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Motivation

Some of the most exciting 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 not possible to directly 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. This will require a synthesis of ideas, techniques and analysis spanning the traditionally defined disciplines of Molecular biology, Biochemistry, Physics, Chemistry and Computational analysis.

Focus

The living cell is an assembly of specialized factories with a constant give-and-take of material occurring within them. Tiny 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 material of different size and shapes can be observed under the microscope, and in many cases appears to happen in a highly regulated and disciplined manner. To achieve this transport, the cell uses a network of roadways that are actually polymerized protein filaments. The motors attach to specific cargo, and walk in a step-like manner on these roads 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). Thus, most of the action happens at the nanoscale.

Different motors with inclination to walk in different directions may be simultaneously present as a protein complex on a given cargo, along with a host of other non-motor proteins (Ref 6,7).  It is this protein complex that we are probing with biophysical assays. The big questions that drive our research are :-

  1. All molecular motors are not designed the same, and therefore do not have similar capabilities. Yet, they work together on a single cargo. What is the advantage of this heterogeneity? What are the specific adaptations that allow a particular motor to work with another class of motors, and do something useful in the cell? (Ref. 6; also see Soppina et al, PNAS 2009)
  2. How is the relative activity of antagonistic motors regulated to achieve directed transport ?  (Ref. 7,8)
  3. What is the role of various motor-associated non-motor proteins resident on the cargo?

Why is this important ?

To the biologist :- How motors are regulated to achieve organized transport could provide some answers to a fundamental biological question: How does organization and structure arise from microscopic complexity in the living animal? Transport driven by Molecular motors is crucial for almost every conceivable cellular process, and how the activity of motors is regulated directly impacts all these processes.

To the physicist :- Understanding the architecture and function of molecular motors can elucidate basic design mechanisms in nature for implementing functional machines at the size of nanometers (for example, see Ref 5). Biophysical measurements like what we do are unique in that they directly measure biological function in real-time in an in vivo context.

Health :- Molecular motors are essential proteins, critically important for a myriad biological processes. Motors are involved in Golgi positioning, chromosomal segregation, mRNA and protein transport, neuronal transport, development of the central nervous system, localization of cytoplasmic determinants to establish embryonic body axis etc. For all the above processes to occur normally, regulation of the relative activity of different motors is essential. Dysfunctional molecular motors result in numerous diseases (Cancer, Alzheimers, Motor neuron degeneration etc; see Ref 9).

How is this research being done ?                … For a short summary of our recent papers, go to the Publications page …

The 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 are studying the microtubule-motor dependent motion of endosomes and phagosomes as a model system. Cells are constantly acquiring extracellular material (e.g. nutrients) through invaginations of the plasma membrane that break off and then move along microtubules through the cytoplasm. This motion is bidirectional due to the presence of opposing kinesin and dynein motors on an endosome. This achieves fusion of the endosome with late endosome/lysosomes, and also results in recycling of membrane-bound endocytic receptors to the plasma membrane. Using an optical trap (a highly focused laser tractor beam), we can measure forces generated by motors during such motion, which has allowed us to count the number of motors on a single endosome in real-time. This tells us how motors of different kinds come on and off from the endosome, and how they respond to a “load” applied by the opposing motor. Such experiments are being done at several levels of complexity to understand how cooperative function emerges within interacting motor-ensembles

        Directly inside living cells.

        Semi in vitro experiments with organelles (e.g. endosome, phagosomes) extracted from cells.

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

        Computer simulations using parameters directly obtained from the above experiments.

We have recently shown that dynein and kinesin engage in a tug-of-war on single endosomes inside the cell (see figure below). The motors function very differently at the single-molecule level, and this difference may help in regulating endosome biogenesis. For details, see Soppina et al, PNAS 2009

 

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