Damon C. Shutt

Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassembly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and chemotaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-assisted methods in buffer, in a spatial gradient of cAMP, and after the rapid addition of cAMP. 3XALA cells crawling in buffer exhibit distinct abnormalities in cellular shape, the maintenance of polarity and the complexity of the pseudopod perimeter. 3XALA cells crawling in buffer also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which the shape of each cell changes abnormally from elongate to extremely wide with lateral, opposing pseudopods. At the end of each cycle, 3XALA cells turn 90 degrees into the left or right lateral pseudopod, resulting in a dramatic depression in chemotactic efficiency, even though 3XALA cells are chemotactically responsive to cAMP. These results demonstrate that the phosphorylation of myosin II heavy chain plays a critical role in the maintenance of cell shape and in persistent translocation in a spatial gradient of chemoattractant.

Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis.

Previous studies have demonstrated that overexpression of the carboxyl-terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359-368]. To assess the consequences of having CaD39-stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39-expressing lines. We here found that CaD39-expressing cells adhered faster to plastic, glass, fibronectin-coated glass, and collagen-coated glass than control cells. Moreover, the CaD39-expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39-expressing cells were more elongate and encompassed less area than non-expressing cells during migration in a wound-healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin-enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39-induced advantage for attachment and spreading was lost during mitosis. In CaD39-expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39-expressing lines.

T cells and HIV-induced T cell syncytia exhibit the same motility cycle.

Ameboid cells ranging in complexity from Dictyostelsum amebas to human polymorphonuclear leu kocytes (PMNs) translocate in a cyclical fashion. Using computer‐assisted motion analysis, we have analyzed the motility of human lymphocytes of the immortal SupTl cell line and of a peripheral blood mononuclear cell popu lation highly enriched for CD4‐positive cells (CD4‐enriched PBMCs) on four substrates—plastic, dehydrated rat tail collagen, hydrated rat tail collagen, and bovine aortic en dothelium. In addition, we have analyzed the motility on these substrates of syncytia induced by human immuno deficiency virus (HIV) in cultures of both cell types. It is demonstrated that both SupTl cells and CD4‐enriched PBMCs exhibit a motility cycle with a period of 1.6 min that is independent of substrate, independent of average cell velocity, and similar to the periods of translocating Dictyostelium amebas and PMNs. More surprisingly, it is demonstrated that HIV‐induced SupTl and PBMG syn cytia with volumes 10 to 100 times those of single cells ex hibit the same motility cycle as their single‐cell progeni tors. These observations support the generality of the motility cycle in animal cells and, for the first time, demonstrate that the cycic is independent of cell size. J. Lcukoc. BioL 57; 643–650; 1995.

2-deoxy-D-glucose induces oxidative stress and cell killing in human neuroblastoma cells.

Malignant cells have a demonstrably greater sensitivity to glucose deprivation-induced cytotoxicity than normal cells (Biochem. J. 418:29). This has been hypothesized to be due to a higher level of reactive oxygen species (ROS) production in cancer cells leading to the increased need for reducing equivalents, produced by glucose metabolism, to detoxify hydroperoxides. Because complete glucose deprivation cannot be achieved in vivo, it has been proposed that agents that antagonize glucose metabolism, such as 2-deoxy-D-glucose (2DG), can mimic in vitro glucose deprivation that selectively kills cancer cells by oxidative stress. To test this hypothesis, neuroblastoma cell lines were treated with 2DG and the effects on clonogenic survival and the distribution of cellular phenotypes among surviving colonies was determined. The results showed that all three major cell types found in neuroblastoma (Schwann, Neuronal, and Intermediate) were sensitive to 2DG-induced clonogenic cell killing. Furthermore, treatment with the thiol antioxidant, N-acetyl cysteine, or with polyethylene glycol-conjugated superoxide dismutase and catalase, protected neuroblastoma cells from 2DG-induced cell killing. Finally normal non-immortalized neural precursor cells were relatively resistant to 2DG-induced cell killing when compared to neuroblastoma cell lines. These results support the hypothesis that inhibitors of glucose metabolism could represent useful adjuvants in the treatment of neuroblastoma by selectively enhancing metabolic oxidative stress.

Characterization of Intestinal and Pancreatic Dysfunction in VPAC1-Null Mutant Mouse

Objectives: These studies examined the effect of homozygous deletion of vasoactive intestinal peptide receptor type 1 (VPAC1) on development and function of intestines and pancreas. Methods: Genetically engineered VPAC1-null mutant mice were monitored for growth, development, and glucose homeostasis. Expression of VPAC1 was examined during embryonic development using VPAC1 promoter-driven &bgr;-galactosidase transgenic mice. Results: Homozygous deletion of VPAC1 resulted in fetal, neonatal, and postweaning death owing to failure to thrive, intestinal obstruction, and hypoglycemia. Histological findings demonstrated disorganized hyperproliferation of intestinal epithelial cells with mucus deposition and bowel wall thickening. The pancreas demonstrated small dysmorphic islets of Langerhans containing &agr;, &bgr;, and &dgr; cells. Expression of a VPAC1 promoter-driven transgene was observed in E12.5 and E14.5 intestinal epithelial and pancreatic endocrine cells. Vasoactive intestinal peptide receptor type 1-null mutant animals had lower baseline blood glucose levels compared to both heterozygous and wild-type littermates. Vasoactive intestinal peptide receptor type 1-deficient mice responded to oral glucose challenge with normal rise in blood glucose followed by rapid hypoglycemia and failure to restore baseline glucose levels. Insulin challenge resulted in profound hypoglycemia and inadequate glucose homeostasis in VPAC1-null mutant animals. Conclusions: These observations support a role for VPAC1 during embryonic and neonatal development of intestines and endocrine pancreas.

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