Chester E. Chamberlain

Spatial and Temporal Analysis of Rac Activation during Live Neutrophil Chemotaxis

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.

Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning

Sonic hedgehog (Shh) ligand secreted by the notochord induces distinct ventral cell identities in the adjacent neural tube by a concentration-dependent mechanism. To study this process, we genetically engineered mice that produce bioactive, fluorescently labeled Shh from the endogenous locus. We show that Shh ligand concentrates in close association with the apically positioned basal body of neural target cells, forming a dynamic, punctate gradient in the ventral neural tube. Both ligand lipidation and target field response influence the gradient profile, but not the ability of Shh to concentrate around the basal body. Further, subcellular analysis suggests that Shh from the notochord might traffic into the neural target field by means of an apical-to-basal-oriented microtubule scaffold. This study, in which we directly observe, measure, localize and modify notochord-derived Shh ligand in the context of neural patterning, provides several new insights into mechanisms of Shh morphogen action.

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells.

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a proteins dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site‐specific protein labeling.

Gαi3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins

Gαi3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Gαi3 and Ca2+ and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Gαi3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Gαi3 and calnuc in COS-7 cells expressing Gαi3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Gαi3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when Gαi3 is coexpressed with cytosolic calnuc(ΔN2–25)-CFP lacking its signal sequence. No FRET signal is detected when Gαi3(ΔC12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Gαi3-YFP and calnuc(ΔEF-1,2)-CFP, which is unable to bind Gαi3, are coexpressed. Gαi3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to Gαi3 on Golgi membranes in living cells and that Gαi3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca2+ sensor protein capable of binding to and potentially regulating interactions of Gαi3 on Golgi membranes.

Menin determines K-RAS proliferative outputs in endocrine cells.

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.

Degradation of C/EBPβ in cultured myotubes is calpain‐dependent

Members of the C/EBP transcription factor family regulate cell differentiation and multiple other cellular functions. The cellular levels of C/EBPα, γ, δ, ε, and Gadd153/CHOP are regulated in part by proteasome‐dependent degradation. In contrast, mechanisms regulating the degradation of C/EBPβ are poorly understood. We tested the hypothesis that the degradation of C/EBPβ is calpain‐dependent. Studies were performed in cultured L6 myotubes (a rat skeletal muscle cell line) because we have found previously that C/EBPβ may be involved in the regulation of muscle proteolysis. Treatment of cultured L6 myotubes with the calpain inhibitors calpeptin and Calpain Inhibitor I and II resulted in increased C/EBPβ concentrations but did not influence cellular levels of the other C/EBP transcription factor family members. Transfection of myoblasts with a plasmid expressing the endogenous calpain inhibitor calpastatin resulted in increased cellular levels of C/EBPβ whereas the opposite result was observed in myoblasts overexpressing µ‐ or m‐calpain. Co‐immunoprecipitation provided evidence for protein–protein interaction between C/EBPβ and µ‐ and m‐calpain suggesting that C/EBPβ may be a calpain substrate. This notion was supported by experiments in which immunoprecipitated C/EBPβ was incubated with purified µ‐calpain in a cell‐free system. The increase in C/EBPβ levels caused by inhibition of calpain activity was accompanied by increased C/EBPβ DNA‐binding and gene activation. The present results suggest that C/EBPβ is degraded by a calpain‐dependent mechanism in skeletal muscle cells and that the role of calpains is specific for C/EBPβ among different members of the C/EBP transcription factor family. J. Cell. Physiol. 208: 386–398, 2006.

Follicle dynamics and global organization in the intact mouse ovary.

Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.

Abstract A185: Menin functions as a gatekeeper of the proliferative MAPK arm of K-Ras signaling.

Activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas, but have never been found in pancreatic endocrine tumors, which more commonly inactivate tumor suppressors such as MEN1, the gene mutated in the autosomal dominant cancer syndrome Multiple Endocrine Neoplasia type 1, showing that outcomes of K-Ras signaling depend on cellular context. We have shown that K-Ras does not promote but instead strongly suppresses endocrine cell growth and that this paradoxical activity derives from the Men1 gene product Menin. We have proposed a model in which K-Ras activates both the pro-proliferative MAPK pathway, and the anti-proliferative RASSF1 pathway. In tissues susceptible to MEN1 gene mutation, Menin normally prevents the MAPK effector pathway from driving proliferation while leaving the inhibitory RASSF1 effector pathway intact. In this model, loss of Menin causes proliferation due to removal of the block in MAPK-driven proliferation downstream of K-Ras, while loss of K-Ras signaling increases proliferation by decreasing the unopposed RASSF1 activity in susceptible cells. To test this model, we assessed the effect of various inhibitors of the K-Ras pathway on beta cell proliferation in cultured mouse islets. We found that treatment with a combination of farnesyl- and geranyl-geranyl-transferase inhibitors, which block K-Ras signaling, stimulated beta cell proliferation; but blocking MAPK signaling with an inhibitor of Mek1/2 had no effect on proliferation rate. However, in beta-cells with reduced Men1 gene dosage, both K-Ras and Mek1/2 inhibition reduced the increased basal replication rate, consistent with our model that Menin specifically blocks pro-proliferative outputs of the MAPK arm of K-Ras signaling. Remarkably, complete loss of Men1 created a synthetic lethal interaction with Mek1/2 inhibition. Our study suggests that inhibitors of the Raf/MEK/ERK pathway offer a rational therapy for tumors with germline or somatic MEN1 mutations. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A185. Citation Format: Chester Chamberlain, Michael German. Menin functions as a gatekeeper of the proliferative MAPK arm of K-Ras signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A185.

Abstract A11: KRAS inhibition of pancreatic beta-cell growth occurs through a MENINRASSF1 regulatory network.

KRAS is a regulated GDP/GTP molecular switch that controls several signaling networks. Although activating missense mutations in the KRAS gene are a major cause of accelerated growth in many epithelial cancers, KRAS signaling can elicit diverse, cell-type specific responses. We discovered that mice heterozygous for a Kras null allele (KrasLSL-G12D) have an expanded pancreatic beta-cell population at birth and improved glucose tolerance as adults. We showed that the additional beta-cells come from two sources: increased numbers of Neurogenin3expressing endocrine progenitors during embryogenesis and increased beta-cell proliferation during the perinatal period. Expression of KRASG12D directly suppressed both sources of new beta-cells (neogenesis from non-beta-cell progenitors, and proliferation of pre-existing beta-cells) and activated the opposing MAPK and RASSF1 downstream effector pathways in beta-cells. The block of beta-cell growth caused by KRAS activity depends on the expression of the endocrine tumor suppressor MENIN. In support of this conclusion, endocrine cells in the parathyroid and pituitary glands, two tissues known to develop tumors in patients carrying heterozygous mutations in MENIN (Multiple Endocrine Neoplasia Type 1, MEN1), similarly showed increased proliferation in KRAS heterozygous null mutants. Our data suggest a model in which KRAS activates both the pro-proliferative MAPK pathway, and the antiproliferative RASSF1 pathway. In the tissues susceptible to the MENIN gene mutation, MENIN prevents the MAPK effector pathway from driving beta-cell proliferation while leaving the inhibitory RASSF1 effector pathway intact. In this model, loss of MENIN causes proliferation due to removal of the block in the proliferative drive mediated by MAPK signaling, while a reduction in KRAS signaling increases proliferation by decreasing the unopposed RASSF1 activity in susceptible cells. Citation Format: Chester E. Chamberlain, Michael S. German. KRAS inhibition of pancreatic beta-cell growth occurs through a MENINRASSF1 regulatory network. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A11.