Tata Institute of
Fundamental Research, Mumbai ,
India
Mallik Lab
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 make single-molecule-like
measurements to understand how protein complexes function inside living cells.
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 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. Thus, most of the action happens at the nanoscale.
Different motors with inclination to walk
in different directions may be simultaneously present on a given cargo along
with a host of other non-motor proteins. We are working to understand the
cooperative function of interacting motors within this protein complex. The big
questions that will drive our research are:
- How is the relative activity of antagonistic motors
regulated to achieve directed transport ?
(Ref. 8, 9)
- What is the role of various motor and non-motor
proteins resident on the cargo?
- All molecular motors are not designed the same, and
therefore do not have similar capabilities. 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. 7; also see Soppina et al, PNAS 2009)
Why this is 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 8). 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 Hirokawa et al, Ref 4).
How this research is being done
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… For a short
summary of our recent papers, please 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, see image on right), we
can measure forces generated 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. 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 to understand the
bare bones behaviour of motors. −
Computer simulations using parameters
directly obtained from the above experiments. |
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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.
Hirokawa N, Takemura R: Biochemical and molecular characterization of diseases
linked to motor proteins. Trends
in Biochemical Sciences 2003, 28: 558
5.
Visscher K, Schnitzer MJ, Block SM: Single kinesin molecules studied with a molecular force
clamp.
Nature 1999, 400:184-189.
6.
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.
7.
Mallik R, Gross SP: Molecular motors: strategies to get along. Curr
Biol 2004, 14:R971-982.
8.
M. Welte. Bidirectional transport along microtubules
Current Biology, Vol 14, R525-37 (2004)
9.
S.P. Gross. Hither and yon: a review of bi-directional
microtubule-based transport
Physical Biology, Vol 1, R1-11 (2004)
10.
A.R.
Reilein et al. Regulation of molecular motor proteins Int
Rev Cytol. Vol 204:179-238 (2001)