Difference: CemDualBeamInvestigators ( vs. 1)

-

Dave Hall/Scott Emmons

Hall: biosketch, othersupport

Emmons:

I have forwarded this idea to Scott Emmons here at AECOM. He and I have a project that may be perfect for this application. We are reconstructing the male worm nervous system from thousands of serial thin sections. We are currently trying both the old way (very hard) and the Atlum approach with Jeff Lichtman.

Other likely interested parties here in NYC would include Cori Bargman and Shai Shaham, both with labs at Rockefeller, and interested in 3D reconstruction of worm nervous system elements in mutants.

My only problem with the device as you describe it is that Scott and I would want to mill away from a broader mesa - from a zone of about 80 x 80 microns to encompass an adult nematode head. Younger animals (also of interest ) would be smaller - but still about 20 x 20 microns. Does that seem feasible?

Also, I talked today to Ann Wehman from the Nance lab. She also has a project that seems perfect for electron tomography (now), and maybe for ion beam milling when it becomes available. Working on a portion of the young worm embryo where even the 8 x 8 micron mesa might be large enough to encompass the zone of interest.

Fred Maxfield

biosketch, othersupport, project

I am very interested! I had a description handy from a request for a conventional EM to check samples for our tomography studies.

Please suggest revisions that would be appropriate for this.

I think that Tim Ryan <TARyan@med.cornell.edu> would also be interested. I've attached a description of his project.

[Fred Maxfield Project]

Tim Ryan

biosketch, othersupport, project

Understanding the Heterogeneity of Synaptic function Timothy A. Ryan, Ph.D. The main goal of this project is to develop and apply methods that will allow one to correlate functional properties of individual synaptic contacts between neurons with their ultrastructural organization. Recently we developed successful approaches that allow one to robustly characterize functional properties of individual synaptic terminals in the same neuron. Our goal is to see how individual ultrastructural characteristics correlate with functional properties at the single synapse level.

Dear David:

I have been meaning to contact you for several years now about our potential interests in using the NYSBC. Our potential use has always shifted to the back burner as I have never had a pair of hands to dedicate to this anyway. However I am supportive of your efforts to secure a new microscope for the facility. I don't think at present I need to discuss the details more. Certainly one can imagine using a smaller area than the 150 x 150 I previously mentioned. In particular we have just started using a mcherry-NCAM-HRP construct that when expressed labels the plasma membrane quite well. Running a DAB reaction would allow us to much more easily identify the single transfected neuron we had previously done opto-physiology on (and the nasty DAB polymer would be all extracellular, and hopefully not interfere with what we want to see which is the synaptic vesicle distribution). Given this it may be possible to use much smaller areas than I first mentioned.

Let me know what you need.

Tim

[Tim Ryan Project]

Jeremy Nance

I’m a postdoc in Jeremy Nance’s lab and we chatted about my poster on a P4-ATPase at the last Skirball retreat. I’ve recently started doing EM of my P4-ATPase mutant with the Skirball EM facility and we discovered a really interesting phenotype. It appears that the cell-cell junctions are full of protrusions or vesicles instead of being a flat contact. I’ve attached a picture of a tri-cell junction from a 4-cell embryo. We’d like to know whether these are protrusions or vesicles and find out whether they’re open to the cytoplasm. We thought that Electron Tomography would be a great way to visualize this.

I met with Dave Hall today and he suggested that we use the NYSBC facilities. He mentioned that he’s been working with KD Derr and has had great success with 150 nm sections for tomography. We were also hoping to do high-pressure freezing of our embryos, potentially in utero. Is there a high pressure freezer here at the Skirball or would we be able to use the one at NYSBC? Dave mentioned that the HPFs at NYSBC were currently down. Can we meet sometime to get your take on the phenotype and talk about how best to use the facility?

Xuejun Jiang

biosketch, othersupport, project

Autophagy, a cellular process mediated by lysosomal activity and unique intracellular membrane trafficking and reorganization, is conserved in all eukaryotic cells and crucial for normal development and cell growth. Autophagy functions to degrade long-lived proteins and organelles in order to maintain cellular homeostasis and to promote survival under stressful conditions. Deregulation of autophagy is involved in human diseases such as cancer, neurodegenerative disorders, infectious diseases and cardiac diseases. Although many autophagy genes (Atg) have been identified, in mammals, how autophagy is induced and regulated, and more importantly, how the gene products of Atg's dictate the specific intracellular membrane events are not defined. One of such fundamental questions is the mechanism underlying maturation of autophagosome, which is the intracellular membrane structure that defines autophagy and is essential for the process. Recently, we identified a new multi-component protein complex containing ATG1 that regulates mammalian autophagy, and obtained strong evidence suggesting that this protein complex controls the transition of pre-autophagosome/isolation membrane to mature autophagosome. To further examine/validate this model, we propose to perform a systematic electron microscopic study to examine pre-autophagosome and mature autophagosome structures in a series of mammalian cell lines with defined genetic background (overexpression and RNAi knockdown of individual components of the protein complex). We also propose to determine the Cryo-EM structure of this multimeric protein complex. We believe the proposed EM study in collaboration with NYSBC will be critical not only for this specific project but also for our long-term study of autophagy.

We have confirmed the involvement of the protein complex in autophagy, characterized biochemical interaction of individual components both in cells and using purified recombinant proteins. We also investigated how nutrient starvation regulates activity and cellular localization of the complex. Based on an exhaustive immunofluorecence study of this complex and other autophagic factors using cells with various genetic background, we conclude that this protein complex dictates switch from pre-autophagosome to mature autophagosome (which needs final confirmation from the proposed EM study). Since we have already generated recombinant proteins for all components of this complex, the possibility to determine its cryo-EM structure is under consideration.

Wenbiao Gan

biosketch, othersupport, project

Specific Aim. Determine structural plasticity of presynaptic terminals in the mouse cerebral cortex using correlated light and serial EM (Gan)

Introduction Changes in synaptic connections are fundamental to the development and plasticity of the nervous system. Determining the degree of synaptic plasticity would significantly increase our understanding of how neural circuits are established and maintained (Grutzendler et al., 2002; Pan and Gan, 2008; Xu et al., 2007; Zuo et al., 2005). It is well-established that axonal terminals are highly dynamic early during development as neurons make proper synaptic connections to form functional neural networks. However, it remains unclear to what degree presynaptic terminals change in adulthood, largely because it is difficult to identify presynaptic terminals as they have fewer unambiguous structural marks at the optical level.

Recent generation of Green/Yellow Fluorescent Protein (GFP/YFP)-expressing mice and two-photon microscopy permit, for the first time, long-term high-resolution imaging of axonal structures in vivo (Grutzendler et al., 2002; Pan and Gan, 2008). In such transgenic mouse lines, there are many fluorescently labeled axonal varicosities along the axonal shaft (Grutzendler et al., 2002). Using the in vivo transcranial imaging approach, we have been able to follow YFP-labeled varicosities over time to determine changes in their number, size, and location in different cortical regions of mice at various ages. Our preliminary studies using serial electron microscopy reconstruction revealed that these varicosities indeed contain a large number of synaptic vesicles and are in contact with dendritic spines, suggesting most YFP-labeled axonal varicosities are excellent indicators of sites of synaptic contact. However, because a small percentage (~11%) of axonal varicosities contain only mitochondria (Shepherd and Harris, 1998), disappearance of YFP-labeled varicosities could either mean elimination of the presynaptic site or simply the movement of mitochondria.

To determine the precise identities of the varicosities, we need do examine axonal varicosity at the ultrastructural level after in vivo imaging by using a correlated light and serial electron microscopy approach. Such an approach will allow us to answer several fundamental questions about ultrastructural changes of presynaptic terminals in vivo: What are the internal constituents of axonal varicosities in the cerebral cortex? How do presynaptic boutons change over time at different developmental stages? Do newly formed boutons and preexisting ones differ in their stability?

Kartik Chandran

ebola virus

Shai Shaham

Glial cell development and function: Glia make up over 90% of the cells populating the human brain, and glia-derived tumors are the most common and most lethal human brain tumors. Surprisingly, compared to their neuronal counterparts, little is known about glial cell development, function, or morphogenesis. Growing evidence suggests that glia play active roles in regulating synaptic transmission and neuronal current propagation, and are likely to play key roles in many, if not all, aspects of nervous system function.

C. elegans contains 24 neuron-associated cells termed sheath cells that bear striking similarities to vertebrate glia. Reminiscent of astrocytes, they possess a large cell body with multiple small projections and a long main projection that enwraps the dendritic processes of sensory neurons. Recent availability of C. elegans glial reporter genes, the sequencing of the entire C. elegans genome, and the facility with which genetic studies can be undertaken in this organism make it an excellent system to study basic aspects of glial cell biology. C. elegans sensory structures are composed of three cell types: sensory neurons responsive to odor, osmolarity, mechanical stimuli and heat; sheath cells that associate with the neurons and envelope the distal ends of their dendritic processes; and socket cells involved, in part, in generating a pore through which chemical signals can be sensed by the ensheathed neurons. Two bilaterally symmetric sensory structures, the amphids and phasmids, seem to be important for most chemosensation in C. elegans. Because the neurons of the amphids and phasmids have been well characterized, we have chosen to study the functions of their accompanying glia as a model for understanding glial cell development and function. We are interested in two aspects of support cell biology: how ensheathing cells modulate neuronal function, and how they acquire their unique morphologies. We have begun to address these questions using a combination of laser ablation, time-lapse microscopy, genomic and genetic approaches.

Xiang-Peng Kong

urothelia bacteria pathogenesis

Mike Rout

STEM detector for mass quantitation (Iban, Mancilla-Soto, Sheetz-Biais)

Seth Darst

phase plate for single particle

Aneel Aggrawal

phase plate for single particles

biosketch; othersupport

Our lab focuses on the mechanisms of cell-cell fusion between myoblasts. Myoblast fusion is responsible for forming the mature multinucleate myotubes that are the basic unit of muscle. Using our traditional approaches of genetics, molecular biology and cell biology, we have identified specific genes that are critical to the process. Moreover, our use of confocal imaging in live embryos has allowed us to define specific cellular and subcellular behaviors required for the process. However, some of our questions need techniques beyond our standard approaches. To this end, I have found that researchers working on viral fusion have successfully used EM tomography to describe macromolecular structures that appear at the sites of virus-membrane fusion (specifically, the work of Dr. Margret Kielian, see ref. below). Her lab and the labs of others in the field have identified structurally similar motifs between different fusion proteins from different viruses (via crystallography) that encoded by very divergent primary sequences. This field, as well as other membrane fusion fields (vesicle fusion) have been able to identify the specific proteins involved in fusing two membranses, but this machinery has eluded the myogenesis field. I am hoping that EM may be able to provide a new angle from which to approach this problem. I am interested to find out whether there are a number of proteins at the fusion interface between two apposed cells (similar to the schematics on Dr. Stokes website of desmosomes), or if there is an isolated fusion complex similar to that employed by viruses. I do not know what type of “starting material” is necessary for such an investigation. To look at myoblast fusion the currently lab uses the intact drosophila embryo, but I have also developed a primary drosophila myoblast cell culture system where myotubes form on a 2D surface (in a dish). I am curious if either will work for performing EM tomography.

I will not be answering email or other requests until May 15. If your message is important, please resend it then.

We are interested in the 3-D high-resolution structure of mammalian photoreceptor (a modified cilium). We are specifically interested in resolving the structure/morphology of the proximal region of outer segment (the light sensing organelle of visual cell), connecting cilium (a motile cilium like structure that link the outer segment to cell body), and/or types of vesicles within these compartments.

• Set ALLOWTOPICVIEW =

-- DavidStokes - 02 Oct 2009