Understanding the role of Clathrin light chain in Development

Misri, Gunjan (2021) Understanding the role of Clathrin light chain in Development. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Gunjan Misri (16MS116)) 16MS116_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Clathrin is a major coat protein required for the formation of vesicles inside a cell. The process of forming highly curved vesicles from a planar membrane requires a well-balanced interplay of over 40 known different proteins. In this study we try to understand the role of Clathrin light chain, governing various development and signaling pathways. In our study we use Drosophila melanogaster as our model system, which has a single Clathrin light chain and heavy chain coding gene. We used CRISPR Cas9 to make Clathrin light chain knockouts. The adult Clathrin light chain knockouts showed a severe defect in locomotion. A negative geotaxis behavioural climbing assay was performed to quantify the defective locomotion in adult knockout flies. This behaviour was attributed to the neuron-specific role of Clathrin light chain in recycling synaptic vesicles, following the neuron-specific knockout of the protein. A larval crawling assay was also performed which showed less mobility in the Actin promoter driven (ubiquitous) Clathrin light chain knockout larvae. Although, no significant defect in movement was seen in larvae of the neuron-specific, ELAV promoter driven protein knockouts. The mortality of knockouts was considerably high, compared to WT-CsBz adult flies. At a lower rearing and assay temperature of 18°C, the mortality and climbing ability of Actin promoter driven adult Clathrin light chain knockouts was relatively lower and better, compared to knockouts reared and assayed at 25°C. We also showed using the in-vitro platform of supported membrane tubes how one can reconstitute the process of Clathrin polymerization on membranes in real time, using fluorescence-based microscopy to understand the effect of Clathrin light chain on the kinetics of Clathrin assembly.

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