Principles of subcellular organization and transport

Figure: An overview of the known organisational details of membranous endosomes, cytoskeletons and the motor proteins in a single cell.

We are interested in the dynamic organisation of the cellular processes. We want to understand how the itinerary of cargoes such as endogenous proteins and pathogenic entities are operated from the plasma membrane to their intracellular destinations. Although the identities and general distributions of individual machines and regulatory elements in endocytic pathways are known, how different protein machinery, membrane organisation and cargo biochemistry integrate to orchestrate protein and lipid transport and cargo delivery is unclear. Imaging these fast intracellular dynamics require an imaging system that can image multiple fluorescent species at high 3D volumes per second at an appropriate spatial resolution. We are focused on using the lattice light sheet microscope (Betzig lab, Chen et al. Science, 2013), to image the fast moving membranous organelles – endosomes of the cell. We want to measure the precise parameters of endosomal dynamics – fusion and fission events, cargo exclusivity, organelle identity, mobility characteristics, in order to understand how do molecules in space and time organise cargo transport in a noisy environment.

Currently, we are investigating the early endosomal dynamics. Generally, cargoes from the plasma membrane enter the early endosomes, from where they are sorted by different mechanisms to distinct intracellular targets. Shown on the left is a simplified view depicting the different processes necessary for endosomal functioning – Anchoring (brown), sorting (blue), membrane scission (pink) and transport (green). An imaging based mechanistic approach has been largely inefficient owing to the fast dynamics of  endosomes. With lattice light sheet, which enables rapid imaging of cellular volumes, we aim to catch these endosomes in action. A recent review from our lab describing the early endosomal biochemistry and open questions can be found here.

The second approach in the lab compliments the live cell techniques. The cytoplasm of the cell is extremely crowded and the microarchitecture of the dense cytoskeletal network is unclear.  These factors influence the motion of an endosomal vesicle in unpredictable manner. A semi-reductionist approach, where purified membranous organelles are reconstituted in a lipid bilayer to study reactions or on cytoskeletal elements to probe properties of associated motor proteins and mobilities is a powerful tool to extract biophysical and biochemical proterties without noise from dense intracellular milieu. This approach  permits quantitative interrogation of phenomena involved in trafficking processes at near-cellular complexities.

(left) the lattice light sheet microscope. (right) movie of Rab 5 (blue) and Rab11 (yellow) in living cells. (below) movie showing tracking of endosomes in 3d. 



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