Wade Sigurdson

Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di‐, mono‐ and unliganded receptors.

1. The voltage dependence of binding and gating in wild‐type and mutant recombinant mouse nicotinic acetylcholine receptors (AChRs) was examined at the single‐channel level. 2. The closing rate constant of diliganded receptors decreased e‐fold with approximately 66 mV hyperpolarization in both wild‐type (adult and embryonic) and mutant receptors. The opening rate constant of a mutant receptor (alpha Y93F) was not voltage dependent. 3. The voltage dependence of closing in monoliganded receptors was examined in several receptors having a mutation in the binding site (alpha G153S) or pore region (alpha L251C and epsilon T264P). The closing rate constant of these monoliganded receptors decreased e‐fold with approximately 124 mV hyperpolarization. 4. The voltage dependence of closing and opening in unliganded receptors was examined in two receptors having a mutation in the pore region (alpha L251C and epsilon T264P). Neither the closing nor the opening rate constants of unliganded receptors were voltage dependent. 5. If z if the amount of charge that moves during channel closure and delta is the distance (as a fraction of the electric field) that the charge moves, we conclude that z delta = 0.4 in diliganded receptors, 0.2 in monoliganded receptors, and 0.0 in unliganded receptors. It is likely that charges on the protein, rather than the agonist molecule, move z delta = 0.2 after each ACh molecule has bound. 6. The results suggest that unliganded openings arise from a local, concerted change in the structure of the pore (channel opening) that does not involve the net movement of charged residues. We speculate that as a consequence of agonist binding, charged moieties in the protein change their disposition so that they move with respect to the electric field when the channel gates. The results are consistent with the idea that there is semi‐independent movement of distinct domains during AChR gating.

Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells

Abstract. Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca2+. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca2+ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca2+.In these three cell types, no influx of ions is required for Ca2+ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca2+. The Ca2+ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP3-activated channels.Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn2+ influx pathway. This Mn2+ pathway may be mechanosensitive channels, Ca2+-activated cation channels or depletion-activated Ca2+ channels.

Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase.

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of iNOS + Neutrophil-like ring cell in tumor-bearing mice

BackgroundMyeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset.MethodsSingle-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro.ResultsThe results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells.ConclusionsIn this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel

Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly accumulate Lucifer Yellow lumenally, it is very likely that processes other than organic anion accumulation are involved in the process of cystogenesis, including the Na,K-ATPase.

Proteasome inhibitors suppress formation of polyglutamine-induced nuclear inclusions in cultured postmitotic neurons

At least nine neurodegenerative disorders are caused by expansion of polyglutamine repeats in various genes. This expansion induces the formation of nuclear inclusions (NI) within various cell types. In this study, we developed a model for polyglutamine diseases using primary cultures of sympathetic neurons from the superior cervical ganglia of prenatal rat pups. Transfection with a plasmid encoding 127 glutamine repeats causes NI to develop in ∼70% of the sympathetic neurons within 6 days. In addition, it causes somatic atrophy and inhibits dendritic growth. The NIs contain ubiquitinated proteins and sequester the molecular chaperone heat shock protein 70 (Hsp70). We found that two specific proteasome inhibitors, lactacystin and CEP1612, suppress thezformation of polyglutamine‐induced NI. In addition, lactacystin treatment induced the removal of preexisting NI. Western blotting and immunocytochemistry revealed that lactacystin and CEP1612 strongly induce the expression of Hsp70, whereas less specific proteasome inhibitor such as N‐acetyl‐Leu‐Leu‐Norleucinal does not. Coexpression of 127 glutamines with a plasmid encoding wild‐type Hsp70 gene resulted in a marked reduction of the percentage of neurons containing NI. In addition, transfection with plasmids encoding mutant Hsp70 blocked the effects of lactacystin. These findings further implicate Hsp70 as a neuroprotective molecule and they suggest the potential utility of certain proteasome inhibitors in the treatment of polyglutamine diseases.

Storing analog data in a video record

We have designed a simple device that will encode, in machine readable form, multiple analog data in a video record. The analog data is visible to the user within the video frame permitting visual correlation of these signals with activity observed in the image. By superimposition of both analog and video data sets, the two are tightly synchronized and remain so in all copies of the data. The analog data can be separated from the image following digitization by a frame grabber. The bandwidth for each of the five analog channels is approximately 5 kHz. The device, which is essentially an eight channel video multiplexor, includes a video channel, a field counter, an amplitude calibration signal and five analog data channels. The amplitude calibrator allows corrections for gain errors that are particularly prevalent when data is stored on video tape.

Role of Ponticulin in Pseudopod Dynamics, Cell-Cell Adhesion, and Mechanical Stability of an Amoeboid Membrane Skeleton

ELIZABETH J. LUNA’, ANNE L. HITT’, DAMON SHUTT*, DEBORAH WESSELS*, DAVID SOLL*, PAT JAY3, CHRIS HUG3, ELLIOT L. ELSON3, ALEX VESLEY4, GREGORY P. DOWNEY4, MICHAEL WANG’, STEVEN M. BLOCK’, WADE SIGURDSON6, AND FREDERICK SACHS6 ’ University of Massachusetts Medical Center, Worcester Foundation Campus, 222 Maple Ave, Shrewsbury, Massachusetts 01545; * Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242; 3 Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63 110; 4 Department of Medicine, University of Toronto, Ontario, ON M5S lA8, Canada; 5 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544; and 6 Department of Biophysics, SUNY at Buffalo, Buffalo, New York 14214 Ponticulin is a transmembrane protein that constitutes the major high-affinity link between the actin cytoskele- ton and the plasma membrane of the soil amoeba Dictyo- stelium discoideum. As one of the few membrane proteins known to contain both a cytoplasmic domain and a glyco- syl anchor, ponticulin accounts for about 90% of the actin- binding and actin-nucleating activities of isolated plasma membranes. The function of ponticulin in vivo is being deduced by analyzing mutant amoebae in which the single-copy ponticulin gene has been disrupted by homologous recom- bination. These cells are deficient in high-affinity actin- membrane binding, as determined by co-sedimentation of actin and membrane vesicles from freshly broken cells, electron microscopic analysis, and in vitro assays of actin- membrane binding and membrane-mediated actin nucle- ation. Thus ponticulin’s role as a major link between the actin cytoskeleton and the plasma membrane has been confirmed both in vivo and in vitro. Because ponticulin