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person:stoked01
Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse
Milstein, Oren; Tseng, Su-Yi; Starr, Toby; Llodra, Jaime; Nans, Andrea; Liu, Mengling; Wild, Martin K; van der Merwe, P Anton; Stokes, David L; Reisner, Yair; Dustin, Michael L
The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity
PMCID:2590684
PMID: 18826951
ISSN: 0021-9258
CID: 92165
Plakoglobin is required for effective intermediate filament anchorage to desmosomes
Acehan, Devrim; Petzold, Christopher; Gumper, Iwona; Sabatini, David D; Muller, Eliane J; Cowin, Pamela; Stokes, David L
Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength
PMID: 18496566
ISSN: 1523-1747
CID: 93304
Structure of a copper pump suggests a regulatory role for its metal-binding domain
Wu, Chen-Chou; Rice, William J; Stokes, David L
P-type ATPases play an important role in Cu homeostasis, which provides sufficient Cu for metalloenzyme biosynthesis but prevents oxidative damage of free Cu to the cell. The P(IB) group of P-type ATPases includes ATP-dependent pumps of Cu and other transition metal ions, and it is distinguished from other family members by the presence of N-terminal metal-binding domains (MBD). We have determined structures of two constructs of a Cu pump from Archaeoglobus fulgidus (CopA) by cryoelectron microscopy of tubular crystals, which reveal the overall architecture and domain organization of the molecule. By comparing these structures, we localized its N-terminal MBD within the cytoplasmic domains that use ATP hydrolysis to drive the transport cycle. We have built a pseudoatomic model by fitting existing crystallographic structures into the cryoelectron microscopy maps for CopA, which suggest a Cu-dependent regulatory role for the MBD
PMCID:2705936
PMID: 18547529
ISSN: 0969-2126
CID: 94876
Cryoelectron tomography of isolated desmosomes
Owen, Gethin Rh; Acehan, Devrim; Derr, K D; Rice, William J; Stokes, David L
Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell-cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin
PMID: 18363558
ISSN: 0300-5127
CID: 94877
Electron cryo-tomographic structure of cystovirus phi 12
Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul
Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed 'the donut' and appears to inhibit BHT-induced viral envelope fusion
PMCID:2692713
PMID: 18022662
ISSN: 0042-6822
CID: 76385
Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals
Hu, Guo-Bin; Rice, William J; Drose, Stefan; Altendorf, Karlheinz; Stokes, David L
The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis
PMCID:2322856
PMID: 17945510
ISSN: 1095-8657
CID: 78734
A high-throughput strategy to screen 2D crystallization trials of membrane proteins
Vink, Martin; Derr, Kd; Love, James; Stokes, David L; Ubarretxena-Belandia, Iban
Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography
PMCID:2211413
PMID: 17951070
ISSN: 1095-8657
CID: 94878
Desmosomes from a structural perspective
Stokes, David L
Desmosomes are cell-cell junctions responsible for maintaining the structural integrity of tissues by resisting shear forces. Defects result in diseases of mechanically challenged tissues such as skin and heart. The architectural design represents the key to understanding the strength and durability inherent to desmosomes. A number of different proteins contribute to this architecture, and X-ray crystallography has made considerable progress in defining the atomic structure of various isolated domains. Electron tomography has been used to determine the three-dimensional structure of intact desmosomes in situ. By combining information from X-ray crystallography, cell and molecular biology and electron tomography, it should ultimately be possible to deduce the specific protein interactions that define the mechanical properties of this important adhesive junction
PMCID:2211412
PMID: 17945476
ISSN: 0955-0674
CID: 75667
Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases
Pomfret, Andrew J; Rice, William J; Stokes, David L
Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozen-hydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na(+)/K(+)-ATPase, rabbit skeletal Ca(2+)-ATPase and scallop Ca(2+)-ATPase in order to evaluate the potential for sub-nanometer resolution as well as the robustness in the presence of disorder. We found that Fourier-Bessel methods perform better for well-ordered samples of skeletal Ca(2+)-ATPase and Na(+)/K(+)-ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca(2+)-ATPase, which was too disordered to analyze by Fourier-Bessel methods
PMCID:4040983
PMID: 16879984
ISSN: 1047-8477
CID: 71141
Reconstitution and crystallization of CopA from archaeoglolms fulgidis [Meeting Abstract]
Wu, CC; Stokes, DL
ISI:000243972400031
ISSN: 0006-3495
CID: 71386