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1.
Methods Mol Biol ; 2551: 575-593, 2023.
Article in English | MEDLINE | ID: mdl-36310226

ABSTRACT

Liquid-liquid phase separation (LLPS) has emerged as a common biophysical event that facilitates the formation of non-membrane-bound cellular compartments, also termed biomolecular condensates. Since the first report of a biomolecular condensate in the germline of C. elegans, many regulatory hubs have been shown to have similar liquid-like features. With the wealth of molecules now being reported to possess liquid-like features, an impetus has been placed on reconciling LLPS with regulation of specific biological properties in vivo. Herein, we report a methodology used to study LLPS-associated features in C. elegans neurons, illustrated using the RNA granule protein TIAR-2. In axons, TIAR-2 forms liquid-like granules, which following injury are inhibitory to the regeneration process. Measuring the dynamics of TIAR-2 granules provides a tractable biological output to study LLPS function. In conjunction with other established methods to assess LLPS, the results from the protocol outlined provide comprehensive insight regarding this important biophysical property.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/genetics , Biomolecular Condensates , Caenorhabditis elegans Proteins/genetics , Germ Cells/metabolism , Axons/metabolism
2.
Neuron ; 104(2): 290-304.e8, 2019 10 23.
Article in English | MEDLINE | ID: mdl-31378567

ABSTRACT

Phase separation into liquid-like compartments is an emerging property of proteins containing prion-like domains (PrLDs), yet the in vivo roles of phase separation remain poorly understood. TIA proteins contain a C-terminal PrLD, and mutations in the PrLD are associated with several diseases. Here, we show that the C. elegans TIAR-2/TIA protein functions cell autonomously to inhibit axon regeneration. TIAR-2 undergoes liquid-liquid phase separation in vitro and forms granules with liquid-like properties in vivo. Axon injury induces a transient increase in TIAR-2 granule number. The PrLD is necessary and sufficient for granule formation and inhibiting regeneration. Tyrosine residues within the PrLD are important for granule formation and inhibition of regeneration. TIAR-2 is also serine phosphorylated in vivo. Non-phosphorylatable TIAR-2 variants do not form granules and are unable to inhibit axon regeneration. Our data demonstrate an in vivo function for phase-separated TIAR-2 and identify features critical for its function in axon regeneration.


Subject(s)
Axons/metabolism , Caenorhabditis elegans Proteins/metabolism , Nerve Regeneration/physiology , RNA Recognition Motif Proteins/metabolism , Animals , Axons/physiology , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Cell Compartmentation , Cytoplasmic Granules , Protein Domains , RNA Recognition Motif Proteins/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , T-Cell Intracellular Antigen-1/genetics , T-Cell Intracellular Antigen-1/metabolism
3.
Elife ; 72018 11 21.
Article in English | MEDLINE | ID: mdl-30461420

ABSTRACT

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.


Subject(s)
Axons/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , NAD/metabolism , Nerve Regeneration/genetics , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Animals , Axons/ultrastructure , Axotomy , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Genetic Testing , Kelch Repeat , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microtubules/metabolism , Microtubules/ultrastructure , Molecular Sequence Annotation , Nicotinamide-Nucleotide Adenylyltransferase/metabolism
4.
Neuron ; 97(3): 511-519.e6, 2018 02 07.
Article in English | MEDLINE | ID: mdl-29395906

ABSTRACT

The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells. The piRNA pathway inhibits axon regrowth independent of nuclear transcriptional silencing but dependent on the slicer domain of PRG-1/PIWI, suggesting that post-transcriptional gene silencing is involved. Our results reveal the neuronal piRNA pathway as a novel intrinsic repressor of axon regeneration.


Subject(s)
Argonaute Proteins/metabolism , Axons/metabolism , Caenorhabditis elegans Proteins/metabolism , RNA, Small Interfering/metabolism , Regeneration , Animals , Caenorhabditis elegans , Germ Cells/metabolism , Signal Transduction
5.
J Biol Chem ; 291(15): 7796-804, 2016 Apr 08.
Article in English | MEDLINE | ID: mdl-26907690

ABSTRACT

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Stress, Physiological , Animals , Caenorhabditis elegans/immunology , Caenorhabditis elegans Proteins/immunology , Immunity, Innate , Janus Kinases/immunology , Janus Kinases/metabolism , Mitogen-Activated Protein Kinases/immunology
6.
Cell Cycle ; 12(9): 1416-23, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23574720

ABSTRACT

Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling.


Subject(s)
Cell Cycle , Chromatin Assembly and Disassembly , E2F Transcription Factors/metabolism , Neurons/cytology , Neurons/metabolism , Retinoblastoma Protein/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Survival , Chromatin/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary
7.
Cell Stem Cell ; 12(4): 440-52, 2013 Apr 04.
Article in English | MEDLINE | ID: mdl-23499385

ABSTRACT

The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations.


Subject(s)
Cell Cycle , E2F3 Transcription Factor/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , Aging/metabolism , Animals , Base Sequence , Cell Count , Cell Cycle/genetics , Cell Lineage/genetics , Cell Proliferation , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Mice , Models, Biological , Molecular Sequence Data , Neurogenesis , Promoter Regions, Genetic/genetics , Protein Isoforms/metabolism , SOXB1 Transcription Factors/metabolism
8.
J Neurosci ; 32(42): 14809-14, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-23077065

ABSTRACT

The retinoblastoma protein (Rb) family members are essential regulators of cell cycle progression, principally through regulation of the E2f transcription factors. Growing evidence indicates that abnormal cell cycle signals can participate in neuronal death. In this regard, the role of Rb (p105) itself has been controversial. Germline Rb deletion leads to massive neuronal loss, but initial reports argue that death is non-cell autonomous. To more definitively resolve this question, we generated acute murine knock-out models of Rb in terminally differentiated neurons in vitro and in vivo. Surprisingly, we report that acute inactivation of Rb in postmitotic neurons results in ectopic cell cycle protein expression and neuronal loss without concurrent induction of classical E2f-mediated apoptotic genes, such as Apaf1 or Puma. These results suggest that terminally differentiated neurons require Rb for continuous cell cycle repression and survival.


Subject(s)
Cell Survival/physiology , Mitosis/physiology , Neurons/physiology , Retinoblastoma Protein/physiology , Animals , Cell Death/genetics , Cell Death/physiology , Cell Survival/genetics , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitosis/genetics , Retinoblastoma Protein/deficiency
9.
J Neurosci ; 32(24): 8219-30, 2012 Jun 13.
Article in English | MEDLINE | ID: mdl-22699903

ABSTRACT

During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.


Subject(s)
E2F Transcription Factors/physiology , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/biosynthesis , Neurogenesis/physiology , Retinoblastoma Protein/physiology , Transcription Factors/biosynthesis , Animals , Brain/growth & development , Brain/physiology , Cell Count/methods , Female , Interneurons/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Pregnancy , Signal Transduction/physiology
10.
Mol Cell Biol ; 31(2): 238-47, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21059867

ABSTRACT

The Rb/E2F pathway has long been appreciated for its role in regulating cell cycle progression. Emerging evidence indicates that it also influences physiological events beyond regulation of the cell cycle. We have previously described a requirement for Rb/E2F mediating neuronal migration; however, the molecular mechanisms remain unknown, making this an ideal system to identify Rb/E2F-mediated atypical gene regulation in vivo. Here, we report that Rb regulates the expression of neogenin, a gene encoding a receptor involved in cell migration and axon guidance. Rb is capable of repressing E2F-mediated neogenin expression while E2F3 occupies a region containing E2F consensus sites on the neogenin promoter in native chromatin. Absence of Rb results in aberrant neuronal migration and adhesion in response to netrin-1, a known ligand for neogenin. Increased expression of neogenin through ex vivo electroporation results in impaired neuronal migration similar to that detected in forebrain-specific Rb deficiency. These findings show direct regulation of neogenin by the Rb/E2F pathway and demonstrate that regulation of neogenin expression is required for neural precursor migration. These studies identify a novel mechanism through which Rb regulates transcription of a gene beyond the classical E2F targets to regulate events distinct from cell cycle progression.


Subject(s)
Cell Movement/physiology , E2F3 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Neurons/physiology , Retinoblastoma Protein/metabolism , Animals , Cell Adhesion/physiology , E2F3 Transcription Factor/genetics , HEK293 Cells , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Netrin-1 , Neurons/cytology , Promoter Regions, Genetic , Prosencephalon/anatomy & histology , Prosencephalon/embryology , Prosencephalon/metabolism , Retinoblastoma Protein/genetics , Transcription, Genetic , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
11.
Mol Cell Biol ; 29(17): 4701-13, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19564414

ABSTRACT

We have previously shown that p107, a member of the retinoblastoma (Rb) cell cycle regulatory family, has a unique function in regulating the pool of neural precursor cells. As the pool of progenitors is regulated by a limiting supply of trophic factors, we asked if the Rb/E2F pathway may control the size of the progenitor population by regulating the levels of growth factors or their receptors. Here, we demonstrate that fibroblast growth factor 2 (FGF2) is aberrantly upregulated in the brains of animals lacking Rb family proteins and that the gene encoding the FGF2 ligand is directly regulated by p107 and E2F3. Chromatin immunoprecipitation assays demonstrated that E2F3 and p107 occupy E2F consensus sites on the FGF2 promoter in the context of native chromatin. To evaluate the physiological consequence of FGF2 deregulation in both p107 and E2F3 mutants, we measured neural progenitor responsiveness to growth factors. Our results demonstrate that E2F3 and p107 are each mediators of FGF2 growth factor responsiveness in neural progenitor cells. These results support a model whereby p107 regulates the pool of FGF-responsive progenitors by directly regulating FGF2 gene expression in vivo. By identifying novel roles for p107/E2F in regulating genes outside of the classical cell cycle machinery targets, we uncover a new mechanism whereby Rb/E2F mediates proliferation through regulating growth factor responsiveness.


Subject(s)
E2F3 Transcription Factor/metabolism , Fibroblast Growth Factor 2/metabolism , Neurons/physiology , Signal Transduction/physiology , Stem Cells/physiology , Animals , Base Sequence , Cell Proliferation , Cells, Cultured , E2F3 Transcription Factor/genetics , Female , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation, Developmental , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Neurons/cytology , Pregnancy , Promoter Regions, Genetic , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Retinoblastoma-Like Protein p107/genetics , Retinoblastoma-Like Protein p107/metabolism , Sequence Alignment , Stem Cells/cytology
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