In order to comprehend the role of spatial heterogeneity in synapses, it is important to determine structural organization and real-time regulation of molecular components of micron and nano sized protein domains. This was particularly challenging in 2 aspects. a) Technical limitations to image below the diffraction limit at single molecule resolution b) Feasibility for long term live cell imaging in physiological conditions particularly for primary cultures of hippocampal neuronal cells which are highly sensitive to the imaging conditions. We were successful in developing and adapting novel microscopy and spectroscopy paradigms to understand the molecular changes in live neurons at the spatial scale of 20-100 nm (Photoactivation Localization Microscopy (PALM), Stochastic Optical reconstruction Microscopy(STORM), super-resolution radial fluctuations (SRRF)) and temporal scale from 1ms-20ms (Single Particle Tracking (SPT), Fluorescence Correlation spectroscopy (FCS). Furthermore, our lab has recently developed two novel technologies combining the recent advances in molecular labelling and microscopic analysis namely Stochastic Fluorescence Correlation Spectroscopy (sFCS) and Fluorogen activated SRRF (FA-SRRF)).
Collaboration
Recent advances in microscopy have changed the way we understand the role of molecular organization during information coding and processing at single synapses. Super-resolution and electron microscopic imaging of excitatory synapses confirm that synapses are organized into discrete functional domains where molecules assemble and disperse, controlling the efficacy of synaptic communication. This paint a new picture of synapses were several discrete nanomachineries function in tandem to modulate signals in real-time. Despite such a novel idea, little is known about how this molecular organization helps the synapse to communicate or their role in coding information in synapse to generate complex cognitive processes like learning and memory. To understand this, we probed the role of presynaptic organization involved in neurotransmitter release and how this organization aids in controlling local synaptic transmission as well as homeostatic scaling of synapses upon global activity modulation.
Collaboration
Synthesis of new proteins is found to be a vital step controlling the strength of a synapse. The strength of synaptic connections is determined mainly by the repertoire and expression levels of pre- and postsynaptic proteins. Presence of mRNAs coding for synaptic and signalling molecules inside the synapses has been well documented. Novel synthesis of synaptic molecules is initiated few minutes after the synaptic activity sets in. Current consensus is that after synaptic activity, naïve molecules are recruited to synapse in time scales of minutes. It is hard to conceive the notion that selective synapses can be strengthened by long range transport of newly synthesized molecules. The presence of ribosomes/polysomes in synapses indicate that in many cases new proteins can be synthesized locally at synapses. Not much is known about the machinery involved in the local synthesis of molecules at synapses. A key process by which neuronal activity in long term regulates synaptic proteome is via modulation of mRNA translation efficiency. This is achieved by stimulus-dependent removal of repression of transported mRNAs at synapses and activation of signalling pathways regulating mRNA translation. Though there are evidences indicating local translation in spines, little is known about the distribution and activity of translation machinery in dendritic spines or other dendritic sub compartments. We have incorporated a strategy of looking at the activity of translation factors and the organization of translating ribosomal complexes in morphologically similar and distinct sets of dendritic spines in trying to understand this problem.
Collaboration
Emerging evidences over the last two decades of research shows that many neurodenerative diseases begin as synaptic dysfunction. It is of immense value to understand how the fine organization of synapses are affected during the onset of diseases. Alzheimer’s disease (AD) is described as a progressive neurodegenerative disorder characterised by impairment in higher cognitive abilities like memory and decision. AD is characterized by the presence of neuropathological markers like senile plaques formed by the accumulation of a 39-43 amino acids peptide known as β-amyloid i.e. Aβ. Aβ is derived from a type 1 trans membrane protein called β-Amyloid Precursor Protein (APP) by sequential proteolytic processing involving multiple secretases. In the last decade, a paradigm shift was observed towards understanding the molecular and biochemical pathways implicated in AD. It was postulated that early onset of AD originates at the level of individual synapses much before the manifestation of external symptoms in AD. This has led to profound interest in understanding the dysfunction of synapses at the early stages of AD. In a seminal study from Wilhelm et. al, it was observed that about 1% of the total synaptic protein content was APP. A careful evaluation of presynapse indicated around 7000 molecules of APP distributed only on the presynaptic surface with a molarity of 29µM. Molecular changes in APP and its regulation is a major risk factor towards development of AD. Presence of such high concentrations of APP prompts better evaluation of APP distribution and instantaneous regulation which might provide us fundamental understanding that lead up to a shift in the processing of APP away from its normal regulation and function.
Collaboration