Antara Mukhopadhyay

Abstract

Neurons are the brain cells which communicates through transmission of some neurotransmitters such as adrenalin, nor adrenalin, glutamate etc. One of the major neurotransmitter Glutamate which is essential for learning and memory is received through some ion-channel receptors in dendrites of target neuron cells. Transmission of glutamate causes the positive ion (ex: ca²⁺) influx into target cells through ion channel receptors which in turn releases of neuro-transmitters to next cell. Thus they enable the communication between brain cells which if hampered causes diseases (ex: Hutington’s disease) and in some extreme cases death. This ion channel receptors are of several types such as NMDA receptor, Kainate receptor, AMPA receptor. All of these receptors are made of many subunits and several other auxiliary subunits. The major subunits of these ion channel receptors comprise of four domains i.e N-terminal domain, ligand binding domain, trans-membrane domain, C-terminal domain. Now in order to evaluate the basis of their functions, there is a need of detailed analysis of their structures and how their subunits assembles to make a complete receptor. Although some studies have been going on at proteomics level to decipher the stoichiometry, there is very little study done at single molecule level and a major reason for that is the absence of proper labels for protein labelling for microscopic analysis. GFP is a good fluorescent protein which can be fused to target protein, expressed in a model system like Xenopus laevis oocytes and used for counting of the bleaching steps in a TIRF microscopy system to do the stoichiometry analysis. As of now GFP bleaches in a well manner which is sufficient for bleaching step counting but other fluorescent proteins like YFP, RFP, m-cherry etc. do not bleach regularly and bleach very fast. Hence the bleaching steps of many fluorescent proteins, the basis of stoichiometry analysis at single molecule level are difficult to count. Therefore it is very difficult to decipher complex multimeric receptor structures with only one fluorescent label with good photo-bleaching property. Hence in order to understand the stoichiometry of multimeric receptors at single molecule level it is necessary to find good photo-stable fluorescent labels whose bleaching steps will be easy to count. Therefore we plan to screen through some fusion tags, fluorescent proteins with overlapping spectra, express them in frog oocytes and evaluating their quality and efficiency to be used as multicolour labels for single molecule imaging for future stoichiometry analysis.

Methods

Cloning, Agarose gel electrophoresis, RNA preparation, RNA injection, Protein labeling, TIRF microscopy.