Intercellular
coupling i.e. coupling of intracellular biochemical pathways
plays an important role in syncyctial coordination. The long term
objective is a quantitative understanding of the role of intercellular
gap junction communication in tissue function. Sub aims are (1) To
measure the intrinsic permeability of a variety of fluorescent probes
across homo- and hetero- oligomeric gap junction (GJ) channels in
transfected cells. (2) To build artificial double-bilayer systems, incorporate
whole GJ channels, and study their properties in controllable external
conditions.
a) Permeation of macromolecules
through gap junction channels. We will explore the dependence of permeability
on channel composition. Absolute rates will be determined by simultaneous
electrical (patch-clamp) and fluorescent measurements. Transfer rates of
probes will delimit the bounds on the permeation rates of messenger molecules
with similar size and charge.
b) Forming lipid bilayers, incorporating whole
GJ channels: In collaboration with Dr. S. Tripathi (TIFR, Bombay), we have
(i) incorporated GJ hemichannels (half a GJ channel) in a single bilayer,
mimicking previous work (ii) tried various methods to form artificial double
bilayers, including multiple patch pipette membranes picked from bilayer
“bubbles” and multiple “tip dips”, with some success already (unpublished
results). We will next attempt to incorporate intact GJ channels in double
bilayers.
Single molecule research
- DNA translocation across nanopore: Study of single molecule DNA has
remained focus of interest in recent years. Progress made in the method of
analyzing sequence information from ssDNA translocation across membrane
pores has attracted interest of many researchers. We are interested in the
study of DNA translocation across a -hemolysin
channel. We will be exploring the combination of single molecule
manipulation and detection methods along with single channel
patch-clamp recording, to study DNA translocation along the nanopore
and develop more sensitive methods to extract sequence information.
- Genetic Circuits: Knowledge of spatio-temporal information of protein
molecule invivo is precious to biologist. We are interested in the
simulation of heat-shock response of a bacterial network and extract
spatio-temporal pattern of gene expression products (mRNAs and proteins).
Fluorescence based experiments will be devised to capture real-time
dynamics of mRNA and protein synthesis in single cells hosting
signal-response networks. To this end, sensitive fluorescence
microscopy and fluorescence correlation spectroscopy will be
developed.
Development of biosensors
Molecules
spontaneously organize on metal surfaces forming monolayers. Surface
modification can enable molecular recognition (chemical sensing). One possible
approach towards the development of bio-sensors is by using the modified
metal surfaces and engineering (functionalizing) the monolayer for effective
interaction with the target molecules by using the inherent selectivity
of biomolecules such as enzymes and antibodies. The interaction of the monolayer
with the target molecule can be monitored by fluorescence, or changes in
electrical conductivity or refractive index.
a.) Adsorption
of Lipases on nanoparticles (gold, silver) for enhancing their catalytical
properties This will be studied by Raman spectroscopy. The lipase enzyme
as well as the enzyme complexed with inhibitor will be adsorbed on a nanosurface.
The function of these molecules will be followed spectroscopically.
b.) Quantum dots in monolayers: Immobilization of
quantum dots on atomically flat surfaces (eg.. mica) and on near atomically
flat surfaces (eg., silicon wafers) to study the interplay between molecules
and the surfaces of nanoparticles. Additionally these surfaces are tailored
to produce monolayers of desired functionality. Such modifications of the
functionality might lead to changes of optical properties of quantum dots.