UVM Theses and Dissertations
Format:
Print
Author:
Lodh, Sukanya
Dept./Program:
Biology
Year:
2012
Degree:
PhD
Abstract:
Paramecium, a unicellular ciliate, swims forward in normal culture medium and occasionally swims backward for short periods. Wild type cells perform long backward swimming in depolarizing solutions [e.g. BaC1₂]. Some mutants of Paramecium do not swim backward at all due to the absence of Ca² conductance. These cells are called pawns. Untill now three genes [PWA, PWB, PWC] have been found to be mutated in pawn cells and only PWA and PWB were cloned. As PWA and PWB are not channel proteins, our aim is to find their association with voltage gated Ca²⁺ channel. To do that it is important to find the location ofboth PWA, PWB proteins and to identify whether they interact with the calcium channels.
We used the RNAi feeding method with PWA and PWB constructs in the RNAi vector L4440 to downregulate PWA or PWBmessages in wild type cells and observed swimming behavior. We found that we could produce a pawn phenotype with RNAi. We expressed the FLAG epitope tagged PWA and PWB to follow the protein product to find its location. FLAG epitope tagged PWA and PWB constructs were microinjected into the macronuclei of pawnA-nd6 and pwB mutant cells respectively in order to test whether the cloned sequence rescued the mutant phenotype and to find the location of functional FLAG epitope tagged protein products by immunostaining. The cells expressing PWAFLAG showed intracellular and surface immuno-fluorescence. Western blots of fractional preparations probed with an anti-FLAG antibody showed specific staining in pellicle-rich and endoplasmic reticulum-rich fractions.
PWA-FLAG was also detected in cilia after performing an immunoprecipitation. PWB-FLAG was detected only from ER rich fraction in the Western blots containing immunoprecipitated proteins from pellicle, ER and cilia. We found that PWB interacts with one VGCC. We also observed that plasma membrane calcium ATPase18 (PMCA18), one of the binding partners of VGCC, is significantly lower in the pwA cilia than that of the wild type cells using quantitative mass spectrometry. We have also expressed the pwA constructs with mutated omega sites in the mutant cells. The constructs were able to partially rescue the pawn phenotype in the mutants. We propose that PWA and PWB play important roles in sorting of VGCCs in the ER and trafficking the channels to their final destination, cilia.
We used the RNAi feeding method with PWA and PWB constructs in the RNAi vector L4440 to downregulate PWA or PWBmessages in wild type cells and observed swimming behavior. We found that we could produce a pawn phenotype with RNAi. We expressed the FLAG epitope tagged PWA and PWB to follow the protein product to find its location. FLAG epitope tagged PWA and PWB constructs were microinjected into the macronuclei of pawnA-nd6 and pwB mutant cells respectively in order to test whether the cloned sequence rescued the mutant phenotype and to find the location of functional FLAG epitope tagged protein products by immunostaining. The cells expressing PWAFLAG showed intracellular and surface immuno-fluorescence. Western blots of fractional preparations probed with an anti-FLAG antibody showed specific staining in pellicle-rich and endoplasmic reticulum-rich fractions.
PWA-FLAG was also detected in cilia after performing an immunoprecipitation. PWB-FLAG was detected only from ER rich fraction in the Western blots containing immunoprecipitated proteins from pellicle, ER and cilia. We found that PWB interacts with one VGCC. We also observed that plasma membrane calcium ATPase18 (PMCA18), one of the binding partners of VGCC, is significantly lower in the pwA cilia than that of the wild type cells using quantitative mass spectrometry. We have also expressed the pwA constructs with mutated omega sites in the mutant cells. The constructs were able to partially rescue the pawn phenotype in the mutants. We propose that PWA and PWB play important roles in sorting of VGCCs in the ER and trafficking the channels to their final destination, cilia.