J Pulokas

An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Applications of Automated Electron Microscopy: Using Leginon to Study the Structure of COPII Protein Complexes

COPII proteins are responsible for forming the vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII proteins form a coat around the budding vesicle and are responsible for both selecting the protein cargo and drawing the ER membrane up and pinching it into a vesicle. COPII coats consist of three components: Sar1, a GTPase; Sec23/24, a GTPase activating protein (GAP); and Sec13/31, a GAP stimulator which has also been implicated in inducing membrane curvature. While structures of Sar1 and Sec23/24 have been solved, little is known about the structure of Sec13/31 or the structure of the COPII lattice. We are investigating the structure of the COPII lattice using cryo-electron microscopy (cryoEM).

Automated TEM Data Acquisition Applications Using Robotic Grid Loading

Data acquisition spanning multiple specimen grids using a TEM is conventionally done by an operator manually loading and imaging each grid. During the previous meeting we reported on using a robotic grid loading system to collect low resolution atlases for sets of 96 grids without the need for human interaction [1,2]. Here we report on new developments of the system for routinely collecting higher resolution images selected from areas of interest in the atlas.

Automated cryo-electron microscopy

Cryo-electron microscopy is widely viewed as a uniquely powerful method for the study of membrane proteins and large macromolecular complexes - subjects that are viewed as extremely challenging or impossible to study using x-ray or NMR methods. Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. Our group has done extensive work to automate image acquisition and processing for cryo-EM. In this paper we will provide an overview of our automated system, called Leginon, and present results where we used tobacco mosaic virus (TMV) as a proof of concept.