Our lab studies macromolecular machines in their native cellular environment by electron microscope tomography. Our aim is to visualize their architecture at molecular resolution, and to identify their protein composition through novel labeling approaches. The models derived from our structural studies serve as a platform for cell biological and pharmacological approaches. Among the animal model systems we are using for our studies, we favor zebrafish embryos, because of their unique characteristics with respect to sample preparation, and their excellent genetic and pharmacological accessibility. Moreover, zebrafish have been widely recognized as excellent models for studying a number of human diseases.
Although the task of studying supramolecular complexes in their native cellular environment is challenging, we feel rewarded by the discovery of the fascinating complexity of molecular machines. In particular we are captivated by the way cells organize themselves into tissues and intrigued by the question how they transduce mechanical signals into an electrical or biochemical response. Such mechanosensitive behavior is found in a large variety of cells, and is crucial for physiological functions such as hearing and balance, kidney regulation and cancer metastasis. Our research aims to answer these important biological questions using structural cell biology methods.
Macromolecular Machines
The field of structural biology has an impressive record of 3D structures of proteins at atomic resolution, owing mostly to X-ray crystallography and NMR, with a small but significant contribution from cryo-electron microscopy. In recent years, biology at large has witnessed a paradigm shift: It is now widely recognized that fundamental cellular processes do not occur as the result of protein molecules colliding randomly within the cytoplasm or cell membrane, but that they are carried out by molecular machines which are composed of large numbers of individual proteins. As Richard Feynman put it in 1960: "It is very easy to answer many fundamental questions, you just need look at the thing!"
This is in essence our goal, to look at such molecular machines in sufficient detail in order to understand how they perform the function for which they have been optimized. For a cell at work, we can assume that a fair number of these complexes are transient, and variable in composition and conformation, and that they may not be abundant and stable enough to be isolated. Moreover, some of these complexes need to be in their exact cellular environment to function properly, e.g. they may need to be attached extracellularly to the surrounding matrix and intracellularly to the cytoskeleton. Although the exact composition and therefore the 3D structure of such complexes may vary within one cell at any given time, we would expect to detect an underlying building principle, which is necessary for their function. Such flexibility, scarcity and fragility excludes most structural techniques that explicitly or implicitly rely on averaging of identical components. Hence, for the study of such molecular machines, electron tomography is the most suitable technique to obtain 3D structural information, for it can visualize macromolecular assemblies in their natural cellular context, without the need for averaging. Tomographic imaging can retrieve this 3D information by capturing a series of images of the same object at different tilt angles. The different views of the projected volume are combined and projected back into a volume, a tomogram, that represents the mass density.
Mechanosensation
Most cells are capable to mechanically sense their environment by ways that are largely unexplored. Mechanosensation is crucial for embryogenesis, for the organization of cells into tissues, as well as for the regulation of the function of a number of organs, e.g. the kidney, bones, muscle, etc. Mechanical clues are important in keeping cells alive and within their proper tissue context. Loss of contact of a cell with their surrounding cells or extracellular matrix often leads to apoptosis and central to the mechanism of cancer metastasis.
Mechanosensation lies at the core of the detection of sound, touch, pressure, gravity or magnetic fields. We have focused our attention on inner ear hair cells, which are central to our sense of hearing and balance as well as kidney podocytes which are crucial for the ultrafiltration of blood.
Deafness and kidney failure - Inner Ear Hair Cells and Glomerular Podocytes:
Hair cells and podocytes are -arguably- the most fascinating cell types present in vertebrates. Their malfunction lies at the heart of such devastating diseases as hearing loss and kidney failure. One in 1000 children is born deaf, and about 10% of the population is affected by severe hearing loss. Hearing loss is also common in people with kidney disease, e.g. in case of the Alport Syndrome, which affects one in 5000 Americans.
Hair cell stereocilia and podocyte pedicils are highly specialized actin-based cell organelles and host several distinct cell-cell and cell-matrix adhesion complexes, whose integrity is crucial for the physiological function of the respective organs. Despite their clinical and biological importance, very little is known about the exact molecular composition and 3D architecture of these mechanosensitive signaling complexes. While each complex may be unique in its exact 3D structure, one expects a conserved architectural motif that allows these complexes to carry out their biological function.
Figure: Stereocilia
The mechanoelectrical transduction and adaptation machinery of hair cell stereocilia consists of extracellularly located fine filaments that connect two adjacent stereocilia and whose stretching results in the direct opening of mechanoelectrical transduction channels without the involvement of chemical messengers. The direct nature of mechanoelectrical transduction implies that adaptation to sustained stimuli is achieved by adjustment of the tension in the tip links via a movement of a non-conventional myosin along the actin filament bundle.
Podocytes are highly specialized cells of the kidney, which form multiple interdigitating foot processes and completely cover the outer surface of the glomerular capillaries. They stabilize the glomerular architecture, provide hydraulic resistance of the filtration barrier, and maintain a large, yet selective filtration surface through the slit diaphragms that interconnect foot processes of adjacent podocytes. These highly specialized cells possess a contractile structure based on actin and myosin, and are connected to the glomerular basement membrane at focal contacts through integrins.
Figure: Kidney podocyte
Electron Tomography and Labeling - The Case for Zebrafish
We employ electron tomography as a tool to investigate the architectural organization of macromolecular complexes, also known as molecular machines. Electron tomography is presumably the most generally applicable method that reaches to the resolution of individual protein molecules, while allowing the complex to reside in their native cellular context and is particularly well suited for the study of multi-protein complexes that are too rare or fragile to be purified. Examining electron tomograms reveals the complexity of cellular organization.
We have helped to develop sophisticated tools for the visualization, segmentation and analysis of tomograms. Recently developed protocols avoid time-consuming manual segmentation and therefore speed up the analysis of the large density maps.
We are also developing novel labeling approaches that are based on genetically encoded tags that can be recognized by universal labels, therefore avoiding the uncertainties often encountered by immuno-labeling methods. The identification of molecular components would tremendously aid in the interpretation of tomograms. This approach would allow the 3D localization of any candidate protein with high precision, provided the proteins can be genetically altered to include a tag.
We have recognized the potential of transgenic zebrafish, and have started to establish it as our model system. Zebrafish is a particularly well-suited organism because transient and stable transgenics can be obtained, the expression of proteins can be temporarily interfered with using morpholinos and the entire genome sequence is emerging. Moreover zebrafish has been recognized as a model system for a variety of human diseases and is a model system for vertebrate development. From an electron microscopic point of view, zebrafish embryos are ideal: while they are fully functional animals with all vital organs, zebrafish embryos are thin enough to be instantly frozen and low-temperature processed to ensure excellent sample preparation, and may even allow to to perform cryo-sectioning. The fact that we can take a live animal and cryo-immobilize the entire tissue within milliseconds, therefore avoiding possible artifacts that may arise from the need to dissect the organs of interest. Not only does this approach overcome the long training period necessary for optimal dissection, it would also allows us use the same specimen to look at more than one organ system. This approach is particularly efficient when dealing with transgenic animals.
The Lab's Infrastructure
Our lab is housed in the Donner building at the north-east corner of the UC Berkeley campus (home of the "Free Speach Movement"), at the bottom of Cyclotron Road that leads to the main site of LBNL on the hill overlooking the Bay Area. Four groups in the Donner lab are dedicated to electron microscopy of macromolecules, covering a variety of data acquisition and 3D reconstruction schemes, and when combined offer an almost unique atmosphere of technical know-how. The cordial and informal atmosphere of the Berkeley lab and the strong sense of community make LBNL a great place to get outstanding science done.
Since we research demands an expertise in a broad range of science, spanning from developmental and cell and neurobiology to chemical and bichemical sample preparation, physcial TEM imaging and computational data reconstruction, visualization and analysis, we collaborate with some of the best groups in their respective fields. For instance, we are collaborating with Kent McDonald (UC Berkeley) with respect to high pressure freezing and freeze-substitution as well as cryo-sectioning.
Figure: Jeol 3100 Microscope
The microscope equipment we share is among the best in the world, featuring a Jeol 4000, a Jeol 3000, a Jeol 3100 equipped with an in-column energy filter, as well as a Philips CM200FEG microscope. Each microscope is capable to record high-resolution data, both for tomography and single particle analysis.
The Lab's Philosophy
Our efforts are fueled by the excitement of scientific discovery, and we believe that studying important biological problems requires an atmosphere of true team spirit where everybody's contribution is important and welcome. We consider a true passion for science as the most important ingredient for doing good science, but also aim keep a healthy balance between work and life outside the lab.
