Martha Constantine-Paton - US grants

Six principal investigators in Building 68 of the MIT Department of Biology, proposed to use high-performance digital microscopy to analyze protein distribution, axon guidance, cell motility, chromosome segregation and bacterial cell division in live and fixed cells. This work will be undertaken with an applied Precision DeltaVision Multi-Model Microscope (MMM) that combines laser-scanning and deconvolution- based wide field imaging into a particularly powerful instrument. The microscope will be integrated into a powerful server-based environment so that data can be effectively gathered and analyzed by various users. The Constatine Paton lab will use the MIT MMM in place of a confocal previously available at Yale to examine axon targeting in live visual cortexes fro rodents and amphibians. The goal of the work is to understand how neuronal activity affects axonal development. The Garrity lab will use the microscope to examine axon guidance in live Drosophila tissues that carry GFP markers in specific neurons. Analysis of targeting by these neurons in different genetic backgrounds will reveal the molecular basis of axon guidance in the fly eye. The Gertler lab uses liver-cell imaging to examine actin-based motility in cells derived from wild type and mutant mice. Its immediate goal is to determine how the Mena protein regulates the actin cytoskeleton. The Grossman lab will use the MMM facility is to examine the spatial relationship among proteins involved in chromosomal replication in B. subtilis. Three is a remarkable degree of localization of subtilis, replication proteins and high-resolution imaging of live cells is expected to reveal how this localization varies with the cell division cycle. The Sinskey laboratory will exploit spatially resolved and quantitative information t derived from deconvolved images to examine the biosynthesis of the critical PHA polymer in R. eutropha. PHA is of central importance to the metabolism of cells and has potential application in degradable plastics. The Sinskey lab's work is an early used of advance imaging in metabolic engineering. The Sorger Lab will use the high acquisition speed of the MMM to capture three-dimensional time lapse data on chromosome segregation in wild type and knockout mouse and yeast cells. The goal is to uncover mechanisms responsible for the high accuracy of chromosome segregation. Finally, the users group will, in conjunction with Applied Precision, present two advanced training courses per year at MIT. These will be critical for the long-term operation of the Multi-Model Microscope facility and should be of general benefit to microscopists in the Boston area.

[unreadable] DESCRIPTION (provided by applicant): Abnormal activity-mediated developmental regulation of glutamatergic neurotransmission is implicated in amblyopia, disruption of stereopsis and changes in retinal function. Thus, an understanding of molecular bases of synaptic plasticity during visual development is central to the mission of the National Eye Institute. Data suggest that myosin VA (myo VA) moves membrane vesicles on actin and delivers glutamate receptors (GRs) to synapses. Our recent data indicate that this process is rapidly regulated by light-driven synaptic activity following eye-opening. We hope to establish the mouse mutant strain flailer as a model for mechanistic studies of myo VA in synaptogenesis and for visual activity-dependent trafficking of the NMDA receptor and its mature scaffolding complex, the postsynaptic density proteins PSD-95 and GKAP-95. Flailer mice express a fusion gene containing the promoter and first two exons of a brain-specific G protein plus the C-terminal of myo VA (Jones et al. 2000). The flailer protein appears to act as a dominant-negative myo VA in the central nervous system. Flailer mice have neural abnormalities similar to those of myo VA null mutants, but unlike the null mice homozygous flailer mice survive and breed normally. Functional changes caused by the flailer mutation have not yet been explored at synaptic levels. We propose to determine (l) if flailer protein and normal myo VA are present in retina, superficial visual, layers of the superior colliculus (sSC), and visual cortex (VC) of homozygous flailer mice, and (II) if flailer mice show activity-dependent transport of the PSD-95/GKAP NR scaffolding complex to visual synapses within 6 hours of eye-opening. We will perform quantitative immunoblotting of homogenates of these regions and of fractions enriched for dendritic or whole-lysate protein from VC and sSC using antibodies that distinguish normal and flailer myo VA, PSD-95, GKAP-130 and GKAP-95. If flailer protein is present in retina, we will (Ill) determine whether flailer and WT retina are similar in the distribution of PSD-95 after eye-opening and in ganglion cell responses to light. Retinal function will be analyzed by our collaborator Dr. Niang Tian at Yale University. He will use ERG, mutielectrode arrays, and whole-cell patch clamping to study retinal ganglion cell responses to light. We will perform quantitative confocal analyses of retinas processed for PSD-95 immunocytochemistry before and after eye-opening. Finally, (IV) we will determine, central visual pathway glutamate receptor function is normal in flailer mouse brain using whole-cell voltage recordings of glutamate receptor currents in slice preparations of the sSC.

[unreadable] DESCRIPTION (provided by applicant): N-methyl-D-aspartate glutamate receptors (NRs) and glutamate circuit development have been implicated in neurological diseases with initiating events in perinatal periods when glutamate networks are developing. Two prevalent dysfunctions of this type, autism spectrum disorders and schizophrenia, are good examples. For both autism and schizophrenia, genetic linkage analyses have identified molecules involved in normal NR function. Our goal is to understand NR function in early circuit formation and provide information that would help mitigate or eliminate such devastating diseases. Recently, NRs containing NR2B subunits were shown to produce long-term synaptic depression (LTD) at Schaffer collateral-CAl synapses, and activation of NRs containing NR2A subunits produced long-term synaptic potentiation (LTP) at the same contacts. These studies did not reveal the differences between the two subunits responsible for their abilities to produce dichotomous outcomes. We hypothesize that the NR2A and NR2B subunit tails are responsible for the subunit-specific effect of the NR on synaptic plasticity. The tails contain phosphorylation and interaction domains and also have a differential ability to anchor the NR to PSD-95 and SAP102, the predominant membrane-associated guanylate kinases (MAGUK) that bind the NR and its signaling complex to post- synaptic densities in fore-and mid-brain regions. To test this hypothesis, we made two chimeric subunits. The coding regions of the external and transmembrane domains ( heads) of the NR2A and NR2B subunits were separated from the cytoplasmic tail regions, and the head sequences for each subunit were recombined with the tail sequences of the other, thereby producing two chimeric proteins. We propose to examine HEK293 cells co-transfected with each of these constructs and NR1. NR function will be examined with Ca++ imaging and with whole-cell patch-clamping to examine the currents and dynamic properties of the NRs containing each hybrid subunit. These responses will be compared with those from HEK cells expressing wild-type subunits. We will also test the chimeras after transfection into NR2AKO hippocampal neurons. Finally, using lentiviral transfection of each chimeric and each wild-type protein into NR2AKO mice and patch-clamping infected neurons in hippocampal slices, we will determine if the NR2 subunit tails dictate LTP versus LTD. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Two photon excitation offers significant advantages over other types of microscopy-based imaging, especially for imaging living cells either in vivo or in thick slices. Indeed, the technology underlying two photon imaging has rapidly advanced over the last few years. The objective of this proposal is to provide access to this technology to the growing MIT Neuroscience community. This instrument will enable researchers to perform simultaneous imaging of two or more fluorescent proteins in experiments combining scanning with sophisticated techniques, including electrophysiological recording, stimulation of cells containing channelrhodopsins or halorhodopsins, or rapid uncaging of bioactive molecules. Access to these cutting-edge techniques will enhance a broad variety of research programs, and facilitate an expansive array of NIH-funded research, including studies addressing the following: molecular regulation of synapse formation in the developing brain, factors controlling the migration and integration of newly born neurons in the adult brain, neurite regeneration following injury in C. elegans, structural changes following fear learning and extinction, factors governing inhibitory synapse formation and stabilization, the role of protein misfolding in the progression of mouse models of Parkinson's and prion disease, factors regulating the development of brain ventricles, studies of the role of inhibitory interneurons and astrocytes in visual cortex dynamics, spine dynamics in mouse models of autism and schizophrenia, and the contribution of postsynaptic scaffolding proteins to synaptic plasticity. This instrument will also be used to further the advancement of two photon imaging technologies, by enabling research aimed at developing software scanning protocols for rapid two-photon activation of cells and rapid readout of neural activity, and particularly facilitating the development of algorithms for rapid identification of cells during the imaging process.