MOTOR PROTEINS,LIPIDS,AND THEIR ALLIANCES

 

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Welcome

 

Namaste, and welcome to our website. We are trying to understand how very small things move over very small distances inside the cells of the body. Our work spans the traditionally defined disciplines of Biology, Physics, Chemistry and Computation. You can explore our website using the links above. If you want to know why we are excited about what we do, read on below…  

 

Background

 

Some of the most insightful research in biology has come by applying ideas and tools from the physical sciences on biological systems. Quantitative answers have been obtained through novel techniques, such as those invented to measure function of a single biological molecule (for example, a protein). Such “single molecule” experiments are technically challenging, and are typically done with the molecule placed in an artificial (in vitro) environment. While these experiments are pioneering in their own right, it is often not possible to relate their results to real-world biology. This is because inside a cell (in vivo), there is almost never anything like a “single molecule”. Biological molecules usually work as part of a larger complex, where many of them come together to achieve a common function. It is the NET function of this complex that is relevant, and not just that of the single constituent proteins. Our goal is to extend the precision of single-molecule techniques inside living cells, and directly measure function of a protein-complex in real-time in a cellular environment.

The living cell is an assembly of specialized factories with a constant give-and-take of material occurring within them. Nanoscale proteins called Molecular motors (Ref 1,2,3) carry cellular material as “cargo” from one factory to the other (e.g. mRNA, vesicles, endosomes). This transport of material is essential for the cell and its factories to function. A heavy traffic of cargoes of different size and shapes can be observed under the microscope. The motors attach to specific cargo, and walk in a step-like manner on pre-laid tracks inside the cell. In order to walk, the motors generate forces about a million-million times smaller than what we use in our day-to-day life (Ref 4,5). These machines are indeed the unit generator of force for most cellular processes. As with everything in biology, complexity throws up a challenge -- different motors with inclination to walk in different directions are usually present together on a given cargo, along with a host of other non-motor proteins (Ref 6,7). How do these antagonistic motors work (or not work) together??

And, then, there are the lipids on the membrane of the cargo which provide a platform for the motors to assemble before they can generate force (read more). Lipid-motor interactions are therefore likely to be a central determinant in many mechano-biological processes. Very little, however, is known about this aspect of mechanobiology. Our goal is to understand this alliance between force-generating motors and lipids. In this context, we are working on two problems of direct relevance to human health that are described below.

 

Projects

 

Transport and Degradation of pathogens inside immune cells

Sanghavi et al, Current Biology 2018;    Rai et al, Cell 2016 ;    Rai et al, Cell 2013 ;    Soppina et al, PNAS 2009

We recently demonstrated how cholesterol dictates dynein motor function inside cells during phagosome maturation. The geometrical organization of dynein on a phagosome's lipid membrane was shown to be modified by clustering dynein into cholesterol-rich lipid rafts. This dramatically altered dynein’s cooperative function with no apparent effect on it’s single molecule properties. These results are relevant to immune evasion by pathogens (e.g. Mycobacterium, Salmonella and Leishmania). Building on this finding, we will try to address how lipids and proteins secreted by pathogens influence the organization of motors on pathogen-containing phagosomes, and if this is relevant to immune evasion by these pathogens.  Popular articles on this project:-     LINK   LINK    

 

What your Liver does when you sleep

Rai et al, PNAS 2017;     Sadh et al PLoS ONE 2017;    Barak et al Nature Methods 2013

Excess fat circulating around in blood means obesity, diabetes and heart disease. We recently found a mechanism that allows the liver to control fat secretion into blood. When we fast, for example we go to bed after dinner, glucose is in plenty but soon it runs out and the body must burn fat to keep going. Fat-containing tissues respond by sending off fat to the liver, which gets bloated with fat, but still continues to secrete this fat at a constant rate. This remarkable elasticity of the liver prevents dangerous fat overload in the blood. But, how does all this work at the level of cells and molecules? We showed that insulin recruits the motor protein kinesin to fat bodies. Kinesin propels fat to the periphery of liver cells, from where the fat is secreted out. As the night wears on, fat accumulates in the liver, but insulin levels also diminish. Kinesin now falls off from the fat bodies, secretion is toned down and systemic fat homeostasis can be achieved. So, you should probably thank your liver when you get up fresh as a flower tomorrow morning. Popular articles on this project:-  LINK    

 

 

 

How we do the Research

 

The obvious, yet most remarkable thing about molecular motors is that they actually move !! This motion can be directly observed under a microscope, and interpreted in terms of the motor’s function. We record and analyze this motion, measure the forces generated during motion using optical tweezers, try to identify the molecules driving such motion using protein biochemistry, and also interfere with the motion using genetic/biochemical techniques. We use the microtubule-motor dependent motion of endosomes, phagosomes and lipid droplets as our model systems, and interrogate these systems at multiple levels of complexity:-

−        Directly inside living cells.

−        Semi in vitro experiments in cell extracts.

−        With purified single motor proteins coated on sub-micron plastic beads.

 

To read more, see Summary of our papers on the Publications page

 

Further Reading

 

1.    Vale RD: The molecular motor toolbox for intracellular transport. Cell 2003, 112:467-480.

2.    Vale RD, Milligan RA: The way things move: looking under the hood of molecular motor proteins. Science 2000, 288:88-95.

3.    Schliwa M, Woehlke G: Molecular motors. Nature 2003, 422:759-765.

4.    Visscher K, Schnitzer MJ, Block SM: Single kinesin molecules studied with a molecular force clamp. Nature 1999, 400:184-189.

5.    Mallik R, Carter BC, Lex SA, King SJ, Gross SP: Cytoplasmic dynein functions as a gear in response to load. Nature 2004, 427:649-652.

6.    Mallik R, Gross SP: Molecular motors: strategies to get along. Curr Biol 2004, 14:R971-982.  

7.    M. Welte. Bidirectional transport along microtubules   Current Biology, Vol 14, R525-37 (2004)

8.    S.P. Gross.  Hither and yon: a review of bi-directional microtubule-based transport   Physical Biology, Vol 1, R1-11 (2004)

9.    Hirokawa N, Takemura R: Biochemical and molecular characterization of diseases linked to motor proteins. Trends in Biochemical Sciences 2003, 28: 558