Master’s seminar 1 Biochemical and Immunological characterization of polyclonal antibodies generated against RIBEYE, Tctex-1, Cav1.4 and Cre protein in retina Praveen Kumar Under guidance of Prof. Dr. Frank Schmitz Institute for Anatomy and Cell Biology

CONTENTS 2 Aim Introduction Molecular and biochemical techniques Application Benefits References

AIM 3 Aim of this project is to express and purify RIBEYE, Tctex-1, Cav1.4 and Cre-recombinase fusion protein and to generate polyclonal antibodies against these proteins. Further, these antibodies will be used for various Biochemical and Immunological characterization of above mentioned proteins in retina.

INTRODUCTION

THE VISUAL SYSTEM 5 The part of the central nervous system, which interprets the information from visible light to build a representation of the world surrounding the body.

RETINA 6 There are six classes of neuron in the mammalian retina: rods (1), cones (2), horizontal cells (3), bipolar cells (4), amacrine cells (5) and retinal ganglion cells (RGCs) (6). They have a laminar distribution (OS/IS, outer and inner segments of rods and cones; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, optic nerve fibre layer)

SYNAPTIC RIBBONS Ultrastructure of synaptic ribbons. (A) Conventional transmission electron micrographs of the photoreceptor synapse from (Carassius carassius). Synaptic ribbon (sr), synaptic vesicle (sv), horizontal cell (hc) and bipolar cell (bc). The synaptic vesicles are shown by arrows; the arrowheads denote the site of endocytosis (Schmitz, 1996). (B) A synaptic ribbon (sr) is tangentially cut, thus making the plate-like character of synaptic. sr = synaptic ribbon; sv = synaptic vesicle; pr = presynaptic terminal; en = large, tubular compartment possibly of endosomal origin; ad = arciform density; sri = synaptic ridge; hc, bc = horizontal/bipolar cell dendrites. Scale bars: 100 nm (A, B). 7

RIBEYE, a component of synaptic ribbons RIBEYE is composed of a unique A domain specific for ribbons, and a B domain identical with CtBP 2, a transcriptional repressor. Evolved in vertebrates under utilization of a preexisting protein to build a unique scaffold for a specialized synapse A domain: Mediates assembly of Ribeye into large structures. B domain: Binds to NAD(H) with high affinity. 8

CaV1.4 calcium channels 9 Cellular function:- Release neurotransmitter from photoreceptors, mediate the entry of calcium ions into excitable cells.

TCTEX-1, a light chain of the dynein complex 10 The major microtubule minus end–directed motor protein of the cell; it is involved in many essential cellular processes such as membrane trafficking and mitosis. Function: (1) end-directed organelle movement, (2) endosomal transport, (3) centrosomal localization of the Golgi complex, (4) mitotic spindle alignment, (5) anaphase chromosome segregation, (6) and nuclear distribution,

CRE-RECOMBINASE 11 - Cre- recombinase, often abbreviated to Cre, is a Type1 topoisomerase form P1 bacteriophage that catalyzes site specific recombination of DNA between loxP sites. - Does not require any energy cofactors and Cre-mediated recombination, quickly reaches equilibrium between substrate and reaction products. - Capable of creating any desired modification of the mouse genome (addition of functional genome pieces, remove exciting gene from genome, excise or invert loxP flanking DNA segment, create intramolecular recombination between different DNA molecules)

MOLECULAR AND BIOCHEMICAL TECHNIQUES 6/8/15

(A) Cloning, Expression and Purification of Cre- recombinase 6/8/15 DNA cloning into plasmid Transformation of competent BL 21 bacteria with different techniques(heat shock, electroporation) PCR DNA Amplification Protein quantification with colorimetric assay such as Brodford assay Expression and purification of BL 21 bacterial fusion proteins

(A) Cloning, Expression and Purification of Cre- recombinase 14 Gel images of plamid vector pMAL-C2 (66 2 6bp) contain Cre-recombinase DNA insert ( 1 Kb). Showing successful cloning in 20 E.coli colonies whose chosed for heterologous expression.

(A) Cloning, Expression and Purification of Cre- recombinase 6/8/15

(B) GENERATION AND CHARACTERIZATION OF POLYCLONAL ANTISIRA 16 Generate anti rabbit polyclonal antibodies using fusion protein Identify specific proteins from a complex mixture of proteins extracted form rabbit serum by using western blotting

IMMUNOHISTOCHEMISTRY OF RETINA TISSUE Visualize the location of proteins on tissue sections by using immunofluorescence 2D9Ribeye+ 2D9mergephaseRibeye INL ONL OPL

APPLICATION Research applications such as: western blot, ELISA, flow cytometry, immunohistochemistry, immunofluroscence etc. Disease case studies such as: Retinitis pigmentosa, Night blindness, Blind vision, Myopia, retinal degeneration, color vision defect etc. Pathway studies such as: signal transmission, membrane depolarization, cell death, T-cell activation, transport, cell activation, muscle contraction, phototransduction, segmentation, lymphocyte activation and differentiation etc. 18

BENEFITS Provide beneficial antibodies at affordable prices and in large amount. Check reactivity in variety of model species such as: Human, Mouse, Rat, Bovine, Porcine etc. Open new areas of research in disease diagnosis and therapeutic drug studies. 19

REFERENCES Alpadi, K., Magupalli, V.G., Käppel, S., Köblitz, L., Schwarz, K., Seigel, G.M., Sung, C.H and Schmitz, F. (2008). RIBEYE recruits Munc119, a mammalian ortholog of the C. elegans protein unc119, to synaptic ribbons of photoreceptor ribbon synapses. J. Biol. Chem. 283, Mohammad Hossein Maghami, MS; Amir Masoud Sodagar, PhD; Alireza Lashay, MD Hamid Riazi-Esfahni, Mohammad Riazi-Esfahani, MD. Visual Prostheses: The Enabling Technology to give Sight to the Blind. Jean Defourny, François Lallemend, Brigitte Malgrange. Structure and development of cochlear afferent innervation in mammals, American Journal of Physiology - Cell Physiology Published 1 October 2011 Vol. 301 no. 4, C750-C761 DOI: /ajpcell Andrew W. Tai, Jen-Zen Chuang and Ching-Hwa Sung. Localization of Tctex-1, a Cytoplasmic Dynein Light Chain, to the Golgi Apparatus and Evidence for Dynein Complex Heterogeneity oductionAndPurification.aspx 20

REFERENCES

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