R-3 Repository :: Browsing by Author "Gomer, Richard H."

Browsing by Author "Gomer, Richard H."

Now showing 1 - 10 of 10

A cell number-counting factor regulates group size in Dictyostelium discoideum
(2002) Tang, Lei; Gomer, Richard H.
Little is known about how a morphogenetic rearrangement of a tissue is effected by the individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams which then break up into groups of ∼2 x 104 cells. Cell number is sensed using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF causes an aggregation stream to break up. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. On the other hand, CF significantly potentiates the cAMP-induced cAMP pulse size through the up-regulation of the adenylyl cyclase. Both the cAMP-induced cAMP and cGMP pulses are dramatically smaller in REMI-11 cells, which over-express REMI-11 protein, a smlA second site suppressor. CF does not affect the cAMP receptor, its interaction with its associated G proteins, or the translocation of the CRAC to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. We also show that Ca 2+ is required for CF's function although the mechanism is not clear. Computer simulations indicated that stream breakup could be caused by CF decreasing cell-cell adhesion and/or increasing cell motility, and we have previously observed that CF does indeed decrease cell-cell adhesion. We further demonstrated that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and ABP-120. Reducing motility using drugs which interfere with actin polymerization inhibits stream dissipation resulting in larger groups. CF also potentiates the phosphorylation and redistribution of myosin. Our results indicate that CF may regulate myosin function and cell polarity, at least in part, through activating protein kinases Akt/PKB and PAKa. CF thus appears to induce the morphogenesis of streams into evenly-sized groups by decreasing adhesion, increasing actin polymerization, increasing myosin phosphorylation, and increasing cell polarity.
A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum
(National Academy of Sciences, 2012) Phillips, Jonathan E.; Gomer, Richard H.; National Institutes of Health
Chemorepellants may play multiple roles in physiological and pathological processes.However, few endogenous chemorepellants have been identified, and how they function is unclear. We found that the autocrine signal AprA, which is produced by growing Dictyostelium discoideum cells and inhibits their proliferation, also functions as a chemorepellant.Wild-type cells at the edge of a colony show directed movement outward from the colony, whereas cells lacking AprA do not. Cells show directed movement away from a source of recombinant AprA and dialyzed conditioned media from wild-type cells, but not dialyzed conditionedmedia fromaprA− cells. The secreted protein CfaD, the G protein Gα8, and the kinase QkgA are necessary for the chemorepellant activity of AprA as well as its proliferation-inhibiting activity, whereas the putative transcription factor BzpN is dispensable for the chemorepellant activity of AprA but necessary for inhibition of proliferation. Phospholipase C and PI3 kinases 1 and 2, which are necessary for the activity of at least one other chemorepellant in Dictyostelium, are not necessary for recombinant AprA chemorepellant activity. Starved cells are not repelled by recombinant AprA, suggesting that aggregation-phase cells are not sensitive to the chemorepellant effect. Cell tracking indicates that AprA affects the directional bias of cell movement, but not cell velocity or the persistence of cell movement. Together, our data indicate that the endogenous signal AprA acts as an autocrine chemorepellant for Dictyostelium cells.
Characterization of CnrN-mediated size regulation in Dictyostelium
(2008) Tang, Yitai; Gomer, Richard H.
An interesting but largely unanswered question in biology is how eukaryotic organisms precisely regulate the size of multicellular tissues or groups of cells. Developing Dictyostelium cells aggregate in dendritic streams using relayed pulses of adenosine 3'5'-cyclic monophosphate (cAMP) as a chemoattractant to form ∼20,000-cell fruiting bodies. Counting factor (CF), a secreted protein complex, regulates group size by increasing cell motility and decreasing cell-cell adhesion to induce the breakup of excessively large aggregation streams. We used a second-site suppressor screen by conducting random insertional mutagenesis in smlA- cells which over-secrete CF to search for CF signal transduction components, and found that an insertion in the cnrN gene affects group size. Cells lacking CnrN (cnrN- cells) form small aggregation territories with few streams, which then form small fruiting bodies. Expressing CnrN in cnrN- cells rescues the abnormal phenotype. Computer simulations suggested that in the absence of stream formation, CF should be unable to affect group size. As predicted, cnrN- group size is insensitive to the addition or depletion of CF, even though CF regulates the motility of cnrN - cells. cnrN- cells have excessively large cAMP-stimulated cAMP pulses, and the small territory phenotype can be rescued by developing cells in the presence of the cAMP-hydrolyzing enzyme cAMP phosphodiesterase or simply by starving cells at low densities. The predicted amino acid sequence of CnrN has similarity to phosphatase and tensin homologs (PTENs). PTENs play a key role in inhibiting phosphatidylinositol 3' kinase (PI3K) dependent pathways. In Dictyostelium, cAMP pulses activate PI3Ks, and activated PI3Ks in turn stimulate adenylyl cyclase to produce cAMP. As indicated by the sequence similarity of CnrN to PTEN, in response to cAMP stimulation, cnrN- cells show elevated and extended activation of PI3K-dependent pathways, including PIP3 accumulation, Akt activation, actin polymerization, and adenylyl cyclase-catalysed cAMP production. Our results suggest that CnrN has functional similarity to PTEN and regulates the cAMP pulse size by negatively regulating PI3K-dependent pathways, and that CnrN-mediated regulation on territory and stream formation may use a CF-independent mechanism.
Characterization of serum anyloid P and Fc gamma receptor: A critical engagement implicated in fibrosing diseases
(2012) Crawford, Jeffrey Ray; Gomer, Richard H.
Fibrotic diseases have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells called fibrocytes participate in the formation of fibrotic lesions. The conserved pentraxin protein serum amyloid P (SAP) inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of immunoglobulin G (FcγR), and has been crystallized bound to FcγRIIa. The in vivo activity of SAP appears to be dependent on the common γ chain (FcγR) of activating Fc receptors. The goal of my project is to elucidate the functional domains of SAP and the receptor responsible for SAP bioactivity, which could lead to refinements for SAP as a therapeutic agent and additional drug targets. I found that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases SAP's ability to inhibit fibrocyte differentiation. In murine cells, deletion of FcγR or FcγRI significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb/FcγRIIIa/FcγRIV did not significantly affect sensitivity to SAP, while deletion of just the inhibitory receptor FcγRIIb increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcγR or FcγRI levels significantly decreased sensitivity to SAP, while reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcγR to inhibit fibrocyte differentiation. I am also interested in how SAP functions in various disease states. SAP is known to be elevated in Alzheimer's disease (AD) and binds to amyloid plaques in the brain, a key hallmark of AD. There is a significant population of individuals that have key hallmarks of AD but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). I evaluated SAP levels in post mortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels, while NDAN samples had no significant difference in SAP levels compared to controls. These results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques.
Developmental mechanisms of the cellular slime mold, Dictyostelium discoideum
(2000) Bishop, John David; Gomer, Richard H.
Our laboratory has shown that an extracellular signalling protein, Conditioned Medium Factor (CMF), acts as a cell-type specific cell-density sensing factor that is required for cells of Dictyostelium discoideum to undergo both differentiation and morphogenesis during development. The work pursued in this thesis was initiated both to determine how CMF controls development and to discover additional components of the developmental pathway. Chapter one describes investigations into the CMF signal transduction pathway. In the first section, it is demonstrated that CMF null cells fail to produce viable spores and furthermore that application of exogenous cAMP pulses rescues the inability of the CMF null cells to develop. It is further demonstrated that in CMF antisense cells CMF is required for cAMP-induced cGMP production. In the second section it is demonstrated that CMF does not affect the phosphorylation state of the cAMP receptor cAR1, nor does CMF affect the ability of cAR1 to adopt either the low affinity AL form or the super slow disassociating BSS form. However, CMF does slightly increase the ability of high levels of cAMP to induce a loss of ligand binding to cAR1. These data are contrary to previous reports, and reasons for these discrepancies are discussed. In the third section, it is demonstrated that the cAR1 phosphorylation sites are not required for the CMF effect on cAMP stimulated GTPase activity, cAMP-stimulated calcium influx, or low cell density aggregation. Finally, in the fourth section, it is shown that although CMF binds a G-protein coupled receptor that is linked to Galpha1, and the cAMP and CMF cell surface binding sites are very tightly linked, the sites must be two separate proteins because the linkage between the binding sites is disrupted in Gbeta-mutants. Furthermore, heterologously expressed cAR1 binds cAMP but not CMF. Chapter two describes the search for novel developmental mutants. The initiation of a mutagenesis screen is described, as are the cloning and partial characterization of five mutants. These mutants are caused by disruptions of genes encoding a novel deubiquitinating enzyme, an extracellular signal regulated kinase, a novel uridine diphosphoglucose pyrophosphorylase isozyme, and two completely novel proteins.
Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation
(BioMed Central, 2009) Choe, Jonathan M.; Bakthavatsalam, Deenadayalan; Phillips, Jonathan E.; Gomer, Richard H.
Background: Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA. Results: We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a K D of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a K i of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA. Conclusion: Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.
First evidence of skelemin, a myosin-associated protein, in smooth muscle and its involvement in cell adhesion, and, The role of the cell cycle in cell type choice during mammalian development
(1996) Schwartz, Jeanette Marie; Gomer, Richard H.
Indirect immunofluorescence in conjunction with Northern and Western blot analysis were used to identify the presence of skelemin, a myosin-associated protein, in smooth muscle. Feline uterus was cryosectioned and triple-stained for skelemin, smooth muscle myosin, and desmin. I observed that skelemin antibodies colocalized with the myosin-II filaments as well as the desmin intermediate filament cytoskeleton. This is the first evidence of a myosin-associated protein within smooth muscle, and it raises the possibility that smooth muscle myosin may be more organized than has been assumed. Antisense oligonucleotide treatment was used to analyze the underexpression of skelemin protein in developing embryoid bodies. Skelemin underexpression was seen to cause loss of cell-cell as well as cell-substrate adhesion. Time lapse video microscopy was used to analyze the role of the cell cycle in cell type choice during mammalian development. Discussed are methods employed to improve resolution for accurate analysis.
Regulation of cell number and cell movement in Dictyostelium discoideum
(2013-09-16) Phillips, Jonathan; Gomer, Richard H.; Braam, Janet; Beckingham, Kathleen M.; Farach-Carson, Cindy; Tabor, Jeffrey J.
Little is known about how the size of a tissue is established during development and maintained subsequently. Proliferation-inhibiting signals secreted by cells within a tissue that act specifically on cells within that tissue can provide negative feedback on cell number, thus regulating tissue size. A better understanding of tissue-specific inhibitors of proliferation could be useful for designing therapies for cancer and other diseases. However, few signals of this sort have been identified, and little is known about how these signals function. Two examples of such signals are the proteins AprA and CfaD, which are secreted by the social amoeba Dictyostelium discoideum and inhibit cell proliferation in a concentration-dependent manner. Cells lacking either AprA or CfaD proliferate rapidly, and adding recombinant AprA or CfaD to cells reduces proliferation. However, little is known about the signal transduction pathways downstream of AprA and CfaD. I identified three proteins that are required for the normal function of AprA and CfaD: the kinase QkgA, the putative transcription factor BzpN, and the putative kinase PakD. Cells lacking any one of these proteins proliferate rapidly, and adding AprA or CfaD to cells lacking these proteins does not cause reduced proliferation, indicating that these proteins are involved in AprA/CfaD signal transduction. I also found that, in addition to its proliferation-inhibiting activity, AprA also functions as an autocrine chemorepellant. Colonies of cells lacking AprA expand less rapidly than wild-type colonies, despite the fact that individual cells lacking AprA show a random motility like that of wild-type cells. Further, two independent assays demonstrate that cells show a biased movement away from a source of AprA. The chemorepellant activity of AprA requires CfaD, QkgA, and PakD, but not BzpN, indicating that AprA affects proliferation and chemorepulsion through distinct but overlapping pathways. These results suggest that AprA functions as a readout of local cell density, to which cells respond by slowing proliferation and chemotaxing to regions of lower cell density, where nutrients are more likely to be present. The study of human AprA, CfaD, QkgA, BzpN, and PakD orthologs may serve to guide therapeutic approaches that modulate chemorepulsive or antiproliferative processes.
Theoretical and experimental studies on a cell number counting mechanism
(2004) Jang, Wonhee; Gomer, Richard H.
It is unclear how organisms regulate the size of their components during development. Dictyostelium discoideum form evenly sized groups of ∼2 x 104 cells during aggregation. One mechanism that Dictyostelium use to control group size is by breaking up the aggregation stream when too many cells are present. We developed a computer model in which the cells are treated as discrete points having random motility and adhesion. The model predicted that stream breakup can be regulated by modulating adhesion and motility. The model also showed that a precise group size control can be achieved by having the local concentration of a secreted factor modulating adhesion and motility. In Dictyostelium , a secreted 450 kDa protein complex called counting factor (CF) regulates group size by repressing cell-cell adhesion and increasing cell motility. To identify components involved in the CF signal transduction pathway, we performed random mutagenesis to find possible second-site enhancers or suppressors of a transformant lacking bioactive CF. The analysis of one enhancer (icdh-) suggested that icdh - cells may have a defect in the CF signal transduction pathway. In icdh- cells, one of the isocitrate dehydrogenase genes is disrupted, which will lead to the accumulation of citrate. Since citrate downregulates glycolysis, the internal glucose levels may be altered. Therefore, we examined the possibility that CF decreases group size by regulating internal glucose levels. We found that glucose is a downstream component of the CF signaling pathway, and that CF decreases group size in part by repressing internal glucose levels. CF also repressed the levels of glycogen, pyruvate, lactate, ATP, and oxygen consumption. The ability of CF to decrease internal glucose levels was not linked to the synthesis or degradation of glycogen, but to the repression of gluconeogenesis by decreasing the activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Our working hypothesis is that CF decreases group size by downregulatmg the activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase, resulting in the changes in the signaling cascade, which will in turn regulate adhesion and motility to induce stream to break up in Dictyostelium .
Uncovering the signal transduction pathway that regulates the proliferation of Dictyostelium cells
(2010) Hanson, Nana; Gomer, Richard H.
AprA (Autocrine Proliferation Repressor) is a secreted factor that represses proliferation in the slime mold Diclyostelium discoideum . Although the role AprA plays in the regulation of proliferation is known, the signaling pathway by which it occurs is yet to be determined. To discover the different components of the AprA signaling pathway, I examined 12 REMI mutants which proliferated in a similar manner to aprA - cells. Three of 12 mutants were insensitive to AprA. MetPOE was one of the mutants that were insensitive to AprA, and I attempted to construct a complete knockout of the disrupted gene. From an additional REMI screen that I conducted, I found that disruption of dokA led to a mutant with a phenotype similar to aprA-, suggesting this protein may also have a role in the AprA signal transduction pathway. Preliminary data suggested that G proteins may also play a role in the AprA signal transduction pathway. I further characterized six G protein null mutants. I determined that both galpha8- and gbeta- cells were multinucleate. Also there was an increase in the percentage of cells in S phase in gbeta - cells. The similarities between aprA- cells and ga8- and gbeta - cells suggested that Galpha8- and Gbeta - may be part of the AprA signal transduction pathway.

Browsing by Author "Gomer, Richard H."

Results Per Page

Sort Options