Cryo-Electron Tomography (cryo-ET):

Most natural objects are three-dimensional (3D) in structure. The higher the complexity of

an object for study, the less revealing are its two-dimensional (2D) projections, due to

superposition of multiple structures in single image features. The basic idea of

tomography is to record a series of 2D images that are projections of a 3D object at

different angles of tilt. A 3D image of this object is then reconstructed by projecting all the

2D images back into a common volume with appropriate weighting. Different energy

sources can be used to produce the projections (x-rays, ultrasound, electromagnetic

waves, etc.) and tomography has found wide applications in diverse fields, such as

diagnostic medicine and radio astronomy. Electron microscope tomography (ET) can

provide invaluable and novel information about the 3D ultrastructure of tissues, cells and

macromolecules.

The advantage of using rapidly frozen specimen ("cryo") is the near to native and thus

excellent structure preservation of the biological object. Depending on the thickness of the

sample and the possibilities of post-processing (e.g. by 3D averaging), a resolution better

than 4 nm can be obtained. We are developing new image processing methods to push

the current limit to higher resolution.

Eukaryotic Flagella and the Molecular Motor Dynein

Eukaryotic cilia and flagella are highly conserved motile systems built on a microtubule-

based scaffold called the axoneme. The complexity of this organelle and size of its major

motor protein, dynein, have made it difficult to understand the molecular mechanisms that

underlie flagellar beating.

We are using ET of frozen-hydrated flagella to solve this problem. 3D reconstructions

have enabled us to clarify aspects of the morphology of the outer dynein arms and to

relate details of dynein's structure to its function as the motor that moves each doublet

microtubule relative to its neighbor. We have also identified links that connect dynein arms

with one another along the axoneme, as well as structures that connect inner and outer

dynein arms (Nicastro et al. 2006). Both these novel structural elements are likely to

provide the "hard-wired" controls that help to regulate the speed and propagation of bends

along axonemes.

Moreover, we identify the first periodic structures inside microtubules, objects that

decorate the interior surfaces of both A- and B-subtubules of the doublets at the sites

where stabilizing drugs like Taxol bind to tubulin, suggesting that these objects may be

examples of the long predicted physiological regulators that modulate microtubule

stability.

We also compared mutant with wild type axonemes, which showed the reproducibility of

our methods and allowed us to characterize the differences that result from deletion of a

single gene for the heavy chain component of one of the inner dynein arms.

Correlative Light and Electron Microscopy (CLEM)

As a result of a concerted effort of the science community at Brandeis we are receiving a

NSF major research instrumentation grant to implement a state-of-the-art cellular and

molecular visualization facility of the future. This modern cellular imaging facility will allow

experiments previously not possible and provide structural details with multiscale

resolution from nanostructures to whole cells. In fact, in its combination Brandeis' cellular

visualization facility for correlative light and electron microscopy will be unique in the

nation. The correlative approach will provide a new window into the functional organization

of cells, i.e. new insights into the structure and function of macromolecules, cells, and

tissues, and the dynamic spatiotemporal intra- and intercellular interactions that regulate

cellular function.

The new instrumentation includes a high pressure freezer with rapid specimen transfer

unit, a freeze substitution device, a cryo-ultramicrotome and two confocal light

microscopes. Part of the future vision is to integrate education, training and research to

train a new generation of scientists who will be expert in methods of both light and electron

microscopy, and who will thus be able to pursue innovative approaches in cell biology and

neurobiology, and ultimately provide new fundamental insights into cellular architecture

and function.

Future goals

Many biological structures will greatly profit from studies using cryo-ET, and thus our goal

is to apply this powerful technology to diverse biological problems to gain deeper insights

into the functional organization of cells. Most eukaryotic cells are too thick to be imaged

directly by cryo-ET, thus one focus of our group will be to improve the technique of frozen-

hydrated sectioning to minimize sectioning artifacts