Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness
David J. Gill, Keit Min Tham, Joanne Chia, Shyi Chyi Wang, Catharina Steentoft, Henrik Clausen, Emilie A. Bard-Chapeau, and Frederic A. Bard
How cancer cells become invasive is key to understanding malignancy. Perturbations in O-glycosylation are strongly correlated with invasiveness. Here (pp. E3152–E3161) we report that tumor cells display relocation of O-glycosylation initiating glycosyltransferases from the Golgi apparatus to the endoplasmic reticulum (ER). ER-located O-glycosylation stimulates cell migration and invasiveness, which depend on cell surface O-glycoproteins. Inhibition of the glycosyltransferases in the ER reduces tissue invasion and metastasis formation in mice. Our study suggests that control of glycosylation via the subcellular localization of glycosyltransferases is a critical mechanism driving invasiveness in tumor cells.
Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex
Kang Le, Chun-Chun Li, Guan Ye, Joel Moss, and Martha Vaughan
Phosphorylation of myosin regulatory light chain (RLC) alters actomyosin contractility and cell mobility. Finding that myosin IIA interacted directly with brefeldin A-inhibited guanine nucleotide-exchange factors (BIG)1 and BIG2, best known as activators of ADP ribosylation factor (Arf) GTPases for intracellular vesicular trafficking, we showed (pp. E3162–E3170) that BIG1 or -2 depletion increased RLC phosphorylation, which interfered with cell mobility independent of Arf activation. By acting noncatalytically as scaffolds for the assembly and operation of multimolecular phosphatase complexes that included phosphatase 1δ and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 contribute to regulation of F-actin formation and cytoskeleton dynamics required for cell polarization and directed migration.
Experiments on the role of deleterious mutations as stepping stones in adaptive evolution
Arthur W. Covert III, Richard E. Lenski, Claus O. Wilke, and Charles Ofria
It might seem obvious that deleterious mutations must impede evolution. However, a later mutation may interact with a deleterious predecessor, facilitating otherwise inaccessible adaptations. Although such interactions have been reported before, it is unclear whether they are rare and inconsequential or, alternatively, are important for sustaining adaptation. We studied (pp. E3171–E3178) digital organisms—computer programs that replicate and evolve—to compare adaptation in populations where deleterious mutations were disallowed with unrestricted controls. Control populations achieved higher fitness values because some deleterious mutations acted as stepping stones across otherwise impassable fitness valleys. Deleterious mutations can thus sometimes play a constructive role in adaptive evolution.
Conditionals by inversion provide a universal method for the generation of conditional alleles
Aris N. Economides, David Frendewey, Peter Yang, Melissa G. Dominguez, Anthony T. Dore, Ivan B. Lobov, Trikaldarshi Persaud, Jose Rojas, Joyce McClain, Peter Lengyel, Gustavo Droguett, Rostislav Chernomorsky, Sean Stevens, Wojtek Auerbach, Thomas M. DeChiara, William Pouyemirou, Joseph M. Cruz, Jr., Kieran Feeley, Ian A. Mellis, Jason Yasenchack, Sarah J. Hatsell, LiQin Xie, Esther Latres, Lily Huang, Yuhong Zhang, Evangelos Pefanis, Dimitris Skokos, Ron A. Deckelbaum, Susan D. Croll, Samuel Davis, David M. Valenzuela, Nicholas W. Gale, Andrew J. Murphy, and George D. Yancopoulos
We describe conditional by inversion (COIN), a new design for conditional alleles that uses an optimized conditional gene trap module (COIN module) inserted into the target gene in an orientation opposite to the gene’s direction of transcription. Activation by Cre recombinase inverts the COIN module, resulting in expression of a reporter and termination of transcription, thereby inactivating the target gene while marking the cells where the conditional event has occurred. Creation of COIN alleles for more than 20 genes showed (pp. E3179–E3188) that it is a robust and universal method—applicable to any gene regardless of exon–intron structure—that overcomes the limitations of previous conditional approaches.
Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes
Laure Ysebrant de Lendonck, Sandrine Tonon, Muriel Nguyen, Patricia Vandevenne, Iain Welsby, Valerie Martinet, Céline Molle, Louis-Marie Charbonnier, Oberdan Leo, and Stanislas Goriely
Interferon regulatory factor (IRF) 3 is one of the key transcription factors implicated in innate antiviral responses. In recent years its role in shaping adaptive immune responses through activation of a specific transcriptional program in antigen-presenting cells has been appreciated. In this work we show (pp. E3189–E3197) that within CD8 T cells, IRF3 interacts with the transcriptional network that controls their polarization, thereby limiting the capacity of these cells to produce IL-17. These findings shed light on the functions of IRF3 in adaptive immune responses.
Inhibitory role of the transcription repressor Gfi1 in the generation of thymus-derived regulatory T cells
Lewis Zhichang Shi, Nishan S. Kalupahana, Meghan E. Turnis, Geoffrey Neale, Hanno Hock, Dario A. A. Vignali, and Hongbo Chi
Thymic development of regulatory T cells (Treg) is a crucial event for immune homeostasis. Despite extensive descriptions of molecular pathways functioning within Treg cells, little is known how Treg development is shaped by the control of extrinsic factors. Here (pp. E3198–E3205), we show that the transcription factor growth factor independent 1 (Gfi1) restrains thymic generation of Treg cells. Loss of Gfi1 led to aberrant production of the cytokine IL-2. Elevated IL-2 then drove the expansion of Treg cells. Accordingly, deficiency of Gfi1 dampened antitumor immunity. Our results establish that Gfi1 actively controls thymocyte IL-2 production, thereby shaping thymic generation of Treg cells.
Unifying model for molecular determinants of the preselection Vβ repertoire
Suhasni Gopalakrishnan, Kinjal Majumder, Alexander Predeus, Yue Huang, Olivia I. Koues, Jiyoti Verma-Gaur, Salvatore Loguercio, Andrew I. Su, Ann J. Feeney, Maxim N. Artyomov, and Eugene M. Oltz
The assembly of immunoglobulin and T-cell receptor genes by V(D)J (variable, diversity, joining) recombination must strike a balance between maximum diversification of antigen receptors and favoring gene segments with specialized functions. We quantified the use of V gene segments in the primary T-cell receptor β repertoire, defining the relative contribution of 13 parameters in shaping their recombination efficiencies. Computational analysis of these data (pp. E3206–E3215) provides a unifying model, revealing a minimal set of five parameters that predict Vβ use. This model building approach will help predict how natural alterations of large V clusters impact immune receptor repertoires.
T-box transcription factor T-bet, a key player in a unique type of B-cell activation essential for effective viral clearance
Kira Rubtsova, Anatoly V. Rubtsov, Linda F. van Dyk, John W. Kappler, and Philippa Marrack
Here (pp. E3216–E3224), we show that signals delivered by antigen engagement, IFNγ, and toll-like receptor 7 [TLR7] induce T-box transcription factor T-bet and IgG2a switching in B cells. The IgG2a product of these signals is important for viral immunity. For example, the titers of mouse gammaherpesvirus 68, an Epstein–Barr-related virus, are not well reduced if the B cells cannot express T-bet and consequently cannot switch to production of IgG2a. The T-bet expressing B cells resemble a subset of B cells that appears in autoimmune prone mice and women. Thus, a B-cell differentiation pathway that has evolved to promote immunity to viruses may also contribute to autoimmunity.
UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling
Carlene Moore, Ferda Cevikbas, H. Amalia Pasolli, Yong Chen, Wei Kong, Cordula Kempkes, Puja Parekh, Suk Hee Lee, Nelly-Ange Kontchou, Iwei Ye, Nan Marie Jokerst, Elaine Fuchs, Martin Steinhoff, and Wolfgang B. Liedtke
Skin protects against harmful external cues, one of them UV radiation, which, upon overexposure, causes sunburn as part of the UVB response. Using genetically engineered mice and cultured skin epithelial cells, we have identified the calcium-permeable TRPV4 ion channel in skin epithelial cells as critical for translating the UVB stimulus into intracellular signals and also into signals from epithelial skin cell to sensory nerve cell that innervates the skin, causing pain. These signaling mechanisms (pp. E3225–E3234) underlie sunburn and in particular sunburn-associated pain. Thus, activation of TRPV4 in skin by UVB evokes sunburn pain, highlighting the forefront-signaling role of the skin and TRPV4.
Forebrain dopamine neurons project down to a brainstem region controlling locomotion
Dimitri Ryczko, Swantje Grätsch, François Auclair, Catherine Dubé, Saskia Bergeron, Michael H. Alpert, Jackson J. Cone, Mitchell F. Roitman, Simon Alford, and Réjean Dubuc
We found in lampreys (pp. E3235–E3242) that dopaminergic cells from the posterior tuberculum (homologue of the mammalian substantia nigra pars compacta and/or ventral tegmental area) not only send ascending projections to the striatum, but also have a direct descending projection to a brainstem region controlling locomotion—the mesencephalic locomotor region—where it releases dopamine (DA). DA increased locomotor output through a D1 receptor-dependent mechanism. The presence of this descending dopaminergic projection may have considerable implication for our understanding of the role of DA in motor control under physiological and pathological (i.e. Parkinson disease) conditions.
Prevalent mechanism of membrane bridging by synaptotagmin-1
Alpay B. Seven, Kyle D. Brewer, Liang Shi, Qiu-Xing Jiang, and Josep Rizo
The ability of synaptotagmin-1 to bridge two membranes is likely critical for its function as a Ca2+ sensor in neurotransmitter release. A synaptotagmin-1 fragment spanning its two C2 domains bridges membranes directly, binding simultaneously to both membranes. However, a longer synaptotagmin-1 fragment spanning most of its cytoplasmic region was proposed to bridge membranes through protein–protein interactions between oligomers bound to each membrane. This paper (pp. E3243–E3252) now shows that direct bridging actually constitutes the prevalent mechanism of membrane bridging by the longer synaptotagmin-1 fragment. These findings strongly suggest that direct membrane bridging underlies the function of synaptotagmin-1 in neurotransmitter release.