Open Forum on Cell Signaling

 

Open Forum on Cell Signaling

Receptor Topography of Cell Membranes

Nov 1 2000 6:55AM

Konrad F Koehler

Dear STKE Cell Signaling Forum,

I'm wondering if anyone has ever attempted to map the receptor topography of a particular cell type to determine the numbers and kinds of receptors present on its extracellular surface. What kind of experimental strategies would be most conducive for attaining this kind of information?

Response to Topography of Cell Membranes

Nov 20 2000 10:50AM

James Keefer

Dear Konrad, There are several companies including mine, NovaScreen, that can screenmultiple receptor targets on a cell and could add targets of interest. We currently have over 200 receptor and enzyme assays up and running. Ourlab published the cell profile process in J. of Biomolecular Screening, Vol. 3, No. 3, 1998, entitled "Profiling Established Cell Lines as a Means to Screening Diversity", by Evelyn Good et al. A list ofendogenous GPCR coupled receptors on a few cell lines is available atbiomedcomp.com/GPCR.html from Agi Schonbrunn at Univ of Texas, Houston.

What's your opinion on screening using microarray/DNA technology?

Jim Keefer

AP-1/Egr1 puzzler

Nov 7 2000 5:38AM

Andrey Ryabinin

Here is a puzzle: in neural cells expression of Egr1 (aka Zif268, NGFI-A, krox-24,etc.) is often induced together with that of c-Fos. No surprise here, theirpromoters both have CRE and SRE sites. Sometimes Egr1 is induced, but c- Fos is not. This is also possible to explain: there are four SRE sites inegr1 and only one in c-fos promoter, plus these SRE's are not identical.Now we have found a treatment that strongly induces Fos, but not Egr. What could be involved in this?

We have tried to test for increased CREB phosphorylation (fos hasmore CRE sites than egr), and increased phosphorylation of Stat1, Stat3,Stat5, and Stat6 (fos has SIE elements, and egr not), and did not find anyof them upregulated (well, we could have failed in our technique with some of them). What else could be involved in such differential regulation?

Response to AP-1/Egr1 puzzler

Nov 22 2000 7:29AM

Fabio Romerio

I can think of a few possibilities. First: it is known that c-fos transcription is upregulated in response toMEK/ERK triggering through activation of the transcription factor Elk-1,which binds the c-fos enhancer. Does Elk-1 bind also to the Egrpromoter/enhancer?Second: STAT1 and STAT3 are regulated by tyrosine phosphorylation, but for full activity they require also phosphorylation in serine (see papers by Doreen Cantrell and also by Andrew Larner). If you checked for variations in thelevels of tyrosine phosphorylated STAT1/3, you may have missed thedifferences in the serine phosphorylation. My suggestion would be to runEMSA's or follow the experimental procedures in the Cantrell and Larnerpapers. Third: analysis of the promoter sequences may not be enough. Have youchecked also the enhancers? A detailed analysis of the promoter andenhancer sequences of the two genes may give you additional clues. Followthis link: http://transfac.gbf.de/programs.html You will be able touse the MatInspector software to determine which transcription factors are likely to bind to a given DNA sequence. Clearly this provides only anindication, but it may be a good start.

Modeling Signal Transduction Networks

Nov 22 2000 9:33AM

Allen Taylor

I am interested in modeling signal transduction networks, usingtraditional electronic circuit analysis methods, based on time-series data taken with DNA probe arrays. I have two questions:

1. Who is working in this area, that I might be able to communicatewith?

2. Where can I get time-series data that has not yet been completelyanalyzed?

Allen G. Taylor Portland State University Portland, Oregon USA

Re:modelling circuits

Mar 18 2002 6:36AM

Daniel W Carr

Check Cornell University's website. I recently came across a a jobopening for a company in Ithaca, NY that is based on research conducted atCornell. The research was on modelling signalling networks, and they hadcomplex circuitry diagrams on their web page. Good Luck.

Identifying bound and unbound NFAT

Nov 30 2000 7:31AM

Jauliac Sebastien

I would like to know if it is possible todistinguish, in the nucleus of a cell, the DNA-complexed NFAT from the free NFAT ?

concentration of signal transduction molecules

Jan 16 2001 6:55AM

Eric Fung

I am interested in knowing whether there is a source (or more likely, sources) of data detailing the concentration (e.g. in attamoles or innumber of copies) of various signal transduction molecules in cells,either at baseline or in their activated state. This kind of informationis helpful in assessing requirements for sensitivity of different assays.Your expertise would be greatly appreciated!

adenylate cyclase

Feb 1 2001 10:39AM

Marco Mongillo

i am working on the Adenylate Cyclase-PKA signalling pathway and ihave not found literature on the basal activity af AC. Does anyone know if any of the isoforms of AC have a measureable basal activity ? this would be of great interest to understand the details of cAMP signalling pathway.

"Basal" AC activity

Mar 28 2001 7:05AM

Md. Shahidul Islam

In the cells that I commonly work with, i.e. pancreatic beta cells ortumor cell lines derived from them, it has been reported many times thatif you just add some PDE inhibitors to a cell suspension, cAMP levelincreases. This may mean that there is some basal activity of AC in thesecells, even in the apparent absence of ligand binding.

However the meaning of "basal" is ambiguous here. This may mean that low level ofunidentified ligands secreted from the same cells (autocrine) orneighbouring cells (paracrine) cells are active on the receptors linked to AC. This low level of receptor activation does not increase cAMP a lot but is visibleonce PDE is inhibited. The situation in the cell is complex in this way.

What happens inthe test tube can be speculated. It is possible that there are someendogenous forskolin-like molecules and these may maintain some basalactivity of the enzyme.

basal AC activity

May 3 2001 1:06PM

Zhengui Xia

Although what Dr. Islam says is correct, several ACs have "high" basal activity even in in vitro AC assays.

Thus AC activity may not only be regulated byauto/paracrine activities or leaky GPCRs, but some ACs have high intrinsic activity before "activation".

In particular see Neilsen et al. from Dan Storm's lab. AC8 has a very high basal activity even when expression levels are accounted for.

Mapping topology of receptors in membranes

Mar 8 2002 1:35PM

Janet M Oliver

Konrad, You opened this discussion some time ago but it would be interesting to keep this topic "alive". We use both transmission electron microscopy of membrane sheets and scanning electron microscopy of intact cells tomap receptors on plasma membranes. This shows promise for givingboth 2-dimensional (membrane sheets) and 3-dimensional (scanning) distributions of proteins across membranes that have dramatic changes in surface topography due to microvilli, ruffles, etc. Please see Wilson et al, JCB 154:645 and JCB 149:1131.

What do you say about using 2-D proteomics?

Sep 16 2002 10:33AM

Dk Singh

Hello members, What do you think about the idea ofusing 2-D electrophoresis using the membranefraction of the cells for topology mapping? I do feel thatthis will not only give one an outlook of the resting cellsas such but also provide information about the changes in the cells' membrane status both in normal resting conditions and in activated states. Please comment on the feasibility of the process.

What do you say about using 2-D proteomics?

Jan 1 2006 7:09PM

Dennis Kreinfeldt

Whilst potentially very powerful, I think the early pioneers of 2Delectrophoresis underestimated the sheer number of permutations of posttranslational modifications. So, instead of having to map just severalthousand membrane protein spots on a 2D gel (which could be manageable), I think you could have to map 30-300,000 protein spots. To elaborate onthis, there's a company called apollo cytokine research which routinelyanalyses it's soluble versions of membrane receptors by 2D electrophoresis - the results are quite spectacular, you can have as many as 20 differentspots on a 2D gel, each reflecting unique glycosylation species (orglycoforms). And this is just one protein!

No role for RTK in GPCR activation of the Ras-MAPK pathway?

Mar 29 2001 7:55AM

Peter Lockyer

I would be interested in opinions about the recent work by theSchlessinger group (JBC, "in press", but full text available at this link) that shows by genetic manipulation thatPyk2, Src, and the EGFR are dispensable for GPCR-induced MAPK activation.

Clearly the results shows the importance of using a genetic approach toresolve a complex issue, and this highlights the context and cell type- specific nature of cell signalling. Does this mean that the these mechanisms are less important thanpreviously thought? What are the dominant mechanisms then regulating GPCR-induced MAPKactivation? And how might these knock-out cells be bypassing these routes?

No role for RTK in GPCR activation of the Ras-MAPK pathway?

May 6 2010 8:16AM

Coralie A. C. Carraway

To my way of thinking, genetic approaches alone cannot begin to answer such critical questions as involvement of known important signaling proteins in a given pathway, especially when crosstalk is known to be involved. There are very few, if any, cases in complex organisms in which crosstalk is not involved. This holds also for simple organisms; even single-cell organisms respond to a variety of stimuli, and the vectorial responses under any set of conditions are tightly regulated by crosstalk.

Such reductionist thinking also does not take into account critically important post-transcriptional processes which are vital to cellular readout. Major players not predictable by genetics include stability of message, all posttranslational modifications, including proteolysis, stability of proteins, protein biosynthesis and turnover, and protein interactions which direct their cellular localization. Superimposed upon all of these factors are cell context considerations.

Mother Nature can be simplified only to a point; careful regulation is complex, and organismal survival depends on that complexity.

No role for RTK in GPCR activation of the Ras-MAPK pathway?

Jun 30 2010 12:46PM

Bradley T. Andresen

I do agree with Dr. Carraway. However, I have previously published results showing that in thoracic aortic smooth muscle cells (TASMCs) and renal microvascular smooth muscle cells (RμVSMCs) from Wistar-Kyoto rats, angiotensin II in TASMCs, but not RμVSMCs, requires RTK kinase activity to signal to ERK (Escano, Jr. et al., 2008). Thus, I am not surprised by the results since there are alternate pathways for GPCR-mediated activation of ERK independent of RTKs in some cell types. It is likely that the knockouts are "rewired" during development to utilize these alternative pathways, whereas the wild type is not. This is parallel to Dr. Carraway's argument.

Dr. Lockyer did ask some specific questions:

1) Does this mean that these mechanisms are less important than previously thought?

Not necessarily – this has been answered previously by Dr. Carraway and in my first paragraph.

2) What are the dominant mechanisms, then, regulating GPCR-induced MAPK activation, and how might these knockout cells be bypassing these routes?

I am familiar with a few different mechanisms for GPCR activating ERK in addition to the now "canonical" transactivation pathway.

  • GPCRs may activate ERK through β-arrestin-mediated scaffolding (Daaka et al., 1998; Luttrell et al., 2001), but this has also been tied to Src and EGFR (Kim et al., 2008; Rakesh et al., 2010).
  • GPCRs may activate ERK through phospholipase D (Rizzo et al., 1999), reviewed in (Andresen et al., 2002).
  • Alternatively, GPCRs may directly activate ERK (Carroll and May, 1994; Schonwasser et al., 1998), but this pathway also utilizes Src family kinases (Mason et al., 1999; Tapinos and Rambukkana, 2005).

It may be possible for these pathways to act independently of Src and EGFR, or at least independently of Src, Yes, and Fyn, which were the only kinases examined (Andreev et al., 2001). However, one problem that has vexed me with the above theories is that there is not a solidified mechanism for GPCRs to activate Ras in these pathways. This is not true for the transactivation theory and is what makes it so appealing.

References

Andreev, J., Galisteo, M.L., Kranenburg, O., Logan, S.K., Chiu, E.S., Okigaki, M., Cary, L.A., Moolenaar, W.H., and Schlessinger, J. Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade. J. Biol. Chem. 276, 20130-20135 (2001).[Abstract] [Full Text]

Andresen, B.T., Rizzo, M.A., Shome, K., and Romero, G. The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade. FEBS Lett. 531, 65-68 (2002).

Carroll, M.P. and May, W.S. Protein kinase C-mediated serine phosphorylation directly activates Raf- 1 in murine hematopoietic cells. J. Biol. Chem. 269, 1249-1256 (1994).[Abstract]

Daaka, Y., Luttrell, L.M., Ahn, S., Della Rocca, G.J., Ferguson, S.S., Caron, M.G., and Lefkowitz, R.J. Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J. Biol. Chem. 273, 685-688 (1998). [Abstract] [Full Text]

Escano, C.S., Jr., Keever, L.B., Gutweiler, A.A., and Andresen, B.T. Angiotensin II activates extracellular signal-regulated kinase independently of receptor tyrosine kinases in renal smooth muscle cells: implications for blood pressure regulation. J. Pharmacol. Exp. Ther. 324, 34-42 (2008). [Abstract] [Full Text]

Kim, I.M., Tilley, D.G., Chen, J., Salazar, N.C., Whalen, E.J., Violin, J.D., and Rockman, H.A. {beta}-Blockers alprenolol and carvedilol stimulate {beta}-arrestin-mediated EGFR transactivation. Proc . Natl. Acad. Sci. U. S. A. 105, 14555-14560 (2008).[Abstract] [Full Text]

Luttrell, L.M., Roudabush, F.L., Choy, E.W., Miller, W.E., Field, M.E., Pierce, K.L., and Lefkowitz, R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proc . Natl. Acad. Sci. U. S. A. 98, 2449-2454 (2001).[Abstract] [Full Text]

Mason, C.S., Springer, C.J., Cooper, R.G., Superti-Furga, G., Marshall, C.J., and Marais, R. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J. 18, 2137-2148 (1999). [Abstract] [Full Text]

Rakesh, K., Yoo, B., Kim, I.M., Salazar, N., Kim, K.S., and Rockman, H.A. β-Arrestin-biased agonism of the angiotensin receptor induced by mechanical stress. Sci. Signal. 3, ra46 (2010). [Abstract] [Full Text]

Rizzo, M.A., Shome, K., Vasudevan, C., Stolz, D.B., Sung, T.C., Frohman, M.A., Watkins, S.C., and Romero, G. Phospholipase D and its product, phosphatidic acid, mediate agonist- dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J. Biol. Chem. 274, 1131-1139 (1999). [Abstract] [Full Text]

Schonwasser, D.C., Marais, R.M., Marshall, C.J., and Parker, P.J. Activation of the mitogen-activated protein kinase/extracellular signal- regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol. Cell Biol. 18, 790-798 (1998).[Abstract] [Full Text]

Tapinos, N. and Rambukkana, A., 2005. Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli. Proc. Natl. Acad. Sci. U. S. A. 102, 9188-9193. [Abstract] [Full Text]

Cytohesins and signal transduction

Jul 23 2001 7:48AM

Michael Lennick

Is there any information linking Trio, Rho, or Rac with cytohesins?

Vav inhibitor

Oct 5 2001 10:57AM

Anbu Kumar Karuppannan

We are working with primary B cells and I would like to know if there are any chemical inhibitors of Vav.

Request for info concerning receptor classification?

Oct 17 2001 9:15AM

Stelios Papaioanou

Hello all,

I am a medical student and I am currently working on a projectconcerning classification of receptors. Can you please be so kind toindicate me any site which I could start with? The search engines give meonly useless things. Thank you very much.

Classification Sites

Nov 27 2001 7:33AM

Nancy R. Gough

Stelios,

The answer to your question depends very much on the type of receptors that you are trying to classify. You might try the sites listed in the ST on the Web section of the STKE. The (Protein Databases section has links to such places as the GPCRDB: Information System for G Protein-Coupled Receptors ( http://www.gpcr.org/) and the NucleaRDB: Information System for Nuclear Receptors (http://receptors.ucsf.edu/NR/). The Model Organisms section has links to such places as the Mouse Genome Informatics site, which has a browsable ontology ( http://www.informatics.jax.org/go/. Another source for information about classification of receptors is the Gene Ontology group (http://www.geneontology.org/.

Hope this gets you started.

Nancy R. Gough, Ph.D. Managing Editor Science's STKE

Cell signalling in memory-cell/plasma cell developement

Dec 28 2001 7:29AM

Dk Singh

Can anyone working in the area of B-cell signalling give me thedifferent modes of recognition by the B-cells during activation, whichfinally result in two sets of cell population? What exactly determines thedifferentiation of the two in different cell population and reversal back of the memory cells to plasma cells in secondary immune response?

Cysteine Modulation in GPCR Activation

Jan 2 2007 6:10AM

Richard G. Lanzara

Increasing evidence suggests that there exists a free thiol or sulfhydryl residue within the ligand binding domain of many GPCRs (see:http://dx.doi.org/10.1016/j.jmgm.2006.02.008). The importance of this residue in relation to its redox state and to receptor activation needs a larger discussion among receptor scientists and those scientists studying redox regulation.

Cysteine Modulation in GPCR Activation

Jan 10 2007 8:59AM

PAULA, ARACENA,

I am completely ignorant in the field of GPCRs, but I do work inredox signaling field. There is a number of ideas coming to my mind rightnow, but before getting ahead of myself (as I usually do) I would like toask: Is it known what type of GPCR function is altered by this "importantcysteine (e.g. interaction with ligand, with subunits, etc)? And, is itslocation in the sequence known/conserved? I am soon to embark on independent research and I am very interested inthis!

Cysteine Modulation in GPCR Activation

Jan 10 2007 5:08PM

RICHARD G LANZARA

I'm very glad that this post has attracted someone in the redoxsignaling field. There are a number of important questions that may onlybe fully answered by a combined approach from several other perspectives.

To answer your questions about this important cysteine in GPCRs, itis one of the most highly conserved residues in all GPCRs and yes, it isalso known to be important for both ligand binding and receptorexpression. A search on GOOGLE will give you much of this information(such as:Search: GPCR, cysteine, disulfide).

This is a fascinating area that continues to encompass many otherissues in redox signaling (seeReview by M. Reth) forexample.

Highlights from the 2007 ASCB Annual Meeting

Dec 3 2007 12:01PM

STKE Editors

The STKE editors attended several sessions at the 2007 ASCB AnnualMeeting. You will find brief reports from the sessions attended. Click the "Next Message" link to read the editor's reports. Feel free to enter your own comments or share what interested you at the meeting.

Highlights from ASCB Symposium II (02 December 2007)

Dec 5 2007 2:02PM

Nancy R. Gough

At the Sunday morning symposium entitled "Architecture of Signaling Systems", the speakers were Richard Losick (Harvard), Pamela Silver (Harvard), and Tobias Meyer (Stanford).

Dr. Losick led off with a discussion of stochasticity in cell fate determination in which he described four examples from bacteria and one from the fruitfly, Drosophila melanogaster. The Drosophila color perception example and the example that Dr. Losick called "growth versus competence" have been described in detail in the STKE Review by Samoilov et al. The other bacterial examples included "swimming versus chaining", "eating or being eaten" (a story of bacterial cannabilism, a mechanism to regulate sporulation), and "individual versus community" (the formation of biofilms). In each of the bacterial examples, green fluorescent protein (GFP) or its variants was used to report on the expression of a particular gene promoter in individual bacterial cells, which revealed the stochastic differences among individuals in the population. The systems described all involve a transcriptional system with a positive feedback mechanism that allows the system to behave as a bistable switch. Dr. Losick emphasized that stochastic cell fate determination for the bacteria allows the cells to "hedge their bets" so that they are poised to adapt to changing environmental conditions.

Dr. Silver gave a very interesting talk on synthetic biology and posed the question of whether engineering biology can be as exciting as building a robot (see iGEM). After explaining why biological systems are attractive from engineering and design perspectives, she illustrated three applications of synthetic biology. The first she called designing a cellular memory system, in which yeast were created with a synthetic transcription system (a synthetic transcription factor that responded to an external stimulus and reporter gene with an engineered promoter). These engineered yeast activated the reporter gene in response to the cognate stimulus and this response persisted even after the stimulus was removed and after multiple rounds of cell division, thereby providing what Dr. Silver termed a "memory" of the stimulus.

In the second example, she described how the reduction of dimensionality in a biological process can be leveraged to engineer a fusion protein that targeted interferon (IFN) selectively to cancer cells. Biological systems routinely limit the diffusion of molecules to enable specific molecular interactions, for example the interaction of proteins with cellular membranes or residents of cellular membranes (such as receptors) restricts the mobility of the protein. In this example, knowledge of the structure of IFN and its interaction with its receptor allowed point mutations to be introduced that compromised its ability to bind to its receptor and then fusion to ligand specific to certain types of cancer cells (EGF) allowed the interaction of this "crippled" IFN with its receptor to only occur on the cancer cells following recognition of the EGF part of the fusion protein. This example certainly proves that higher affinity isn't always better.

In the final example, a group in her lab has tackled the ambitious goal of solving the energy crisis by engineering a new metabolic pathway in yeast that will allow the production of hydrogen from biomass.

The final speaker, Dr. Meyer, gave a two-part talk. In the beginning, he described his view of the cell signaling landscape in which he identifies ~40 signaling modules, ~3000 signaling components, and 200 unique cell types. He emphasized that fluorescent biosensors have been instrumental in dissecting signaling modules and he described how silencing RNA (siRNA) techniques have provided a genetic toolkit for studying mammalian biology. Using siRNA screens combined with fluorescent biosensors, Dr. Meyer described how a model for a signaling module of store-operated calcium (SOC) entry, which occurs when the calcium in the endoplasmic reticulum (ER) is depleted, was developed. The model consists of 6 core components: (i) calcium, (ii) Orai (the plasma membrane calcium channel activated by store depletion), (iii) PMCA (the plasma membrane calcium pump), (iv) SERCA (the ER calcium pump), (v) STIM proteins (the transmembrane calcium sensors of the ER), and (vi) the IP3 receptor (an endoplasmic reticulum calcium channel activated by inositol trisphosphate).

For the calcium signaling aficionados, STIM1 appears to be the high-affinity calcium sensor that is activated upon ER calcium depletion; whereas STIM2 appears to be a low-affinity calcium sensor that is active under basal conditions. Redistribution of these proteins to puncta of ER and plasma membrane junctions in response to ER calcium depletion requires STIM dimer- or oligomerization. This oligomerization requirement is likely because the polybasic region of STIM proteins that mediates the interaction with the plasma membrane has too few polybasic residues to mediate the interaction of individual STIM proteins.

See the ASCB program for the titles of these presentations and other information about the Saturday and Sunday events.

Everett E. Just Lecture (02 December 2007)

Dec 5 2007 2:02PM

Nancy R. Gough

ASCB president Bruce Alberts presented Alejandro Sánchez Alvarado (University of Utah) with the Everett E. Just Award. Following a few remarks about what the award meant to him, Dr. Alvarado spoke about his work studying regeneration of the planarium Schmidtea meterranea. This organism has remarkable regenerative capacity, which has been known for centuries, and includes the ability for a very small piece (1/279th) of the organism to completely regenerate. The genome of this organism has now been sequenced and annotated and, surprisingly, it contains the same complement of 13 homeobox genes that are found in vertebrates. He described his work to identify and characterize the stem cells responsible for this regenerative response. These stem cells are the only dividing cells in the organism and the two regions that lack these cells are the only ones that do not regenerate. Irradiation of the animals to destroy the stem cells results in wound closure following amputation, but no regeneration. Using RNAi techniques, he found what appears to be a new pathway leading to β-catenin activation that is required for formation and maintenance of tail structures in these organisms. (see the Science article by Alvarado)

Highlights from Minisymposium 4: Host-Pathogen Interactions and Innate Immunity

Dec 10 2007 10:59AM

Nancy R. Gough

I attended four of the talks at this minisymposium on Sunday, 2 December 2007. Joanne Engel (UCSF) described how Exotoxin T (ExoT) from Pseudomonas aeruginosa contributes to virulence. ExoT, which has a domain with GTPase-activating protein (GAP) activity and a domain with ADP ribosyltransferase (ADPRT) activity, is injected into the host cell by the type III secretion system. The GAP domain acts through Rho, Rac, and Cdc42 to cause rounding of the infected epithelial cells. The ADPRT domain ADP ribosylates the adaptors Crk I and Crk II, inactivating their SH2 domains, which causes the cells to lose adherence to the substrate. In addition to the morphological changes, infected cells also exhibit a block in cytokinesis. Analysis of the mechanism by which the ADPRT domain of ExoT inhibited cytokinesis revealed the presence of a focal adhesion-like structure at the midbody. ExoT-infected cells fail to recruit syntaxin to this midbody structure, which blocks cytokinesis.

The remaining three talks that I attended highlighted mechanisms by which plant pathogens interact with their hosts or the plant defense response. Xinyan Li of the lab of Mary Beth Mudgett (Stanford) described an atypical receptor-like kinase (RLK) from tomato that appears to be the target of the virulence protein XopN of Xanthomonas campestris pathovar vesicatoria (Xcv). XopN has multiple α-helical repeats. The N-terminal region interacted with tomato atypical RLK (TARK1), which in vitro had very low kinase activity. TARK1 is missing two critical conserved residues that are important for kinase activity. The C-terminal region of XopN interacted with 14-3-3 proteins. Knockout of TARK1 resulted in termination of the apical meristem, thus this protein may have roles in plant defense and development.

Silke Robatzek (Max Planck Institute for Plant Breeding Research) described her work on the subcellular trafficking of the flagellin receptor FLS2. FLS2 bound to the flagellin peptide Flg22 interacted with BAK1 (brassinosteroid insensitive 1-associated receptor kinase 1), and the cells respond with ion flux, production of reactive oxygen species (ROS), and changes in gene expression. In the presence of BAK1, Flg22 triggers the endocytosis of FLS2 in epidermis and leaf mesophyll. Dr. Robatzek found that some mutations of FLS2 that prevented endocytosis prevented ROS production and some did not block ROS production. Endocytosis of FLS2 appears to involve phosphorylation and ubiquitination. The virulence factor AvrPtoB from Pseudomonas syringae pathovar tomato interacts with FLS2 and this interaction is enhanced in the presence of flagellin. How this virulence factor influence FLS2 function and endocytosis remain open questions. See the Editors' Choice for a summary of some of the published work.

Ho Won Jung of the lab of Jean Greenberg (University of Chicago) described a small molecule, azelaic acid, that was isolated from infected Arabidopsis petioles. Leaves sprayed or injected with this small molecule conferred resistance to infection to nontreated leaves, suggesting that azelaic acid may be a mobile signal or may stimulate the production of a mobile signal involved in systemic acquired resistance.

Highlights from ASCB Symposium V (04 December 2007)

Dec 12 2007 10:44AM

John F. Foley

At the Tuesday morning symposium entitled "Geography of Signaling" the speakers were Deborah Hogan (Dartmouth Medical School), Howard Chang (Stanford University), and Elly Tanaka (Max Planck Institute Dresden).

Dr. Hogan discussed signaling in multicellular microbial communities, in particular about how microbes differentiate and develop within microbial biofilms. Biofilms are formed, for example, at the interface between liquid and air in a neglected bacterial culture, but can also occur when microbes form multicellular communities on surfaces, even on human tissues. Biofilms contain an extracellular matrix component, which is often derived from the microbes themselves, and microbes behave differently within the biofilm compared to their behavior in other environments.

Dr. Hogan explained that two organisms that are often found together in biofilms are the bacterium Pseudomonas aeruginosa and the yeast Candida albicans. P. aeruginosa use filamentous C. albicans as a substrate for biofilm formation, which kills the yeast in a process that is dependent on a form of P. aeruginosa phospholipase C. The unicellular (yeast) form of C. albicans is not bound to or killed by P. aeruginosa and mutant P. aeruginosa that cannot bind to filamentous C. albicans cannot kill it. When P. aeruginosa and filamentous C. albicans are grown together, any new C. albicans grow as single cells, even in the presence of cues for filamentous growth. This implied that C. albicans could sense a substance produced by the bacteria and respond by switching their morphology from a filamentous form, which is killed by P. aeruginosa, to a unicellular form, which can survive. A screen of P. aeruginosa identified 3-oxo-C12-homoserine lactone (3OC12HSL) as a factor that was sensed by C. albicans and prevented their filamentous growth.

In C. albicans, the pathway that determines filamentous growth is dependent on the activation of the small guanosine triphosphatase (GTPase) Ras, the subsequent stimulation of adenylyl cyclase, and the activation of cAMP-dependent protein kinase (PKA), which results in the activation of the transcription factor Efg1. 3OC12HSL inhibited this pathway and also induced stress response genes that were repressed by activated PKA. Examination of the effects of 3OH12HSL illustrated the connection in C. albicans between stress responses and morphology and showed that inhibition of the cAMP-dependent pathway leads to morphological changes and cell survival.

Dr. Chang discussed site-specific differences in the skin and the essential role of stromal cells from the underlying mesenchyme in directing the identities of the overlying epithelial cells. Dr. Chang used microarrays to show that stromal cells from 10 sites on the human adult body had distinct gene expression profiles, thus illustrating the genomic encoding of positional identity. These distinct gene expression patterns were determined by the expression of Hox genes, which encode transcription factors that are necessary for determining the embryonic body plan. Dr. Chang described how a combination of chromatin immunoprecipitation assays and microarray analyses revealed long non-coding RNAs and chromatin modifications contributed to site-specific Hox gene expression in the adult.

Dr. Tanaka spoke on the signaling mechanisms that regulate regeneration in vertebrates, with a focus on limb regeneration in the salamander, Axolotl. After amputation of a limb, a blastema forms under the wound tissue. Tissue-specific cells, from tissues such as skin, muscle, and cartilage, move to the blastema and it is from this zone that tissue regeneration occurs. Whether these tissue-specific cells retain their identity once they enter the blastema or revert to more primitive progenitor cells (in a process known as dedifferentiation) is the subject of much discussion in the field.

Through a series of elegant experiments involving amputation at various locations in the limb, the removal and relocation within the limb of cells of different tissue types, and the fluorescent labeling of tissue-specific cells, Dr. Tanaka argued that dedifferention of cells was not occurring in the blastema and that most, but not all, cell types can give rise only to their own specific tissue type in the regenerating limb. Dr. Tanaka also discussed the role of retinoic acid in the respecification of the blastema; for example, RA causes a blastema found near the hand to behave in a similar way to a blastema from the upper arm, which illustrated the relationship between tissue identity and position in the limb.

Highlights from the Experimental Biology 2008 Meeting

Apr 10 2008 1:03PM

Science Signaling Editors

The Science Signaling editors attended several sessions at the Experimental Biology 2008 Meeting. You will find brief reports from the sessions attended. Click the "Next Message" link to read the editor's reports. Feel free to enter your own comments or share what interested you at the meeting by choosing the "Post a Response" button.

Presidential Address: The American Association of Immunologists

Apr 30 2008 1:23PM

Nancy R. Gough

The Presidential Address was given by Olivera J. Finn from the University of Pittsburgh and was entitled "Immunologic Weapons Acquired Early in Life Win Battles with Cancer Late in Life".

Dr. Finn is an excellent and lively speaker. She started her career in Yugoslavia and at 16 addressed the annual meeting of the communist youth organization of Yugoslavia (now Serbia). She was then--and still is--self-described as revolutionary, controversial, and provocative.

Her lab developed a technique for generating specific T cells from kidney allograft needle biopsies and applied this technique in the late 80's and early 90's to assay T cells that recognize biopsies of tumors and to identify human tumor antigens. They expected to find tumor-specific antigens (mutated genes or viral oncogenic proteins), but instead they found "self" antigens, such as oncofetal proteins, lineage-specific antigens, and overexpressed transformation-related proteins that were not mutated at the peptide level.

Why were these recognized? She used MUC1, which is present on epithelial cells and lines the luminal surface, as an example. Antibodies against normal nontumor MUC1 did not recognize MUC1 from tumors, whereas MUC1 overexpressed in tumor tissue was recognized by a tumor-specific antibody. So, she argues that the tumor-specific antigens are not really "self" and should be renamed from "self/tumor antigens" to "abnormal self/tumor antigens". "Abnormal self" is antigenic; the immune response against "abnormal self" is important for tumor immunosurveillance; she proposes that "abnormal self" represents something that has been encountered prior to tumorigenesis, such as during infection or inflammatory events. Thus, she proposes that the immune system carries a "memory" of these "abnormal self" antigens, which is activated later in response to their reappearance in cancerous cells.

Going back to MUC1 as an example, MUC1 in tumor cells is hypoglycosylated and much more abundant than in normal cells. It is present in 80% of human tumors and is also present on premalignant lesions and putative tumor stem cells (Engelmann et al. 2008). In vitro, the addition of abnormal MUC1 primes CD8+ and CD4+ T cells; in animal models, there is a cellular and humoral immune response and tumor rejection, but no autoimmunity against "self" MUC1. A cancer vaccine based on abnormal MUC1 was effective in mice and has been used in clinical trials in humans in advanced cancer setting (metastatic prostate cancer) and is beginning to be tested for prophylactic use (resected adenomas).

At present, treatments based on antibodies against endogenous antigens can only be applied in the most extreme setting, because of the "self" classification, which generates concerns about autoimmunity. Thus, she argues that this concept of "abnormal self" is very important.

In collaboration with an epidemiologist, her lab found that certain life events that reduce the risk of ovarian cancer promote MUC1 immunity (Terry et al. 2007). These events all could lead to the presentation of abnormal MUC1 prior to cancer formation. There is a good correlation between cancer risk, these life events, and the appearance of antibodies for abnormal MUC1. The chance of recurrence of breast cancer was also negatively correlated with presence of antibodies against abnormal MUC1.

Cyclin B1 is another example of a protein that may be overexpressed in tumors and somehow "abnormal". Cyclin B1 from tumors is antigenic in vitro and in animal models (Kao et al. 2001), eliciting rejection of transplantable or spontaneous tumors. Anti-cyclinB1 IgG is common in the population. Healthy people, both those at high risk for cancer and those not at high risk, have antibodies to cyclin B1. There is immune memory for cyclin B1, which may arise from the induction of "abnormal" cyclin B1 during viral infection. What is unknown is if there a correlation between the abundance of the anti-cyclin B response and tumor protection.

In her model, viral infection, bacterial infection, inflammation, and tumors all have many nonoverlapping antigens, but a subset are common to all (imagine a Venn diagram of the antigens associated with each condition) and these will be the tumor antigens that can participate in immune surveillance of cancer. Identifying this subset may allow the development of "cancer vaccines". She also described how vaccination against viral infections may increase susceptibility to cancer by preventing the immune memory of abnormal self. When mice were vaccinated with a Pox vaccine, then challenged with a tumor 60 days later, the mice succumbed to the tumor rapidly. In contrast, if mice were vaccinated and then infected with the virus to allow them to respond to the viral infection, which is now not lethal, and then challenged with a tumor 60 days later, they either succumb much more slowly to the tumor or are resistant to the tumor (unpublished).

Her model of immune memory raises an interesting health issue. If immune memory for abnormal self-antigens is part of the cancer immune surveillance armamentarium, but these abnormal self-antigens are not occurring naturally and so the immune memory is not acquired naturally, then is it possible to vaccinate to prevent cancer in the future? (If you have been vaccinated against the major viruses, then you won't get a chance to develop these immune memories against abnormal self-antigens). If the overlap in the antigenic profile of infected cells and tumors can be determined, there may be an opportunity for a "universal" vaccine that would allow immune memories to be formed, which may provide a jumpstart to defending against pathogens, chronic disease, and cancer.

Selected reading

K. Engelmann, H. Shen, O. J. Finn, MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res. 68, 2419-2426 (2008).

H. Kao, J. A. Marto, T. K. Hoffmann, J. Shabanowitz, S. D. Finkelstein, T. L. Whiteside, D. F. Hunt, O. J. Finn, Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells. J. Exp. Med. 194, 1313-1323 (2001).

A. W. Silk, O. J. Finn, cancer vaccines: A promising cancer therapy against all odds. Future Oncol. 3, 299-306 (2007).

K. L. Terry, L. Titus-Ernstoff, J. R. McKolanis, W. R. Welch, O. J. Finn, D. W. Cramer, Incessant ovulation, mucin 1 immunity, and risk for ovarian cancer. Cancer Epidemiol. Biomarkers Rev. 16, 30-35 (2007).

Highlights from Toll-Like Receptor Function in the Cancer Microenvironment

May 13 2008 2:05PM

Nancy R. Gough

This session took place Sunday 6 April 2008. The function of the immune system in cancer suppression and promotion is complex with important differences between rodents and humans. There were four speakers: Michael Karin (University of California, San Diego), Xiaoxia Li (Lerner Research Institute, Cleveland Clinic), Rong-Fu Wang (Baylor College of Medicine), and Arthur M. Krieg (Coley Pharmaceutical Group). This series of talks presented intriguing insights into how the immune system both contributes to cancer and how it may be manipulated to treat or possibly prevent cancer.

IKK-Dependent NF-κB Signaling in Inflammation and Cancer- Michael Karin

Although the title of his talk suggested that he was going to discuss NF-κB signaling and cancer, instead for much of his talk Dr. Karin discussed a relationship between metastasis and Toll-like receptor (TLR) signaling. He wanted to address the question of how the immune system may be involved in early stages of metastatic growth, prior to any necrosis that may trigger an inflammatory response.

Cancer cells can release factors [for example, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α)] that activate macrophages, which in turn may release factors that trigger metastases of the cancer cells. Versican, a chondroitin-sulfate proteoglycan that binds hyaluronic acid, was secreted by a metastatic lung carcinoma cell line (LLC), enhanced metastases, activated TLR2 signaling, and stimulated the production of inflammatory cytokines from macrophages.

The importance of TLR2 signaling in cancer metastases of the LLC in mice was confirmed with TLR2-knockout mice. When injected with LLC, the TLR2-knockout mice had fewer or undetectable lung tumors at early stages compared to the wild-type mice and had a longer lifespan than did the wild-type mice.

The importance of versican was verified by silencing versican in the cancer cells and injecting these into the mice. The versican-deficient cells were less metastatic (fewer lung tumors following tail vein injection). Versican may act by binding large fragments of hyaluronic acid produced by tumor cells and then by presenting the hyaluronic acid fragments to macrophages, which stimulates TLR2 signaling. A low metastatic derivative cell line derived from the LLC line was deficient in versican production and did not stimulate macrophage IL-6 production. Thus, the model is that cancer cells with a high metastatic potential release versican, which binds to hyaluronic acid, and this versican-hyaluronic acid complex stimulates TLR2 signaling in macrophages, which respond by releasing factors, such as TNF-α, that feed back onto the cancer cells to stimulate metastasis. It may be that different types of cancers produce different proteoglycans and these have been reported in the literature to be inflammatory.

Selected References

W. W. Lin, M. Karin, A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Invest. 117, 1175-1183 (2207). [PubMed]

The Toll-Like Interleukin-1 Receptor Member SIGIRR Regulates Colonic Epithelial Homeostasis, Inflammation, and Tumorigenesis- XiaoXia Li

SIGIRR is a founding member of a new subgroup of the TLR family, notable for having a single Ig extracellular domain. Characterization of SIGIRR-knockout mice by Wald et al. suggested that SIGIRR inhibits TLR2, TLR9, ST2, IL-1R, IL-18R, and TLR4 signaling by forming complexes with these receptors. The extracellular Ig domain of SIGIRR interferes with receptor dimerization and the intracellular domain of SIGIRR inhibits recruitment of downstream signaling components.

The mRNA expression pattern suggested that SIGIRR is important for commensal microflora survival and may contribute to microbial tolerance, for example, in the intestinal epithelial layer (Xiao et al.). The SIGIRR-knockout mice exhibit altered colon crypt structure (elongated relative to wildtype), which is consistent with the increased cell proliferation and constitutive TLR7 signaling in these animals. Consistent with a role in tolerance for commensal microflora, SIGIRR-knockout mice showed an increased inflammatory response and tissue damage and ultimately death in a chemical-induced intestinal inflammation model (wild-type mice do not die in this model) and exhibited increased colon tumor development in a colitis-associated model of cancer.

Cancer formation in the colon is influenced largely by the Wnt pathway. In SIGIRR-knockout and wild-type mouse tumors, β-catenin is in the nucleus, which indicates that the Wnt pathway is activated. However, SIGIRR deficiency enhances the number of colonic polyps, thus Dr. Li proposes that SIGIRR may serve as the gatekeeper in the intestinal epithelia, allowing commensal microflora to trigger a survival response in the colonic epithelia. In the absence of SIGIRR, cells respond to commensal microflora with an inflammatory response.

However, SIGIRR is also present in two subsets of CD4+ T cells: Th17 and Th2 cells, which are both important in epithelial immunity. In Th2 cells, SIGIRR appears to negatively regulate ST2 signaling and SIGIRR-knockout animals exhibit an allergic pulmonary inflammation in a mouse model of asthma. The function of SIGIRR in Th17 cells appears to be to decrease the production of interleukin-17 (IL-17) and thus negatively regulate Th17 function. This latest work means that the interpretation that the cancer and inflammatory effects of loss of SIGIRR are due to altered signaling in the epithelial cells needs to be reassessed, because SIGIRR is also important in the mucosal T cell population. Thus, it will be interesting to determine how the loss of SIGIRR in the T-cell population is contributing to the phenotypes of the SIGIRR knockout mice.

Selected References

D. Wald, J. Qin, Z. Zhao, Y. Qian, M. Naramura, L. Tian, J. Towne, J. E. Sims, G. R. Stark, X. Li, SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4, 920-927 (2007). [PubMed]

H. Xiao, M. F. Gulen, J. Qin, J. Yao, K. Bulek, D. Kish, C. Z. Altuntas, D. Wald, C. Ma, H. Zhou, V. K. Tuohy, R. L. Fairchild, C. de la Motte, D. Cua, B. A. Vallance, X. Li,The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis. Immunity 26, 461-475 (2007).[PubMed]

TLR Signaling and Regulatory T Cells in Cancer- Rong-Fu Wang

Dr. Wang is interested in how the connections between cancer and the immune system can be applied to the development of a "cancer vaccine." He focuses on human disease because there are some important differences between mouse and human in the function of TLR signaling that impact the use of mice as a model system for manipulating the immune system for the development of human cancer therapies.

TLRs may be present on tumor cells, T cells, and antigen-presenting cells (APCs); thus, there is a complex effect of TLR signaling in cancer progression and treatment. He divides the tumor environment into three immune components: (i) the CD8+ T cells and Th1 effector T cells, (ii) the pro-inflammatory Th17 cells, and (iii) the immunosuppressive Treg cells. The environment may be further complicated because the Treg cells are a diverse population, at least partially due to the activity of the transcription factor Foxp3. A subset of Treg cells, the CD4+ Tregs, recognize some tumor-associated antigens (LAGE1, ARTC1, EBNA1).

Different types of tumors are associated with different types of T cells. Analysis of cells associated with normal prostate tissue and prostate cancer samples indicated that CD8+, Foxp3+ Tregs are only associated with prostate cancer cells. γδ T cells, which inhibit naïve and effector T cell function and dendritic cell maturation through release of soluble factors, are commonly associated with breast and prostate cancers, but not with melanoma.

The tumor environment appears to promote the Treg arm of the immune system, which inhibits the CD8+ and Th1 arm of the immune system to promote tumorigenesis. Th17 cell differentiation also occurs in response to soluble factors in the tumor environment. Dr. Wang suggests that one approach to cancer treatment would be to interfere with the immune response and alter the environment of the tumor tissue. In humans, this might be achieved by stimulating TLR8 signaling, which would decrease the immunosuppressive response by inhibiting Treg function, or by stimulating TLR9 signaling, which would directly stimulate an immune response against the tumor.

It is worth noting that in mice, TLR8 does not appear to have the same functions as in humans. In mice, dendritic cells may be more important for regulating effector T cell function in response to TLR ligands; whereas in humans, TLR8 signaling on Tregs may be the critical connection to regulating effector T cells. Reconstitution of human TLR8 in mice restores similar function as that observed in a human tumor environment; the TLR8 ligand Poly-G becomes effective in the mouse for decreasing tumor size.

Selected References

R. F. Wang, Y. Miyhara, H. Y. Wang, Toll-like receptors and immune regulation: Implication for cancer therapy. Oncogene 27, 181-189 (2008). [PubMed]

Clinical Development of TLR Agonists for Cancer Therapy- Arthur M. Krieg

Dr. Krieg is from the commercial sector and focused on TLR agonists, oligoribonucleotides (ORNs) that are ligands for TLR7 and TLR8 or oligodeoxyribonucleotides (ODNs) that are ligands for TLR9, as adjuvants for cancer vaccines or as monotherapies for cancer. ORNs stimulate B cells, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), monocytes, and natural killer (NK) cells; whereas DNA agonists, of which ODNs would be one type, only stimulate B cells and pDCs. Usually, ODNs are much more effective than ORNs for decreasing tumor load in mice.

TLR9 agonists have reached phase III clinical trials for allergy*, anti-idiotype (Id) vaccine, cancer vaccine, lung cancer therapy* (*terminated). CpG ODN are becoming the "gold standard" for vaccine adjuvants and have been used in greater than 35 human trials. These increase the antibody response and T cell response. There are many examples where addition of a CpG ODN with a tumor-specific antigenic peptide vaccine for cancer patients leads to higher Th1 responses and antibody titers than are produced with peptide alone. Studies by GlaxoSmithKline (poster only, not published) have shown decreased tumor load in response to vaccination. Seven to ten vaccinations were required, whereas many studies stop after four or five, which is an important detail for considering clinical applications.

There are several additional mechanisms by which TLR9 agonists may be useful in the treatment of cancer. Several studies with human cancers (metastatic melanoma, cutaneous T cell lymphoma, and non-Hodgkins lymphoma) suggest that TLR9 activation may be effective not only as an adjuvant, but as a stand-alone therapy (monotherapy). In mice, radiotherapy followed by a TLR9 agonist or combining chemotherapy with a TLR9 agonist improved survival. The additive effect required CD8+ T cells and did not occur in nude mice or in CD4-deficient mice. Unfortunately, in clinical trials, chemotherapy plus a TLR9 agonist did not improve survival of patients with a highly metastatic nonsmall cell lung cancer.

As the previous speaker, Dr. Wang, suggested, suppression or depletion of Tregs may also an effective cancer therapy. Some chemotherapy treatments alter the immune profile. For example, in mice treated with paclitaxel, there was a decreased abundance of Tregs in the blood but not the spleen, decreased abundance of Foxp3+ CD4+ T cells, and an increase in tumor-specific T cells.

Selected References

A. M. Krieg, Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene 27, 161-167 (2008). [PubMed]

Highlights from the Session: New Strategies for Imaging Protein Localization and Dynamics

Jul 1 2008 10:13AM

Nancy R. Gough

This session took place Monday, 7 April, and the speakers described various techniques for detecting the location of molecules within cells, the movement of molecules within cells or at the membrane, the activity of proteins, or the posttranslational modification of proteins. There were six speakers: Jin Zhang (Johns Hopkins University), Brent Martin of the Cravatt lab (Scripps Research Institute), Jay Groves (University of California, Berkeley), May C. Morris (CNRS-CRBM, Research Center of Macromolecular Biochemistry); Sophia Breusegem of theDoctor lab (University of Colorado Health Sciences Center), and Ronald Raines (University of Wisconsin, Madison)

Dynamic Visualization of Signaling Activities inLiving Cells- Jin Zhang

Dr. Zhang is interested in how specificity is achieved in a signaling network. Spatiotemporal regulation is a key to creating signaling specificity. Protein kinase A (PKA) is as an example of a spatially-restricted signaling molecule whose location is controlled by the anchoring proteins known as AKAPs. Traditional biochemical methods or immunocytochemistry do not allow spatiotemporal information to be gathered. However, biosensors (see Gaits and Hahn) were developed to allow monitoring of signals in live cells. For example, a reporter comprised of a phosphobinding domain + substrate peptide that separate two fluorophores will in the presence of a kinase and ATP lead to changes in FRET (fluorescence resonance energy transfer). AKAR1, 2, and 3 are biosensors that report on PKA activity.

Zhang and colleagues use two reporters (one for PKA called AKAR and one for cAMP based on the cAMP-regulated GTPase Epac that is called Epac-ICUE) to monitor both Epac and PKA signaling in pancreatic beta cells. One caveat to using these sensors is that they may alter the cellular signaling properties if they compete for cAMP or PKA.

Beta cells exhibit an oscillatory cAMP signal (see Borodinsky and Spitzer). Oscillatory changes were recorded with Epac-ICUE and AKAR reported that PKA activity also oscillated. If H89 was applied to inhibit PKA, then calcium oscillations ceased. ICUEPID is a three-fluorophore reporter that reports on PKA and has also an Epac domain. Experiments with ICUEPID indicated that these two regions of the reporter were not activated with the same oscillatory pattern, but that one precedes the other.

The AKAR sensors have been used in several other systems. When the AKAR sensor was used to monitor PKA activity in fibroblasts that were migrating into a wounded region of a monolayer, membrane-localized AKAR revealed that PKA was activated at the leading edge of the cell. The AKAR system has been adapted for high-throughput screening assays for drugs that alter PKA activity. There is work underway to engineer reporters for phosphatase activity and other types of posttranslational modifications, as well as new types of "tunable" reporters that use three fluorophores with transfer from the first to the second to the third.

Proteomic Profiling of Dynamic Palmitoylation- B. R. Martin

Palmitoylation occurs on cysteines, and is a dynamic and reversible modification that anchors proteins to membranes. One of the first approaches to do a global analysis of palmitoylation used acyl-biotinyl exchange chemistry [Wan et al., Nature Protocols 2, 1573 (2007)] and identified ~50 proteins in yeast. Palmitoyl acyl transferases (PATs) use C:16 or C:18 fatty acids, whereas myristoylation enzymes use shorter-chain fatty acids (see Resh). 17-ODYA, which is an alkyne that is sold by Sigma as a cytochrome p450 inhibitor, can be used to specifically label palmitoylated proteins because of its size and the presence of the reactive alkyne. When this chemical is added to cells, it becomes incorporated into acyl CoA, and then becomes covalently attached to proteins. If "click chemistry" is then used to attach rhodamine or biotin (which would be used for streptavidin purification) to the 17-ODYA-tagged proteins, then these tagged proteins can be visualized on a gel (rhodamine) or analyzed by mass spectrometry (biotin). This approach, called MudPIT, was used to identify the palmitoylated profile in Jurkat T cells and with this method, the palmitoylated proteins that are known to be part of the T cell receptor signaling complex were identified.

Imaging the Mechanics of Signal Transduction in Membranes- J. T. Groves

Dr. Groves is interested in how lipids and proteins on the surfaces of cells, especially at points of cell-cell contact or cell-substrate contact, contribute to the organization of molecular signaling structures as well as the cell's response to the contact (see Groves). The T cell immune synapse, which is the organized complex of proteins and lipids at the site of contact between a T cell and an antigen-presenting cell, is spatially organized in such a way that the center of the synapse is closely apposed membranes (15 nm), and then the membranes around it are farther apart (42 nm). The overall size is large (close to 5 micrometers).

Dr. Groves is interested in how the position of the T cell receptor (TCR) on the surface influences its signaling. He created a hybrid live-cell and supported-membrane synapse, using artificial membranes on spatially-restricted supports, and then added a cell to interact with this (see Mossman et al.). At early time points following cell contact, the TCRs start to cluster and their movement is unimpeded, but at later times the barriers on the membrane supports influence the organization of the immune synapse.

Various types of imaging can be used in conjunction with these hybrid systems: total internal reflection fluorescence (TIRF) microscopy, epifluorescence microscopy, and fluorescence cross-correlation spectroscopy (FCCS). In real time, TIRF reveals that the immune synapse forms from multiple small TCR signaling complexes and they move around the imposed boundaries readily. TIRF allows single-cluster tracking and then movement of individual clusters can be combined into an ensemble tracking record. The movement of TCR clusters toward the immune synapse is oscillatory, and once they form an immune synapse, the TCRs all show collective oscillations with a period matches that of calcium oscillations.

The actin cytoskeleton appears to flow past the TCR clusters that encounter the barriers, which suggests a frictional coupling mechanism. The adhesion molecules (ICAM and LFA) that ultimately make up the ring around the TCRs at the immune synapse are also driven toward the immune synapse. The migrating TCR clusters and adhesion molecule clusters are mutually exclusive (no TCRs migrate with ICAM and LFA) even when they are moving toward the synapse. Clustering of LFA appears to control where it sorts (Fab-labeled LFA does not form a tight donut around the TCRs at the immune synapse, mAb-labeled LFA forms a tight ring around TCRs at the immune synapse, and crosslinked mAb-labeled LFA goes to the center of the immune synapse with the TCRs.) Actin flow is the only necessary driving force and clustered molecules are "dragged" along more effectively.

Sensors of Mitosis- May C. Morris

Her group is developing sensors of mitotic kinases and phosphatases. CDK1-cyclinB1 is regulated by phosphorylation (phosphorylated forms are inactive, dephosphorylated forms are active). The players in this system also undergo dynamic subcellular localization. Some of the nongenetically-encoded (those not based on proteins) fluorescent sensors include peptide-based or nucleotide-based sensors, as well as sensors that are targeted to a particular protein interaction domain. The structural and sequence information of cyclin B, Cdc25, and CDK1 were used to develop sensors for the CDK1-cyclin B complex, activated CDK2, activated CD25 phosphatases. These sensors were introduced into cells using cell-penetrating peptides.

Microvillar Protein Trafficking and Dynamics Imaged by TIRF Microscopy in Living Cells- Sophia Y. Breusegem

The Doctor lab is interested in phosphate homeostasis in the kidney and in the trafficking of phosphate tansporters. Transporter abundance at the cell surface is regulated by luminal phosphate and hormones, and is controlled by endocytosis and exocytosis. They have developed a way to use TIRF to detect apical proteins and image the microvilli and the clefts between the microvilli. With this system, they determined that if the actin cytoskeleton is poisoned with jasplakinolide, then the two transporters are differentially affected: one has its internalization inhibited and the other has its internalization promoted in response to PTH (parathyroid hormone). They have also monitored the movement of PDZ-containing proteins, some of which interact with the transporters. Shank2E (a PDZ-containing protein) appears to move up and down the microvilli.

Latent Fluorophores for Biomolecular Imaging- R. T. Raines

Ribonuclease A (RNase A) is a secreted protein and is now being developed as a drug for blocking the flow of biochemical information from DNA to protein. For example, Onconase is an RNase found in the Northern leopard frog that is in phase III clinical trials. Onconase is selectively toxic to tumor cells, whereas mammalian RNase A is not toxic. One reason for the lack of toxicity is that mammalian cells have a horseshoe-shaped ribonuclease inhibitor protein. The interaction between RNase A and ribonuclease inhibitor occurs with femtomolar affinity (which is orders of magnitude higher than antigen-antibody interactions). Onconase does not interact with ribonuclease inhibitor at all. So, if one could engineer an RNase A that can evade ribonuclease inhibitor, this protein may kill tumor cells. Onconase treatment of nude mice with an implanted tumor resulted in tumor regression, but also caused massive weight loss, which was related to renal toxicity.

With that introduction to RNase A. Dr. Raines switched gears to discuss how he is developing probes to try to understand the mechanism of toxicity. Onconase and engineered RNase A are selectively toxic to cancer cells and are not toxic to normal cells. An enormous amount of RNase A binds to the surface of cells in culture, thus Raines' group developed probes that are not fluorescent until the protein is taken into the cell by endocytosis. They took a page from the "trimethyl-lock" mechanism of drug delivery to engineer these latent fluorophores that are unlocked by exposure to the intracellular esterases and thus are only visible after they get inside cells. These were then used to show that charge-neutralized RNase A gets into the cells poorly, whereas the normal +6 charged RNase A is readily endocytosed. This may even be a mechanism by which tumor-cell selectivity is achieved, because tumor cells are more anionic than are nontumor cells.

ASBMB-ASPET Symposium: The G-Whizards of GPCR/G-Protein Signaling

May 13 2008 11:45AM

John F. Foley

Lee E. Limbird, Meharry Medical College, gave a stimulating historical overview of how the field of G proteins and Gprotein-coupled receptors (GPCRs) has evolved. She began by discussing the importance of the work of Earl W. Sutherland in characterizing cyclicadenosine monophosphate (cAMP) as a second messenger in hormone signaling, which essentially launched the field. Also important was the work of Martin Rodbell on the effect of guanosinetriphosphate (GTP) on cAMP synthesis. This discovery was followed byexperiments that showed that the hormone receptors and cyclases (enzymesthat synthesized cAMP) in this system were separate molecules. The work of Alfred G. Gilman established that anotherprotein, now known as a G protein, conveyed the effect of GTP on thissystem. The relationships between these three components (receptor, Gprotein, and cyclase) then had to be characterized in terms of theirphysical and functional interactions. Whereas agonists stabilized theassociation between the receptor and the G protein, GTP had the reverse effect. Thomas Pfeuffer then showed that GTP, but not GDP, stabilized interactions between the G protein and the cyclase. Functional interactions between the receptor and G protein were appreciated when it was shown thatcatecholamines, such as epinephrine, stimulated GTPase activity in turkeyerythrocyte membranes. In terms of signals going back the other way,guanine nucleotides decreased receptor affinity for hormones and agonistdrugs. Competition binding studies revealed that GTP regulatedinteractions between the agonist-bound receptor and the G protein. Thiswas the beginning of the use of models to describe what was going on inthis system, perhaps the most famous of which is the ternary complexmodel, which helps describe how agonists and GTP regulate the GPCR-Gprotein-cyclase system. Of course, as studies of other GPCRs, G proteins,and effector molecules have progressed, models have continued to change to address such concepts as the pre-coupled receptor and inverseagonism, in which a ligand binds to a constitutively-active receptor andinhibits its activity. This talk nicely provided the historicalbackground for the other speakers in this session.

Robert J.Lefkowitz, Duke University Medical Center, spoke about thecharacterization of the structures and functions of GPCRs. Initial work in this field was hampered by thedifficulty in isolating β-receptors; the key was getting the right high-affinity chromatography supports to enable receptorpurification. In the 1980s, β-receptors were reconstituted invesicles, which could be fused with cells that had no β-receptors, to restore responsiveness. This work led to the cloning, in 1986, of the β2-adrenergic receptor(β2-AR). What was shocking at the time wasthat the sequence of the β2-AR showed that itcontained 7 transmembrane spans and had some homology with rhodopsin; itwas thought that 7 spans must be a characteristic of light-sensitiveproteins, such as bacteriorhodopsin. Now of course we know that this is ageneral feature of GPCRs. The cloning of theβ2-AR was followed up with the cloning of theα2-ARs. These experimentsthus established the general structure of the GPCRs. Further work by Dr.Lefkowitz and others has shown that the activation and desensitizationmechanisms of all of the various GPCRs are also very well conserved. Itwas the study of the phosphorylation and desensitization of stimulated receptors that led to the isolation of β-adrenergic receptorkinase (βARK), the founding member of the family of GPCR kinases(GRKs), which now has seven members. These researchers then found that the more purified the preparation of GRK was, the less able it was todesensitize the isolated β2-AR. So someimportant component of this system was being lost, but what? The sequenceof visual arrestin was used to help search for the missingcomponents, which turned out to be the β-arrestins 1 and 2.Reconstitution studies then showed that the β-arrestins were verypotent at desensitizing β2-ARs. There are now4 members in the arrestin family. However, recent work has changed our way of thinking about how the β-arrestins work. Not only are theyinvolved in desensitizing GPCRs (by binding to the cytoplasmic tails ofGPCRs thereby blocking their interactions with G proteins), β-arrestins also serve as adaptor proteins during clathrin-mediatedendocytosis of GPCRs. Furthermore, β-arrestins mediate signaling intheir own right, for example in the activation of mitogen-activatedprotein kinases (MAPKs). β-arrestins are also involved in suchprocesses as cell survival and chemotaxis, and act as scaffold proteins to bring together different members of the MAPK cascade.

Heidi E.Hamm, Vanderbilt University Medial Center, spoke on theinteractions between GPCRs and G proteins and on the mechanisms of Gprotein activation. Dr. Hamm started her own research in the field ofvisual transduction. At the time, there was controversy as to the identity of the second messenger involved. Was it calcium, as suggested byelectrophysiologists, or was it cGMP, as put forward by biochemists? Thepurpose of Dr. Hamm’s research was to try to use monoclonal antibodiesto block the activation of the G protein transducin by light. She foundsuch an antibody that also blocked the cGMP response. Peptide mapping ofthe epitopes recognized by the various antibodies that were studiedrevealed one peptide that blocked the receptor independently of thetransducin. These studies and work with Paul Sigler and Joseph Noel led to solving the structure of the G protein α-subunit, transducin in1993. Over the next few years, the structures of the GTPase domain and the helical loop were published and then studies continued to characterize the GTP switch region of the G protein. At about the same time, Al Gilman’sgroup worked on α-subunits and also on βγ dimers. In 1996 the crystal structure of the heterotrimeric G protein was published. Allof these studies showed that the GTPase domain and the N-terminal regionof the α-subunit have interactions with the β-subunit, but that there are no contacts between the α and γ-subunits. In 1998 it was shown that residues of the βγ-dimer that were involved ininteractions with the α-subunit were also important for interactions between the βγ-dimers and effector molecules. Dr. Hammemphasized how important the C-terminal region of the α-subunit ofthe G protein is, as it is through this region that the α-subunitbinds to the GPCR and stabilizes its high-affinity binding state. The work of Henry R.Bourne and Bruce R.Conklin on chimeric G proteins showed that the last five aminoacid residues of the C-terminus of a G protein α-subunit arecritical for GPCR-coupling specificity. Dr. Hamm then outlined more recent research that has helped to understand how activation of the GPCR triggers binding to the G protein. Future challenges include determining what theGPCR-G protein complex really looks like and to find out how the GPCRcatalyzes the release of GDP from the G protein α-subunit.

Alfred G. Gilman, University of TexasSouthwestern Medical School, started his MD PhD program in 1962 in Earl W. Sutherland’s laboratory, where he worked on adenylyl cyclase (AC); cAMPhaving been discovered in 1957. At the time, it was thought that hormonescould not work on anything other than intact cells; a "rule" thatSutherland successfully broke. In his talk, Dr. Gilman also presented anhistorical perspective on how AC and G proteins were discovered; the ACbeing one of the last pieces of the puzzle to be completed. At the end ofhis talk, Dr. Gilman looked to the future of research in this field. Itseems likely that not so many more new components are likely to bediscovered in this system, so now the challenge is to find people who arewilling to do the really hard work of fully understanding the mechanismsinvolved in GPCR-G protein interactions and signaling. Most of ourknowledge has come from relatively simple experimental conditions with asingle ligand-receptor pair, but it is unclear what really happens when multiple ligands are present and when many interactions are happening in the cellsimultaneously. Dr. Gilman emphasized that imaging will be a veryimportant technique to gain information about spatial and temporaldynamics. Another area of concern was how to explain why heterogeneouseffects that are observed in a single population of cells may not havecorrelations within individual cells. In the future, researchers will need to adopt a more quantitative approach and to look moreat the level of the single cell.

Highlights from the 67th Annual Meeting of the Society for Developmental Biology

Aug 25 2008 6:27AM

Annalisa M. VanHook

Society for Developmental Biology 67th Annual Meeting University of Pennsylvania, Philadelphia, PA 26-30 July 2008

26 July, Presidential Symposium, "Developmental Biology in the 21st Century", talks chosen by the outgoing president of the SDB, Eric Wieschaus, Princeton University

Scott Fraser, from the Biological Imaging Center at Caltech, highlighted some of the technological advances that are changing the way researchers can image developmental events. Two approaches have commonly been used to see great detail in 3-dimensional samples such as embryos. The first has been to collect images of optical or physical sections of fixed embryos at specific stages and use these static images to infer kinetic events. The second involves single embryo live-imaging, which, when carried out at high resolution, can take so long that optical sections taken on opposite sides of the embryo essentially represent different developmental stages. Fraser reported on some new technologies that allow microscopists to image an entire fly embryo, for example, in great 3-dimensional detail in 2 seconds. He also talked about using optical resonators, lens-like devices that are used to generate a standing wave of laser light energy, to detect proteins present at extremely low concentrations in extracts from a single cell.

Cynthia Kenyon (University of California, San Francisco) presented an overview of the pathways that affect life span in Caenorhabditis elegans. All of the data she discussed has been published, but she provided an overview of all the inputs -- chemosensation, glucose consumption, temperature, and signals from the reproductive system -- that affect longevity. The effects of most of these inputs on life span are mediated by some or all of the insulin signaling pathway components and the transcription factor DAF-16/FOXO.

27 July, "Evolution and Diversity of Pattern" symposium

Itai Yanai, from Craig Hunter's lab at Harvard University, talked about comparing the transcriptomes of C. elegans and C. briggsae. These twospecies look extremely similar, but they are as distantly related to one another as humans are to mice. He wanted to determine how much and in what ways genomes can diverge but still generate virtually identical phenotypes. The two species use a largely similar set of genes to make the animal. However, the transcriptional profiles of individual genes varied a great deal. About 1/3 of the genes analyzed differed in expression level or in the developmental timing of expression between the two species.

27 July "RNA Localization, Translation, and Regulation" symposium

Marja Timmermans (Cold Spring Harbor) talked about the determination of adaxial (dorsal) versus abaxial (ventral) cell fates in the leaf. She works primarily with Zea mays (corn) to identify genes through mutagenesis but uses Arabidopsis thaliana for genetic analysis. The dorsal epidermis of a leaf is optimized for maximal photosynthesis, whereas the ventral epidermis is optimized for gas exchange. The leaf is only 4 to 6 cells deep, with the micro RNA miR-390 accumulating in dorsal cells and miR-166 in ventral cells. miR-166 promotes ventral cell fate by repressing the homeodomain Zip III transcription factors that promote dorsal cell fate. miR-390 promotes dorsal cell fate by activating the trans-acting small interfering RNA tasiR-ARF, which represses miR-166. leafbladeless 1 (lbl1) mutants make leaves that have only ventral cell fates. Lbl1 is part of the RNAi machinery and is unique to developmental miRNA regulation. The fact that these miRNAs function cell-autonomously distinguishes them from most previously described RNAi pathways in plants, such as those that mediate stress responses and immunity, that function systemically. Developmentally-regulated, cell autonomously-acting siRNAs are 24nt in size and are processed by a mechanism distinct from that by which non-cell autonomous (systemic) siRNAs, which are 21nt long, are processed. Plants have separate RNAi pathways for systemic and developmental (cell-autonomous) functions. This is a unique observation of developmental regulation of an miRNA's mobility.

Eric Lecuyer (from Henry Krause's lab at the University of Toronto) presented results from an expression screen performed in collaboration with Pavel Tomancak at the Max Planck Institute in Dresden that indicate that over 70% of Drosophila embryonic transcripts are subcellularly localized. The screen gave very high-resolution spatial information and identified mRNAs associated with spindle poles, centromeres, specific regions of membranes, and cytoskeletal elements. He said that in most cases message and protein localization correlate, suggesting that localized translation may play a larger role in protein localization than does regulated transport of the protein. The major exception to this trend was nuclearly localized transcripts, which were generally not translated. The earliest zygotically-transcribed mRNAs tended to localize to chromatin, and some of these zygotically-transcribed RNAs are involved in RNAi-mediated chromatin remodeling. Localized transcripts appear to be more common than are ubiquitous transcripts. Data from the screen is available at http://fly-fish.ccbr.utoronto.ca.

27 July Plenary Session

Ken Irvine (Rutgers) gave a review of patterning and growth control by the bone morphogenetic protein (BMP) Decapentaplegic (Dpp) in the Drosophila leg imaginal disc. In cells that give rise to the distal parts of the leg (the middle of the disc), Dpp functions as a classic morphogen, but in the cells that will form the proximal parts of the leg it functions as a mitogen. In the cells where it functions as a morphogen, the shape of the gradient and not the actual concentration of Dpp determines cell fate. Planar cell polarity (PCP) is determined by the vector of the Dpp gradient, but the proliferative response of cells to Dpp is determined by the slope of the gradient. Irvine extended these features of fly leg disc patterning to other limb patterning systems, using them to explain the results of classic amputation experiments in which salamander legs amputated at different points along the proximal-distal axis eventually regenerate a compete leg when grafted together.

Dominique Bergmann (Stanford Univerisity) presented a talk about asymmetric cell divisions in the leaf epithelium and how they differ from asymmetric cell divisions in animals. In animal cells, asymmetric cytokinesis is determined by spindle orientation and results in the unequal inheritance of asymmetrically-localized RNAs and proteins from the mother cell. Plants, however, do not have the genes whose products are typically asymmetrically localized in animal cells (PAR proteins, β-catenin). Her lab identified BASL, a novel membrane-associated protein of unknown function that is asymmetrically localized in guard cell mothers (stem cells), which are irregularly shaped (like jigsaw puzzle pieces). When these stem cells divide, they do so asymmetrically and give rise to a stomatal guard cell, which terminally differentiates, and a stem cell. The daughter cell to which BASL is localized retains the stem cell fate and can continue to divide asymmetrically.

28 July, "Signaling Pathways and Networks" symposium

Alex Schier (Harvard University) talked about miRNA regulation of Nodal signaling in zebrafish. There are two Nodals in fish: Cyclops, which acts as a short-range signal, and Squint, which is a long-range signal. Cyclops and Squint are antagonized by Lefty1 and Lefty2, both of which provide long-range signals. Squint and Cyclops promote mesoderm formation at the vegetal pole of the blastula. miR430 represses Squint, Lefty1, and Lefty2, and is distributed throughout the blastula. His group wanted to know how miR430 functions in vivo given that it represses both the signal and its antagonist. miR430 has at least 1,000 predicted targets, so his group used target protector morpholino antisense oligonucleotides (morpholinos), which bind to miRNA binding sites in target mRNAs to prevent miRNA binding, to block miR430 repression of only these three targets (Squint, Lefty1, and Lefty2). Blocking the miR430-induced repression of either Squint or both of the Leftys resulted in more endoderm. Blocking its repression of all three resulted in less endoderm. Blocking repression of all three in an embryo in which Cyclops function was removed with morpholinos, however, caused more endoderm to be produced. When miR430 action on Squint, Lefty1, and Lefty2 is blocked at the same time Cyclops is overexpressed, then less endoderm is produced. The conclusion is that miR430 modulates the balance of agonist and antagonist. But, given that miR430 is ubiquitously expressed, how is this useful? The answer appears to lie in the fact that Cyclops acts only over short ranges and Squint acts over a long range. If you compare the morphogen activities of fish Squint with fly Dpp, then it is apparent that Squint patterns s larger field of cell, and it does so in a shorter amount of time. Therefore Squint must not diffuse like Dpp. They measured the diffusion kinetics of a biologically active Squint-GFP fusion protein and found that it diffuses much faster than does Dpp. Dpp binds to proteoglycans, which modulates its rate of diffusion across the disc, but fish embryos have no proteoglycans to impede Squint diffusion.

Sreelaja Nair, from Tom Schilling's lab at the University of California, Irvine, talked about the role of the chemokine Cxcl125 and its receptor Cxcr4a in during zebrafish gastrulation. If either is knocked down by morpholinos, either the liver, or the pancreas, or both, are duplicated, but the heart is normal. Both liver and pancreas are endodermal derivatives, whereas the heart comes from mesoderm. Cxcl125-Cxcr4a signaling is specifically required for migration of endoderm during gastrulation, but probably not as a chemokine tether (a process in which a chemokine and its receptor are expressed in different tissues to keep the tissues together during migration) because mesoderm migration is unaffected. Instead, Cxcl125 signaling through Cxcr4a induces expression of integrin receptors in the endoderm. In the absence of chemokine signaling, integrin-mediated cell adhesion is compromised, and the endodermal precursors get split into two groups, hence the duplicated organs. Apparently duplication of endoderm is not often observed independent of mesoderm defects, such as a split heart field.

Arthur Lander (Univeristy of California, Irvine) gave a talk about modeling signaling pathways and how developmental signaling pathways work from an engineering perspective. He focused on the difference between how information is conveyed through a pathway (the pathway's mechanism) and how that flow of information is regulated (the pathway's engineering). He used the generation of olfactory neurons from the olfactory epithelium as a case study for explaining the logic of networks. He started by discussing the mechanism of the growth factor pathway that regulates stem cell proliferation and went through the pathway step-by-step to explain why more and more feedback and cross-talk interactions have to be added to make the system both robust and fast. Lander talked about open versus closed systems, the latter of which do not exist in biology, although "mostly closed" systems do. The olfactory epithelium is a "mostly closed" system since secreted factors eventually leak through the basement membrane. He explained how feedback loops can create a gradient of activity even in the absence of a localized source of morphogen and how computational models can be used to identify the performance objectives that a biological system must meet to be useful.

Philippe Soriano, from the Fred Hutchinson Cancer Research Center, summarized his lab's work on reverse signaling by EphrinB1 (EphB1). B-type Ephrins can mediate both forward signaling, in which they act as ligands to induce signaling in neighboring cells, and reverse signaling, in which they act as receptors to transduce signaling into the cell in which they are expressed. Ephrin signaling has been most commonly studied in axon guidance, where it mediates both attractive and repulsive behaviors, but it is also important in establishing compartment boundaries. In boundary formation, ephrin signaling promotes repulsion between different cell populations. ephB1 knockout mice have polydactyly, fused limbs, cranial abnormalities, cleft palate, and notochord defects. It was unclear whether this was due to forward or reverse signaling, so Soriano's group made a knockout in which only the reverse signaling function was eliminated. The reverse signaling-defective EphB1 receptors still cluster correctly and mediate forward signaling normally, and everything is pretty much normal in the mutants except in the corpus collosum, where neurons form and extend axons but are misrouted. Therefore the defects observed in the ephB1 knockout are due to interruption of forward signaling. ephB is on the X chromosome, and ephB1+/- females are more severely affected than are homozygous females. Because females are mosaics, some cells in a heterozygote have ephB and some do not. The two different types of cells sort themselves apart from one another, resulting in ectopic boundary formation which causes the cranial defects. This implies that boundary formation is a product of forward Ephrin signaling. Gap junction communication does not occur between wild-type and ephB1-deficient cells, and overexpression of Connexin 43 can partially rescue the phenotype, so they hypothesize that the cells sort apart because of impaired gap junction communication.

Scott Weatherbee (Yale Univeristy) gave a talk about Kerouac and Mks1. kerouac mutants have malformed cilia, an ectopic digit on the anterior side of each autopod, and left-right patterning defects in the lung (where they have two left lobes) and in the heart (where the direction of looping is randomized). The gene encoding Meckel Syndrome 1 is mutated in the kerouac mutant, and Mks1 localizes to basal bodies, which are the structural bases of cilia. Knockdown of mks1 in cultured cells caused the production of fewer cilia than normal. These observations point to a role for Kerouac/Mks1 in Hedgehog (Hh) signaling. Indeed, Sonic hedgehog (Shh) signaling is reduced in kerouac mutants, but Smoothened (Smo) localization in cilia is normal, indicating that Kerouac/Mks1 functions downstream of Smo in Hh signaling.

28 July, "Morphogenesis" symposium

John Wallingford (University of Texas at Austin) presented an overview of his lab's work on ciliogenesis and PCP using the mucociliary epithelium of Xenopus laevis embryos as their model. The mucosal epithelium is composed of two interdispersed cell types: Goblet cells, which secrete mucus, and multiciliated cells that beat their cilia in the same direction to keep the mucus moving in one direction. This is similar to other mucosal epithelia, such as airway epithelia, but amphibian skin is easier to study. Dishevelled (Dvl) is one of the core proteins of PCP (along with Strabismus, Flamingo, and Prickle). In dvl morphants, the cilia appear short because a considerable proportion of their length is retained inside the cell. The basal bodies of the cilia are not located under the apical membrane where they should be, but are instead deeper inside the cytoplasm. The basal body forms the base of the cilium; it is the structure from which the actin skeleton of the cilium extends. Normally basal bodies are trafficked to the apical plasma membrane before the cilia are built from them, but not in dvl morphants. Dvl is required to dock basal bodies at the apical membrane. Dvl does not localize to the basal body--it is next to the basal body, on the opposite side from the direction of fluid flow. This is analogous to its asymmetric distribution in PCP models such as the fly wing. That the PCP pathway is required to coordinate the movement of the cilia is not surprising, but the connection between PCP and apical-basal vesicle trafficking that is just starting to emerge from his and other labs is interesting.

Andrew Ewald (from Zena Werb's lab at the University of California, San Francisco) talked about mammary branching morphogenesis in the mouse. Migrating mammary epithelial tip cells do not send out filipodia or any other actin-based protrusions. Instead, they migrate as a sort of tumbling mass of loosely associated cells without distinct leaders or followers. The extending tip of the tube is a transient multi-layered epithelium that exhibits polarization at the tissue, but not cellular, level. As it turns out, metastatic mammary tumors look just like tip cells in terms of their organization, dynamics, and migration behavior. They differ, however, in the growth of their branches. Ewald has made movies of live human mammary tumor explants in 3D culture, where they look like migrating tip cells. He noted that tumor explants behave very different in 3D culture compared to 2D culture. In 3D culture, tumor explants exhibit collective migration, but in 2D culture the tumor dissociates and the cells migrate individually, sending out actin-based protrusions like cultured tumor cell lines.

29 July, "Organ Systems in Vertebrate Development" symposium

Matt Harris, a postdoc from Christiane Nusslein-Volhard's lab, talked about formation of the dermal skeleton in zebrafish and variations in its morphology across different species. Dermal bone is formed from mesenchyme condensations without a cartilage intermediary. For example, skulls and turtle shells are dermal bone. In bony fish, the skull, scales, and fin rays are dermal bone. Harris performed a genetic screen for zebrafish mutants that have dermal bone phenotypes that phenocopy naturally occurring variations in other species. Of 900 genomes screened so far, he has identified 9 adult-viable mutants with dermal bone phenotypes. He got finless mutants, which lack fins, scales, and teeth, all of which are epidermal keratinocyte derivatives. All of the finless mutants identified so far have mutations in ectodysplasin (eda). Human mutations in eda also cause epidermal structure phenotypes. EDAR, the eda receptor, is found in the signaling centers of epidermal placodes, tissues that give rise to scales and other dermal bone. They identified a mutation called kronos that causes a larval-type skull to be retained in the adult. Fibroblast growth factor (FGF) signaling is also important to skeletal variation. He mentioned an example in which he was able to identify a mutation in an FGF receptor that confers the phenotype for which mirror carp are named, a patch of scaleless epidermis near the cheek. The lab is now trying to identify genes that are important in scale variation by mapping traits in interspecies hybrids from related species from lakes in Croatia, an approach similar to that used by other labs to study skeletal variation in sticklebacks.

Molly Ahrens (Andy Dudley's lab, Northwestern University) talked about phosphatidylinositol glycan class A (PIG-A) in chondrogenesis, specifically in the formation of endochondral bone, which proceeds through a cartilege intermediate and includes the long bones of the vertebrate skeleton. PIG-A functions in biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors, and mutants have bones that are patterned and mineralized correctly but do not elongate. Looking closely at bone morphology in these mutants, she noted that the chondrocytes in the growth plates of these mutant bones were not organized into columns as they should be. In the growth plate, chondrocytes divide perpendicular to the long axis of the bone, then the daughter cells change shape and intercalate to form a column. In PIG-A mutants, chondrocytes mature normally and most (75%) divide in the correct orientation, but all fail to intercalate. These mutants also have PCP-type defects in the alignment of hair cells in the inner ear. There is an emerging connection between the polarity of bone growth plates and PCP, but the link is unclear.

29 July, "Mitosis and Cell Polarity" symposium

Claire Tomlin (Electrical Engineering and Computer Science Department, University of California, Berkeley) has been collaborating with Jeff Axelrod at Stanford University to build computational models of PCP signaling in the fly wing disc. They have published a number of papers on their model, which has provided an explanation for some perplexing results with wing cell clones that lack the cadherin Fat. Hair polarity (and therefore PCP signaling) is propagated across some, but not all, fat clones normally. Whether or not PCP is normal across these clones does not correlate with the size of the clone or any other readily apparent characteristic, so it was unclear why some clones had normal PCP and others did not. The computational model suggested that even slight alterations in cell geometry could have a large effect on PCP propagation. When the model was trained to measure cell geometry and correlate this with observed phenotypes, the hair polarity patterns in mutant clones matched the model's predictions. The model even accurately predicted the aberrant hair polarity patterns within the clones across which PCP was not propagated normally. The model has made predictions that have been experimentallyverified and contributed significantly to what is known about PCP.

Yunwei Li, from Andy Dudley's lab at Northwestern University, talked about PCP signaling components in chondrogenesis. Frizzled (Fz) and the PDZ domain of Dvl are required for oriented cell divisions in the growth plate in which cells divide perpendicularly to the long axis of the bone. In this context, Fz and Dvl mediate PCP signaling rather than signaling through a canonical Wnt pathway. PCP mutants have short bones that are patterned normally but the intercalation of cells into columns is defective.

Darren Gilmour (EMBL, Heidelberg) gave a talk on migration of the lateral line primordia in zebrafish. Lateral line precursors move along like a giant slug, leaving lateral line organs behind as they travel from the anterior end of the embryo to the posterior. Cells in the leading edge half of the group jostle and rearrange and move around within the cluster while cells at the back end are progressively organized into rosettes that are left behind as they drop off the migrating cluster. The cells in the anterior part of the group are loosely organized, similar to the mammary tube tip cells Andy Ewald talked about. There are no leaders and no followers in the group, so you can kill leading edge cells and other cells can adopt the forward positions. Even if he kills all the cells in the front half of the group and leaves only the partially patterned rosettes at the posterior, the rosettes break up, and cells from the rosettes reform a leading edge for the migrating mass. If he starts ablating cells from the organ-forming posterior end of the primordium, he reaches a point where the primordium gives up and stops migrating. FGF signaling directs the nucleation of the organs into rosettes, and SDF-CXCR4 signaling drives the movement of the group. If he provides a point source of SDF, some cells will break away from the group and head toward that second source. He has wonderful images of collective migration behavior in normal and experimentally manipulated primordia.

29 July Plenary Session

Eileen Shore (University of Pennsylvania) talked about her work on fibrodysplasia ossificans, a disease in which soft tissues are gradually ossified. This is due to de novo bone synthesis and not overgrowth of existing bone. People with the disease are born with a normal skeleton, and extra ossifications start appearing around the age of 5. The extra bone tissues are endochondral, not dermal, bone, and their growth seem to be initiated when a localized inflammatory response induces tissue damage and repair that then leads to chondrogenesis. Some cases are spontaneous, but there is a rare autosomal-dominant form associated with a mutation in the gene encoding the activin type I receptor. The mutation results in an amino acid substitution in the part of the protein that is both phosphorylated by the type II activin receptor (to active signaling) and mediates binding to FKBP12 (to suppresses signaling). Therefore this mutation could affect BMP signaling both negatively and positively. In cell culture, this mutation causes chondrogenesis but not as much as does constitutively activation of BMP signaling. Shore's lab has made chimeric mice that carry cells with this targeted activin type I receptor allele, and they essentially phenocopy the human disease.

Highlights from a TGF-β Workshop

Nov 18 2008 7:27AM

Nancy R. Gough

TGF-β: Discovery and Promise-- A Symposium Honoring the Memory of Anita B. Roberts

This one-day symposium was held at the Natcher Conference Center at the NIH campus in Bethesda, Maryland, on 19 September 2008. With over 400 registrants and 13 speakers, as well as more than 35 posters, this short meeting was packed with interesting science. The symposium was organized by Kathleen Flanders (National Cancer Institute), Michael Sporn (Dartmouth Medical School) and Lopa Mishra (Georgetown University). The attendees and speakers came from all over the world, including Japan (Kohei Miyazono), Germany (Klaus Unsicker), and the Netherlands (Peter ten Dijke). Many, if not all, of the invited speakers had worked, collaborated, or wrote articles with Anita Roberts (1942-2006) and most included anecdotes and praise for the colleague, scientist, friend, and mentor that she was. Several talks are highlighted here.

Dr. Roberts isolated transforming growth factor-β (TGF-β) and in the 20 years that she worked in the field, she published more than 344 papers, each averaging more than 100 citations. She developed gastric cancer in 2004 and documented her feelings, treatment, and struggles in a blog. Even for those at the symposium who had not had the opportunity to meet or work with Dr. Roberts, the stories and comments from the speakers allowed everyone to get a sense of the person she was. Sporn gave an especially poignant talk that was a historical perspective on the TGF-β field highlighting the contributions made by Dr. Roberts, especially the early ones in the 1970s and 1980s that opened this area of investigation. He also included a personal reflection on the present state of cancer research and proposed that we are failing to cure this disease, citing data that suggested that cancer rates are on the rise rather than decline in the U.S. He blames the commercialization of cancer research, with the issues surrounding intellectual property and the funding problems whereby striking out in new directions is nearly impossible because of the current structure of the grant and peer review processes.

Several speakers emphasized the context-dependent nature of the cellular response to TGF-β and how it can serve as both growth inhibitor and a growth promoter, which means it can serve to both promote and inhibit cancer. Harold L. Moses (Vanderbilt University) described his work on understanding why loss of TGF-β function has different effects on carcinogenesis depending on the type cells in which its activity is lost. For example, knocking out the receptor in epithelial cells does not predispose those cells to becoming cancerous, but knocking out TGF-β signaling in fibroblasts or T lymphocytes does. His lab is also using loss-of-function and knockout models to explore the role of TGF-β in metastases, and is working on developing TGF-β gene expression signatures using microarray data to try to profile cancers to predict the potential for cancer relapse. A final theme of his talk and several others was that TGF-β signaling influences inflammatory signaling, which contributes to cancer progression and metastasis, thus emphasizing the importance of the immune system in cancer.

Joan Massagué (Memorial Sloan-Kettering Cancer Center and Howard Hughes Medical Institute, New York) is also working on identifying TGF-β gene expression signatures and then applying these signatures to understand target site metastasis. For example, TGF-β signaling appears to contribute to the metastasis of breast cancer to the lung and the adaptation of cells for survival in and colonization of this secondary site.

Kohei Miyazono (University of Tokyo) and John Letterio (Case Western Reserve University) also highlighted TGF-β in cancer. Letterio focused on the role that TGF-β has in the immune system and how this affects cancer. Miyazono described his work on the role of TGF-β in scirrhous gastric cancer, which is a diffuse cancer that is associated with fibrosis. TGF-β is produced by the cancer cells, the fibroblasts, and the macrophages that are near the cancer. His lab found that mice injected with OCUM-2MLN cells (a scirrhous cancer cell line) that had been transfected with of a dominant-negative form of the TGF-β receptor type II subunit led to increased tumor size, but the tumors were not associated with fibrosis. Tumor growth was inhibited by thrombospondin-1 (TSP-1), which can trigger the release of TGF-β from the latent complex.

Another theme of the presentations was the role of TGF-β in wound healing and fibrosis. As Peter ten Dijke (The Netherlands Cancer Institute) described, how in addition to serving as a growth regulator, TGF-β also mediates contradictory effects on angiogenesis with ALK5-mediated signaling (inhibitory) and ALK1-mediated signaling (stimulatory) producing opposite effects. ALKs are TGF-β receptor subunits. Ten Dijke highlighted the interactions between cadherins and TGF-β signaling and showed that VE-cadherin appears to inhibit vascular endothelial growth factor (VEGF) signaling and promote TGF-β signaling to enhance endothelial cell proliferation and migration, which improves wound healing (Rudini et al. EMBO 2008). Endoglin, also known as CD105, also interacts with TGF-β and appears to facilitate ALK1 signaling and inhibit ALK5 signaling, which allows TGF-β to promote angiogenesis.

The activity of TGF-β is also regulated by release from a latent complex, which was the topic of talk by Joanne E. Murphy-Ullrich (University of Alabama at Birmingham). She described how TSP-1 displaces active TGF-β from the latent complex and then highlighted that this may be an important mechanism in fibrotic remodeling that occurs in complications of diabetes. Injection of TSP-1-interfering peptides improved heart function in a model of cardiac fibrosis and also improved kidney function in a model of diabetic nephropathy. In each case, the abundance of phosphorylated Smad2 was decreased indicating that TGF-β signaling was disrupted.

Kathleen Flanders discussed her lab's work with Smad3 knockout mice, which show less scarring in response to skin irradiation and faster wound healing in a skin wound assay, less kidney inflammation and fibrosis in a mouse model of rehnal fibrosis, and less secondary cataract formation in a mouse eye lens injury model. Cell culture experiments indicate that Smad3 knockout cells are deficient in chemotaxis toward TGF-β, do not produce TGF-β, and do not undergo the epithelial to mesenchymal transition.

Several talks focused on the details of TGF-β signal transduction. Fang Liu (Cancer Institute of New Jersey) described her work on phosphorylation of Smad3 by kinases other than the TGF-β receptor, such as ERKs, which are mitogen-activated protein kinases (MAPKs), or other kinases. Phosphorylation of Smad3 in the linker region appears to inhibit the transcriptional activity of Smad3 and thus multiple pathways may converge on Smad3 to influence TGF-β signaling. Ryk Derynk (University of California, San Francisco) described his work on sumoylation of the TGF-β receptor (see Editors' Choice by Gough) and experiments suggesting that the receptor may also be a substrate of the protease ADAM17, the activation of which may be controlled by MAPK signaling and thus serve as another point at which MAPK pathways converge on TGF-β signaling. Caroline Hill (Cancer Research, UK) described her mathematical model of Smad signaling, which produced information about the rate-limiting steps in Smad signaling and predicted the existence of a nuclear phosphatase (see Perspective by Shankaran and Wiley).

Klaus Unsicker (University of Heidelberg) described his work on the TGF-β superfamily member GDF-15, which appears to be involved in motoneuron survival and myelination. Mishra described her lab's work on mice that are heterozygous of loss-of-function mutations in the genes that encode the adaptor Elf and the E3 ubiquitin ligase PRAJA, which have been implicated in TGF-β signaling. These mice appear to phenocopy Beckwith Wiedemann Syndrome.

The meeting was successful in presenting interesting new facets of TGF-β signaling, as well as providing a historical perspective on how this interesting molecule and its family members have contributed to the concept of context-dependent signaling and advanced our understanding of multiple diseases of clinical importance.

Barotransmitters: The Neglected Class of Transmitters

Mar 4 2009 1:29PM

Sadollah Mohammadi

Pressure is defined as a perpendicular load or force on a surface, and the bar is a widely used metric unit of measure for pressure. It is a fundamental parameter in thermodynamics. To maintain a homeostatic internal environment, pressure control must be a highly regulated process in complex systems such as the human body. Fluid pressures in different parts of the body, especially in cardiovascular, cerebral and ocular systems, are carefully regulated, indicating the crucial roles of pressure receptors in maintaining whole-body homeostasis (1).

The pressure regulation process involves the release of specific transmitter molecules following the activation of specific signaling pathways. The production of natriuretic peptides, nitric oxide, glutamate, substance P, and acetylcholine have been reported to change in response to pressure fluctuation (2, 3, 4, 5). These transmitters comprise heterogeneous molecules in terms of their structure. However, all of them are controlled by the level of pressure and mediate transference of the signals of pressure changes to target cells or organs.

The classification of transmitters is based on a number of criteria (6). One of the simplest is the type of stimulus that triggers the release of the transmitter. To emphasize the close relationship between transmitters released in response to pressure and pressure receptors, we propose the term "barotransmitter" for this class of molecules to denotes the similarity in transmitters that are endogenously released in response to a change in pressure. The term "barotransmitter" as a descriptive category for transmitters for pressure receptors could be used describe and integrate the ways that pressure affects biological functions.

References

  1. M. J. Lab, Mechanosensitive-Mediated Interaction, Integration, and CardiacControl. Ann. NY Acad. Sci. ,1080, 282-300 (2006).
  2. S. Moncada, E. A. Higgs,,The discovery of nitric oxide and its role invascular biology. Br. J. Pharmacol.147, S193-S201 (2006).
  3. N. Singewald, A. Philippu, Involvement of biogenic amines and amino acidsin the central regulation of cardiovascular homeostasis. Trends Pharmacol.Sci. 17, 356-363 (1996).
  4. J. R. Gontijo, L. A. Smith, U. C. Kopp, CGRP activates renal pelvic substance Preceptors by retarding substance P metabolism. Hypertension 33493- 498 (1999).
  5. H. Schworer, K. Racke, H. Kilbinger, Characterization of the muscarinereceptors involved in the modulation of serotonin release from thevascularly perfused small intestine of guinea pig. Naunyn SchmiedebergsArch. Pharmacol. 339, 263-267 (1989).
  6. D. Hoyer, P. P. Humphrey, Nomenclature and classification of transmitterreceptors: An integrated approach. J. Recept. Signal Transduct. Res. 17, 551-568 (1997).

This response contributed by Sadollah Mohammadi and Masoud Darabi

Katsuhiko Mikoshiba Honored by Karolinska Institutet

May 24 2011 7:00AM

Science Signaling Editors

The editors congratulate Science SignalingEditorial Board and Board ofReviewing Editors member Katsuhiko Mikoshiba on receiving an honorary doctorate fromtheKarolinska Institutet in Stockholm, Sweden. These annual awards recognizepeople who have made outstanding contributions to the university and wereawarded on 13 May 2011 at Stockholm City Hall.