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
:-
- 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)
- How is the relative activity of antagonistic motors
regulated to achieve directed transport ?
(Ref. 7,8)
- 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