The Meyer lab seeks to understand how human cells sense hormones, growth factors and stress and how they integrate and transduce these signals to make decisions to polarize, move or divide. We investigate these cellular regulatory systems by identifying the key signaling components and measuring when and where signaling occurs as we watch cells decide to move forward or enter the cell cycle. We have been intrigued by the near universal importance of locally acting Ca2+ and phosphoinositide lipid second messenger signals, Rho and Ras family small GTPases and protein kinases in controlling these decision processes. Our projects are focused on understanding the general principles of how signal transduction systems work which often requires the development of new experimental and analysis tools involving fluorescent microscopy, small molecule and light perturbations, systematic siRNA screens, bioinformatics, genomics and quantitative modeling of signaling pathways.
July 2016: Seth's paper is out in EMBO
Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.
June 2016: Steve's paper is out in Cell
Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.
February 2016: Amy's paper is out in Elife
Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation.
Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon.
February 2016: Hee Won and Seans's paper is out in Nature Cell Biology
Neutrophils and other amoeboid cells chemotax by steering their front ends towards chemoattractant. Although Ras, Rac, Cdc42 and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, pre-existing local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement on uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, whereas Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.
January 2016: A new postdoc joins the lab
Marielle Koeberlin joined the Meyer lab as a postdoctoral fellow. Welcome aboard Marielle
September 2015: Xuecai's paper is out in Elife
Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.
Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.
August 2015: Gautam's review is out in Cell Systems
Information about functional connections between genes can be derived from patterns of coupled loss of their homologs across multiple species. This comparative approach, termed phylogenetic profiling, has been successfully used to infer genetic interactions in bacteria and eukaryotes. Rapid progress in sequencing eukaryotic species has enabled the recent phylogenetic profiling of the human genome, resulting in systematic functional predictions for uncharacterized human genes. Importantly, groups of co-evolving genes reveal widespread modularity in the underlying genetic network, facilitating experimental analyses in human cells as well as comparative studies of conserved functional modules across species. This strategy is particularly successful in identifying novel metabolic proteins and components of multi-protein complexes. The targeted sequencing of additional key eukaryotes and the incorporation of improved methods to generate and compare phylogenetic profiles will further boost the predictive power and utility of this evolutionary approach to the functional analysis of gene interaction networks.
June 2015: Two new grad students join the lab
Anjali Bisaria and Yilin Fan join the lab as grad students. Welcome aboard Anjali and Yilin
May 2015: New MD/PhD student joins the lab
Leighton Daigh joins the lab as a grad student. Welcome aboard Leighton
April 2015: Sean's paper is out in Molecular Systems Biology
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96‐well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G‐protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low‐ and high‐resolution fluorescence microscopy.
February 2015: Gautam's paper is out in Cell Reports
Systematic Discovery of Human Gene Function and Principles of Modular Organization through Phylogenetic Profiling.
Functional links between genes can be predicted using phylogenetic profiling, by correlating the appearance and loss of homologs in subsets of species. However, effective genome-wide phylogenetic profiling has been hindered by the large fraction of human genes related to each other through historical duplication events. Here, we overcame this challenge by automatically profiling over 30,000 groups of homologous human genes (orthogroups) representing the entire protein-coding genome across 177 eukaryotic species (hOP profiles). By generating a full pairwise orthogroup phylogenetic co-occurrence matrix, we derive unbiased genome-wide predictions of functional modules (hOP modules). Our approach predicts functions for hundreds of poorly characterized genes. The results suggest evolutionary constraints that lead components of protein complexes and metabolic pathways to co-evolve while genes in signaling and transcriptional networks do not. As a proof of principle, we validated two subsets of candidates experimentally for their predicted link to the actin-nucleating WASH complex and cilia/basal body function. Check out the web server accompanying the paper
December 2014: Milos's paper is out in Elife
Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons.
In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.
November 2014: A new postdoc joins the lab!
Damien Garbett joined the Meyer lab as a postdoctoral fellow. Welcome aboard Damien
August 2014: Sean and Sabrina are opening their own labs!
February 2014: Feng Chiao's paper is out in Nature Cell Biology
Ca(2+) signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca(2+)-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca(2+) pulses, local depletion of Ca(2+) in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca(2+) influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca(2+) pump rates in the front relative to the back of the plasma membrane enable effective local Ca(2+) signalling by locally decreasing basal Ca(2+). Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca(2+) control system with polarized Ca(2+) signalling proteins and second messengers to synergistically promote directed cell migration.
January 2014: Milos is opening his own lab!
Contratulations to Milos who got a position in the Universität Münster. Good luck in your new lab!
November 2013: Kyuho's paper is out in Nature Methods
Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5′ terminal oligopyrimidine (5′ TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.
October 2013: Jia-Yun's paper is out in Molecular Cell
Dosage of Dyrk1a shifts cells within a p21-cyclin D1 signaling map to control the decision to enter the cell cycle.
Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels.
September 2013: Sabrina's paper is out in Cell
The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.
Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.
September 2012: Roy is opening his own lab!
Contratulations to Roy who got a position at UC San Diego. Good luck in your new lab!
Last modified Wednesday, 20-Jul-2016 11:02:41 PDT