Biomedical subjects

Mark H Ellisman

Publications and source records attributed to Mark H Ellisman.

52 records · Page 3Linked to original sources

PubMed2003

Examination of the relationship between astrocyte morphology and laminar boundaries in the molecular layer of adult dentate gyrus.

Astrocytes are known to play an integral role in the development of compartmental boundaries in the brain and in the creation of trauma-induced boundaries. However, the physical relationship between astrocytes and such boundaries in the adult brain is less clear. If astrocytes do respect or play an ongoing role in maintaining such boundaries, a correlation between the position of such a boundary and the morphology of neighboring astrocytes might be observable. In this study, we examined the distribution of astrocytes with respect to the laminar boundaries compartmentalizing afferents to the dentate gyrus molecular layer. In addition, we attempted to determine whether astrocyte morphology is influenced by these laminar boundaries. To this end, protoplasmic astrocytes in the adult rat dentate gyrus were revealed with fluorescent tracer dyes and subsequently analyzed with respect to laminar boundaries demarcated by means of immunolabeling for the lamina-specific molecules EphA4 and neural cell adhesion molecule (N-CAM). We find that astrocyte distribution is influenced by the boundary separating the associational/commissural and perforant path afferents. In addition, we show that astrocytes in this region are polarized in their morphology, unlike typically stellate astrocytes, but that the laminar boundaries themselves do not appear to confer this morphology. This polarized morphology, however acquired, may have import for the functioning of astrocytes within the highly organized composition of the dentate gyrus molecular layer and for the overall microphysiology of this and other brain regions.

Age Factors↗

PubMed2003

Three-dimensional analysis of mouse rod and cone mitochondrial cristae architecture: bioenergetic and functional implications.

PURPOSE: Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. METHODS: We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen different measurements of mitochondrial substructures were analyzed. Photoreceptor oxygen consumption was determined in dark- and light-adapted retinas. RESULTS: Rod and cone mitochondria displayed an orthodox conformation. Cone inner segments, compared to rods, contained 2-fold more mitochondria and were more CO reactive. Rod and cone mitochondria had similar outer-inner membrane width, contact site width, diameter and density, crista width, number of cristae/volume, number of cristae segments/volume, and fraction of cristae with multiple segments. In contrast, cone mitochondria had narrower crista junctions, greater cristae connectivity, and approximately 3-fold more cristae membrane surface area compared to rods. The increased cristae membrane surface area in cones was accomplished by connecting more cristae segments together, rather than by creating more cristae. CONCLUSIONS: These results demonstrate that middle wavelength (M) cones have a different bioenergetic signature than do rods and suggest that the aerobic ATP demand and production is greater in light-adapted cones than in light-adapted rods. Cones utilize two complimentary strategies to increase their aerobic ATP production: increase the number of mitochondria and increase the cristae surface membrane area. The greater ATP generation by cones may also provide increased protection against metabolic insults and apoptosis compared to rods.

Adenosine Triphosphate↗

PubMed2003

Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice.

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-x(L) only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca(2+) overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-x(L) overexpression completely blocked all apoptotic events, except Ca(2+) overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-x(L) overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with Pb(2+) and Ca(2+) and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb(2+) and Ca(2+) overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca(2+) overload, lead exposure, andor mitochondrial dysfunction occur.

Animals↗

PubMed2003

JNK1 is required for maintenance of neuronal microtubules and controls phosphorylation of microtubule-associated proteins.

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.

Animals↗

PubMed2003

ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains.

Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles. We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography. We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites. This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles. Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features). These findings provide an alternative framework for analysis of ER-to-Golgi traffic.

2,2'-Dipyridyl↗

PubMed2003

5-HT2a receptors in rat sciatic nerves and Schwann cell cultures.

Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT(2A) receptor (5-HT(2A)R). In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT(2A)R protein mRNA. We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT. Rat Schwann cells of a myelinating phenotype contained both 5-HT(2A)R protein and mRNA. In the healthy adult rat sciatic nerve, 5-HT(2A)Rs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm. The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves. These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells.

Animals↗

PubMed2003

The cell-centered database: a database for multiscale structural and protein localization data from light and electron microscopy.

The creation of structured shared data repositories for molecular data in the form of web-accessible databases like GenBank has been a driving force behind the genomic revolution. These resources serve not only to organize and manage molecular data being created by researchers around the globe, but also provide the starting point for data mining operations to uncover interesting information present in the large amount of sequence and structural data. To realize the full impact of the genomic and proteomic efforts of the last decade, similar resources are needed for structural and biochemical complexity in biological systems beyond the molecular level, where proteins and macromolecular complexes are situated within their cellular and tissue environments. In this review, we discuss our efforts in the development of neuroinformatics resources for managing and mining cell level imaging data derived from light and electron microscopy. We describe the main features of our web-accessible database, the Cell Centered Database (CCDB; http://ncmir.ucsd.edu/CCDB/), designed for structural and protein localization information at scales ranging from large expanses of tissue to cellular microdomains with their associated macromolecular constituents. The CCDB was created to make 3D microscopic imaging data available to the scientific community and to serve as a resource for investigating structural and macromolecular complexity of cells and tissues, particularly in the rodent nervous system.

Brain↗

PubMed2002

Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet.

Evidence that Ca(2+)-activated K(+) (K(Ca)) channels play a role in cell volume changes and K(+) homeostasis led to a prediction that astrocytes would have K(Ca) channels near blood vessels in order to maintain K(+) homeostasis. Consistent with this thinking the present study demonstrates that rSlo K(Ca) channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo K(Ca) channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K(+) modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.

Animals↗

PubMed2002

Depolarization redistributes synaptic membrane and creates a gradient of vesicles on the synaptic body at a ribbon synapse.

We used electron tomography of frog saccular hair cells to reconstruct presynaptic ultrastructure at synapses specialized for sustained transmitter release. Synaptic vesicles at inhibited synapses were abundant in the cytoplasm and covered the synaptic body at high density. Continuous maximal stimulation depleted 73% of the vesicles within 800 nm of the synapse, with a concomitant increase in surface area of intracellular cisterns and plasmalemmal infoldings. Docked vesicles were depleted 60%-80% regardless of their distance from the active zone. Vesicles on the synaptic body were depleted primarily in the hemisphere facing the plasmalemma, creating a gradient of vesicles on its surface. We conclude that formation of new synaptic vesicles from cisterns is rate limiting in the vesicle cycle.

Animals↗

PubMed2002

Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane.

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by antiapoptotic Bcl-x(L). We conclude that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-x(L).

Animals↗

PubMed2002

Multicolor and electron microscopic imaging of connexin trafficking.

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.

3,3'-Diaminobenzidine↗

PubMed2002

Ultrastructure of a somatic spine mat for nicotinic signaling in neurons.

Chick ciliary neurons have somatic spines grouped in discrete clumps or mats tightly folded against the soma and enriched in nicotinic receptors containing alpha7 subunits. An embryonic ciliary neuron has one to two dozen such spine mats, all overlaid by a large presynaptic calyx engulfing the cell. Three-dimensional tomographic reconstruction from serial thick sections revealed 13 somatic spines in one complete spine mat on a ciliary neuron late in embryogenesis. The spines varied in morphology and usually were branched but had numerous similarities to dendritic spines, including mean length, volume, surface area, presence of endoplasmic reticulum, and occasional multivesicular bodies. The spines invariably were connected to the soma via a narrow neck of approximately 0.2 micrometer in diameter as found for dendritic spines, suggesting restricted access from spine lumen to soma. A prominent difference between dendritic and somatic spines is the absence of postsynaptic densities from most somatic spines both on embryonic and adult ciliary neurons. Transmitter access to receptors on the spines may occur either by lateral diffusion from release sites over nearby postsynaptic densities or by release directly onto spines from the overlying calyx lined with vesicles. The latter is less likely in the adult, where some spines are adjacent to but not overlaid by vesicle-enriched presynaptic structures. The anatomical configuration of spine mats suggests coordinate spine activation by transmitter release into a confined volume while spine morphology is used to control the chemical consequences of synaptic signaling.

Aging↗

PubMed2002

Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.

Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominantly on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only approximately 15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.

Animals↗

PubMedDate not supplied

A cell-centered database for electron tomographic data.

Electron tomography is providing a wealth of 3D structural data on biological components ranging from molecules to cells. We are developing a web-accessible database tailored to high-resolution cellular level structural and protein localization data derived from electron tomography. The Cell Centered Database or CCDB is built on an object-relational framework using Oracle 8i and is housed on a server at the San Diego Supercomputer Center at the University of California, San Diego. Data can be deposited and accessed via a web interface. Each volume reconstruction is stored with a full set of descriptors along with tilt images and any derived products such as segmented objects and animations. Tomographic data are supplemented by high-resolution light microscopic data in order to provide correlated data on higher-order cellular and tissue structure. Every object segmented from a reconstruction is included as a distinct entity in the database along with measurements such as volume, surface area, diameter, and length and amount of protein labeling, allowing the querying of image-specific attributes. Data sets obtained in response to a CCDB query are retrieved via the Storage Resource Broker, a data management system for transparent access to local and distributed data collections. The CCDB is designed to provide a resource for structural biologists and to make tomographic data sets available to the scientific community at large.

Animals↗

PubMedDate not supplied

High-performance electron tomography of complex biological specimens.

We have evaluated reconstruction methods using smooth basis functions in the electron tomography of complex biological specimens. In particular, we have investigated series expansion methods, with special emphasis on parallel computation. Among the methods investigated, the component averaging techniques have proven to be most efficient and have generally shown fast convergence rates. The use of smooth basis functions provides the reconstruction algorithms with an implicit regularization mechanism, very appropriate for noisy conditions. Furthermore, we have applied high-performance computing (HPC) techniques to address the computational requirements demanded by the reconstruction of large volumes. One of the standard techniques in parallel computing, domain decomposition, has yielded an effective computational algorithm which hides the latencies due to interprocessor communication. We present comparisons with weighted back-projection (WBP), one of the standard reconstruction methods in the areas of computational demand and reconstruction quality under noisy conditions. These techniques yield better results, according to objective measures of quality, than the weighted backprojection techniques after a very few iterations. As a consequence, the combination of efficient iterative algorithms and HPC techniques has proven to be well suited to the reconstruction of large biological specimens in electron tomography, yielding solutions in reasonable computation times.

Algorithms↗

PubMedDate not supplied

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.

Biological Transport↗