Since the beginning of the Auer Lab in 2004, we have expanded our research portfolio and focus significantly, adding to our initial focus on the 1. structural analysis of inner ear molecular machines a variety of other projects that fall into four main categories:
2. molecular mechanisms in cancer malignancy and metastasis with a focus on cell-cell adhesion patterns, 3. microbial communities (biofilms) including bioremediation, gliding motility, and the roles of vesicles in microbial communities, 4. bioenergy/biofuels including plant cell walls and lignocellulosic degradation, and 5. method development for correlative multiscale, multimodal imaging, including exploring several tag-based labeling approaches.
Our mission is to gain fundamental insight into biology, in part by visualizing molecular machines at molecular resolution, and to identify their protein composition through novel labeling approaches. While 2D electron microscopy and 3D electron tomography continue to be the major tools for the analysis of macromolecular machines in their native cellular environment, our tomographic studies are often complemented by biochemical, cell biological, biophysical and high-end optical and TEM and SEM imaging techniques, as well as computational data analysis including sophisticated visualization, segmentation and quantitative analysis. 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. The models derived from our structural studies then often serve as a platform for further neurobiological, cell biological, pharmacological or microbiological testing. Where we do not possess the expertise in our own laboratory, we do collaborate with a variety of experts, both on the biological as well as the technical side.
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 exclude 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.
Electron Tomography and Labeling
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. However, in complex cellular environment, shape and size may not be sufficient to determine the molecular composition. Moreover, some components may be too small to be identified through a structural approach. Hence, 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.
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 neighboring cells or surrounding extracellular matrix often leads to apoptosis and is likely to be central to the mechanism of cancer pathogenesis. 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.
1. Hearing Loss and Deafness: Inner Ear Hair Cells: Hair cells are -arguably- the most fascinating cell types present in vertebrates. Their malfunction lies at the heart of such devastating diseases as hearing loss deafness. One in 1000 children is born deaf, and about 10% of the population is affected by severe hearing loss. Hair cells are characterized by the hair bundle, which consists of stereocilia, highly specialized actin-based cell organelles. 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. We have determined the 3D architecture of the hair bundle extracellular linkers and measured the exact length of the basal, kinociliary and tip links and found that tip links are found as being either ~110 nm or ~170 nm long, raising questions about their identity. Furthermore we discovered an auxiliary link that has been overlooked so far and that may contribute to hair cell function. [Auer et al, 2008, JARO].
We are currently conducting several studies into the molecular organization of the hair bundle, including the actin bundle organization, the rootlets as well as the transduction and adaptation machinery.
In addition, we have conducted a thorough study on the 3D organization of the guinea pig outer hair cell lateral wall that hosts a prestin-based amplification machinery that consists of the plasma membrane, the underlying cortical lattice, consisting of actin patches and spectrin cross-links, as well as the underlying subsurface cisterna (SSC). Our tomographic results have shown that the SSC is tightly connected to the plasma membrane, through pilar proteins and actin-SSC linkers. From this data we conclude that the SSC, which contains a large number of periodically organized structures, is likely to contain an active element for electromotility. None of the current models so far takes the SSC into account and hence both current models need to be modified to account for the SSC contribution in electromotility [Triffo et al. 2008, submitted]. In collaboration with Jian Zhou we are currently assessing the ultrastructure of prestin k.o.mice.
2. Mammary Gland Biology and Breast Cancer: In collaboration with Mina Bissell's Lab at LBL and Zena Werb's lab at UCSF, we have studied the ultrastructure of human S1/T4 cell line in Matrigel as well as mouse breast organoids in Matrigel, respectively. We have focused our attention on the presence and distribution of cell-cell adhesion complexes and found a breakdown in apical-basolateral organization both for S1/T4 as well as for FGF2-stimulated mouse organoids, suggesting that changes in cell-cell adhesion play a role both for cancer progression as well as embryonal development. [Palsdottir et al, in preparation; Ewald et al, in preparation]
3. Microbial Communities:We have studied the biofilm organization of Desulfovibrio vulgaris (DvH), Myxococcus xanthus, Acid Mine Drainage biofilms, as well as a variety of other microbial communities. We are intrigued by the patterns of extracellular metal depositons and the biofilm organization. We also found evidence of a prominent role of vesicles in such biofilms. [Palsdottir et al, submitted; Wilmes et al, submitted; Remis et al, in preparation]. We are current conducting a study on the termite hindgut community as well as several other environmentally or medically relevant biofilms. Both the sulfate reducing bacteria such as DvH and the myxobacteria display fascinating properties, and one might argue that the exploration of microbial communities/biofilms is one of the main frontiers in understanding microbial life.
4. Biofuels - Plants and lignocellulosic degradation: We are part of both the DOE-funded Joint BioEnergy Institute (JBE: www.jbei.org) as well as the BP-funded Energy Biosciences Institute (EBI: www.energybiosciences.org), both of which aim to overcome the recalcitrance of lignocellulose through basic research of the plant cell wall characteristics as well as of the events of chemical/biochemical as well as microbial lignocellulose degradation. Our group serves as the main liaison for the feedstocks and deconstruction divisions of JBEI, where we use biophysical techniques to characterize plant cell wall properties, as well as imaging to visualize the effect of various treatment on the plant biomass. In the context of EBI we oversee all electron microscopy sample preparation and imaging efforts, with a main focus of a realistic model of plant cell walls through the correlative Raman and EM imaging (electron tomography) of type-I and type-II cell walls. We further aim to develop higher-throughput sample preparation and imaging approaches, as well as adapt conventional immunolabeling as well as novel tag-based labeling approaches.
5. Method Development for Correlative Multiscale Multimodal Imaging: In the course of electron tomographic analysis we learned that 3D structural analysis alone often does not lead to conclusive proof of the molecular composition. As a matter of fact, labeling of certain protein components nicely complements 3D architectural information about protein complexes. As part of the ongoing PCAP project (pcap.lbl.gov) we develop novel tag-based labeling approaches in anaerobic sulfate reducing bacteria, exploring both ReAsH and SNAP-tag labeling followed by photoconversion. We are also exploring Ni-NTA-gold labeling of hexa-His-tagged proteins. Furthermore as part of EBI we are developing technology to conduct correlative Raman microscopy/electron tomography. Beside the novel labeling approaches we are also utilizing recent developments in computational sciences, including immersive 3D visualization, segmentation and quantitative geometric analysis.
One key for correlative imaging in our view is that we study the exact same sample probe/specimen by multipe imaging modalities. This poses some challenges on sample preparation and data registration that we are currently addressing. Since we have recently been able to both retain fluorescence signals as well as Raman signals throughout dehydration and resin-embedding/polymerization, we consider correlative imaging not only doable but also highly desirable.
Multiscale, Multimodal Integrated BioImaging may well be the next frontier in understanding complex systems, and the localization of components through labeling or Raman Imaging, combined with ultrastructural 3D context, will ultimately provide spatial maps that will serve as the foundation for systems biology.
As vertebrate animal models we have been using bullfrogs, zebrafish, mice and guinea pigs. Zebrafish embryos seems to be particularly interesting, 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.
Regarding plants we have been mainly working with Arabidopsis and Rice, but anticipate to also extend our studies to Switchgrass and other potential biofuel feedstocks.
We have also studied a variety of microbial systems, including Myxococcus xanthus, Shewanella oneiidensis, Desulfovibrio vulgaris and Desulfovibrio africanus, Acid Mine Drainage microbial communities, cellulose degraders, termite hindgut microbial communities, and other systems.
The Case for Zebrafish
We have recognized the potential of transgenic zebrafish. 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, is of particular significance, as we can avoid 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 will also allows us use the same specimen to look at more than one organ system. This approach is particularly efficient when dealing with protein localization in 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.
The microscope equipment we share is among the best in the world, featuring two Jeol 4000, a Jeol 3100 equipped with an in-column energy filter, a Tecnai T12, Technai F20 as well as a Philips CM200FEG microscope. Most microscopes are capable to record high-resolution data, both for tomography and single particle analysis. Moreover, we have access to two high pressure freezers (Baltec and Leica Empect 2), two LEICA AMW microwave processors, 5 ultramicrotomes, two of which equipped for cryo-sectioning, as well as equipment for SEM sample preparation and imaging.
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.
Palsdottir H, Remis JP, Schaudinn C, Lux R, Shi W, McDonald KL, Costerton JW, Auer M 3D architecture of cryofixed Myxococcus xanthus biofilms as revealed by EM tomography (manuscript submitted)
Wilmes P, Remis JR, Hwang M, Auer M, Thelen MP, Banfield JF; Natural acidophilic biofilm communities reflect distinct organismal and fucntional organization, (manuscript submitted)
Auer M, Koster AJ, Ziese U, Volkmann N, Bajaj C, Wang DN, Hudspeth AJ (2008) 3D architecture of hair cell stereociliar extracellular links as revealed by electron tomography, JARO, 9:215-224
Triffo WJ, Palsdottir H, McDonald KL, Lee J, Inman J, Bissell M, Raphael R, Auer M (2008) Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography, J. Microcopy, 230: 278-287
Downing KH, Sui H, Auer M (2007) Electron tomography - a new, 3D-view of the subcellular world, Anal Chem 79:7949-7957
Persson S, Paredez A, Carroll A, Palsdottir H, Doblin M, Poindexter P, Khitrov N, Auer M, Somerville C (2007) Genetic evidence for three unique positions in primary cell wall cellulose synthase (CESA) complexes in Arabidopsis, PNAS 104:15566-15571
Chang H, Yang Q, Auer M, Parvin B (2007) Modeling of Front Evolution with Graph Cut Optimization, IEEE International Conference on Image Processing, Texas Signaling, Infection and Immunity, 75:3715-3721
Schaber, JA, Triffo WJ, Suh SJ, Oliver J, Hastert MC, Griswold JA, Auer M, Hamood AN, Rumbaugh KP (2007) Pseudomonas aeruginosa forms Biofilms in Acute Infection Independently of Cell-to-Cell Signaling, Infection and Immunity, 75:3715-3721
McDonald KL & Auer M (2006) High-pressure freezing, cellular tomography and structural cell biology, Biotechniques 41: 137, 139, 141
Bajaj C, Yu Z., Auer M. (2003) Feature extraction, analysis and visualization of tomographic molecular imaging, J Struct Biol.144:132-143.
Huang Y, Lemieux MJ, Song J, Auer M. & Wang D.-N. (2003) Structure and mechanism of the glycerol-3- phosphate transporter from Escherichia coli, Science 301: 616-620.
Auer M. (2000) Electron cryo-microscopy as a powerful tool in molecular medicine, J. Mol Med, 78: 191-202.
Lancaster C.R.D., Kröger A., Auer M. & Michel H. (1999) Structure of fumarate reductase from Wolinella succinogenes at 2.2 Å resolution, Nature, 402:377-385.
Auer M., Scarborough G. & Kühlbrandt W. (1998) Three-dimensional Map of the Plasma Membrane H+-ATPase in the Open Conformation, Nature 392: 840-843.
Two Postdoctoral Fellows (JBEI)
Research Associate (PCAP)
In the News
Apart from a number of projects to which we contribute some high-end electron micrographs, we are predominantly focused on the following projects:
- SNAP tag labeling of the anaerobic sulfate reducer Desulfovibrio vulgaris for subsequent subcellular protein localization in planctonic cells and biofilms
- Ultrastructural analysis of Desulfovibrio vulgaris biofilms
- Ultrastructural analysis of Acid Mine Drainage biofilms
- Ultrastructural analysis of Myxococcus xanthus biofilms
- Study of the molecular composition and role of vesicles in Myxococcus xanthus
- Electron tomography of hair bundle molecular machines
- Electron tomography of outer hair cell lateral walls
- EM analysis of Arabidopsis mutants
- Electron tomography of Arabidopsis Cellulose Synthase
- Correlative Raman microscopy/electron tomography of plant cell walls
- Protein localization in rice by various labeling approaches
- Monitoring the effect of pretreatment on lignocellulose biomass
- Correlative FISH/EM analysis of termite hindgut microbial communities
- Ultrastructural characterization of mouse organoids in Matrigel
- Ultrastructural characterization of S1/T4 cell lines in Matrigel
We collaborate with some of the finest scientists and typically the respective experts in their fields, such as
Chandrajit Bajaj (University of Texas, Austin)
Jill Banfield (UC Berkeley)
Mina Bissell (LBL)
Bill Costerton (USC)
Yuri Gorby (The Venter Institute)
Terry Hazen (LBL)
Jim Hudspeth (The Rockefeller University)
Phil Hugenholtz (Joint Genome Institute)
JBEI team (Joint BioEnergy Institute)
Richard Kollmar (University of Illinois, Urbana Champain)
Bram Koster (University of Leiden, NL)
Jan Liphardt (UC Berkeley)
Kent McDonald (UC Berkeley)
Eva Nogales (UC Berkeley)
PCAP team (pcap.lbl.gov)
Rob Raphael (Rice University)
Wenyuan Shi (UCLA)
Chris Somerville (Energy Biosciences Institute, UC Berkeley)
Niels Volkman (Burnham Institute)
Zena Werb (UCSF)
Jian Zhou (St. Jude's Children Hospital)