˚C upside down until colonies were formed. Yeast 2-Hybrid: Restreak colonies and confirmation of bait-prey interaction by colony-lift filter assay After noticeable colonies grew on the (SD/-Trp-Leu/-Ade/-His), the colonies were re- streaked on another (SD/-Trp-Leu/-Ade/-His) plate and incubated at 30˚C until colonies are formed. The colony-lift filter assay was performed (Clontech). A filter paper was presoaked in 2.5mL of Z buffer/X-gal solution in a clean 100-mm plate. Then a dry filter is placed over the surface of the plate of yeast patches, and the filter was rubbed with forceps to ensure the yeast
- 9. 9 colonies stuck to the filter. This filter was then submerged in liquid nitrogen for about 10sec. The filter paper thawed at room temperature and, with colony side up, placed on presoaked filter and incubated at room temperature until signs of blue patches or dry filter paper. In vitro Pulldown Assay: TnT Transcription/Translation systems In vitro pulldown assay was done to determine protein interactions of Spta1 and MyoGEF. In this part of the In vitro pulldown assay, the positive clones have been previously isolated, and the protein for each clone is expressed through this process. The reaction mixture consists of the following: 20µL TnT Quick Master Mix (Sp6), 0.5µL Methionine, 1µL plasmid, and 3.5µL Nuclease-Free Water. The components were spun down and then incubated for 90min at 30˚C. During this incubation period, the GST- beads were washed with 500µL BC100 binding buffer. In vitro Pulldown Assay The following reaction mixture was made: 10µL of in vitro translated proteins, 20µL GST-beads, 150µL 2x BC buffer, 120µL ddH2O, and 0.5µL 1M DTT. These contents were incubated overnight at 4˚C. The beads were washed with BC100 buffer 3 times and then resuspended in 1xSDS load buffer to elute the bound proteins. In vitro Pulldown Assay: Western Blot The pulldown samples were separated on 8% SDS-PAGE gel at 200volts for 1hr. The gel was then transferred to nitrocellulose membrane. Western Blot was run at 15volts for 40mins. The membrane was then covered with Ponceaus in order to check if there were proteins attached to the membrane. Then water and TBST buffer were used to wash membrane. Once the membrane is cleared of Ponceaus, blocking buffer was added to membrane in container for 30min. Then the primary antibody was added: GFP(1:1000, R). With the primary antibody, the
- 10. 10 membrane soaked overnight at 4˚C. After three washes, the membrane was incubated with the secondary antibody, anti-rabbit (1:5000), for 1hr at 24˚C. Results C-terminal half of MyoGEF interacts with Spta1 2040-2340 only This round of yeast 2-hybrid was performed in order to see which of cloned Spta1 plasmids interact with MyoGEF, specifically whether the plasmids interact with the N-terminal or the C-terminal of MyoGEF. The first yeast 2-hybrid test that was performed revealed Spta1 amino acid residues 2040-2340 interacted with the C-terminal half of MyoGEF, as shown below in Figure 3. The results also indicated there was no interaction between the N-terminal half of MyoGEF with Spta1 2040-2340. There was also no interaction between Spta1 between 2040- 2256 with both the N-terminal and C-terminal half of MyoGEF. Figure 3. (left) This is the result of the colony lift filter assay. The blue yeast colonies (as shown in rectangle) are the amino acid residues 2040-2340 of Spta1 interacting with C-terminal of MyoGEF. The blue colonies indicate a positive interaction between the two proteins, whereas the lack of blue indicates no interaction between the two proteins.
- 11. 11 GST- Pulldown Assay indicates no interaction with Spta1 2040-2256 or 2040-2340 GST-pulldown assay was performed in order to confirm the yeast 2-hybrid results, shown in Figure 4. This assay tested the same Spta1 fragments as the first round of yeast 2-hybrid results. The only difference is that this assay wanted to confirm the interaction through in vitro binding between the Spta1 fragments and MyoGEF amino acid residues 501-790, the C-terminal of MyoGEF. The N-terminal of MyoGEF was not tested through in vitro pulldown assay. The results in Figure 4 show that there was no interaction between Spta1 2040-2256, Spta1 2040- 2340 with MyoGEF 501-790. Figure 4. (left) Western Blot results from GST- pulldown assay. Lanes 1-3 are the results of Spta1 2040-2256. Lanes 4-6 are the results of Spta1 2040- 2340. Lanes 3 and 6 show no interaction between MyoGEF 501-790 and both Spta1 fragments.
- 12. 12 Yeast 2-Hybrid results show Spta1 may interact with MyoGEF between 501-790 The second round of yeast 2-hybrid testing was performed in order to determine which part of the MyoGEF C-terminal interacts with Spta1 C-terminal. Previous studies in Dr. Wei’s lab have shown interaction with MyoGEF 501-790 and C-terminal of Spta1. Therefore this yeast 2- hybrid testing determined the possible interaction with C-terminal half of Spta1 and truncated fragments of MyoGEF’s C-terminal. Table 1 shows that MyoGEF 501-790 interacted with the Spta1 C-terminal, but there was no interaction between Spta1 C-terminal and MyoGEF 501-752 or 501- 590. These results suggest that the region between 752-790 is the region where the interaction occurs. Bait Plasmids Prey X-Gal filter assay GAL4-BD-MyoGEF 501-790 GAL4-AD-Spta1 Positive GAL4-BD-MyoGEF 501-590 GAL4-AD-Spta1 Negative GAL4-BD-MyoGEF 501-752 GAL4-AD-Spta1 Negative Table 1. Summary of yeast 2-hybrid results shown above. The results show that there is an interaction between MyoGEF 501-790 and the C-terminal of Spta1 and no interaction between MyoGEF 501-752 and C-terminal of Spta1, suggesting that the region of MyoGEF 752-790 is important. Discussion In this study, we have shown that the C-terminal half of MyoGEF is important in the interaction with the C-terminal of Spta1. Specifically, it seems that MyoGEF’s amino acid residues 752-790 are important with the interaction of Spta1’s amino acid residues 2256-2340. These results from the yeast 2-hybrid seem to indicate that the EF hand, a calcium-binding motif shown in Figure 2, is important in the MyoGEF-Spta1 interaction. Our findings are consistent
- 13. 13 with previous data collected, because Dr. Wei’s laboratory showed that the Spta1’s amino acid residues 2040-2415 are important in the interaction with MyoGEF’s amino acid residues 501- 790. But there were some contradictory results, as shown with the GST- In vitro Pulldown Assay because there was no interaction with Spta1 2040-2256 and Spta1 2040-2340 with MyoGEF 501-590. One possible explanation for this lack of interaction can be due to posttranslational modifications, which can occur during the process of yeast 2-hybrid protocol, but will not occur during in vitro binding. Further experimentation is necessary in order to determine if the EF hand is actually important in this interaction. This determination can be accomplished through further cloning of Spta1’s C-terminal. Along with characterizing the interaction of MyoGEF-Spta1, the effects of this interaction will be analyzed. It is not well known how exactly this interaction will affect the formation of erythrocytes. The effects of decreased levels of MyoGEF in erythrocytes is unknown, it is only known that MyoGEF activates RhoA, which is present in erythrocytes cytoskeleton. Further, this research is the important beginning to see how MyoGEF-Spta1 interaction can affect the formation of erythrocytes and the maintenance of erythrocyte cytoskeleton as it travels throughout the circulatory system.
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