MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

Mapping Intimacies ◽

10.1101/209221 ◽

2017 ◽

Author(s):

Peishan Yi ◽

Chao Xie ◽

Guangshuo Ou

Keyword(s):

Cell Cycle ◽

Sensory Neurons ◽

Intraflagellar Transport ◽

Time Lapse ◽

Genetic Screen ◽

Forward Genetic Screen ◽

C Elegans ◽

Neuronal Cilia ◽

Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

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Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3

The Journal of Cell Biology ◽

10.1083/jcb.200605179 ◽

2006 ◽

Vol 174 (7) ◽

pp. 931-937 ◽

Cited By ~ 76

Author(s):

Miki Imanishi ◽

Nicholas F. Endres ◽

Arne Gennerich ◽

Ronald D. Vale

Keyword(s):

Atpase Activity ◽

Single Molecule ◽

Intramolecular Interaction ◽

Optical Trap ◽

Coiled Coil ◽

Intraflagellar Transport ◽

Wild Type ◽

C Elegans ◽

Sensory Cilia

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3–null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

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Intraflagellar transport motors in Caenorhabditis elegans neurons

Biochemical Society Transactions ◽

10.1042/bst0320682 ◽

2004 ◽

Vol 32 (5) ◽

pp. 682-684 ◽

Cited By ~ 13

Author(s):

J.M. Scholey ◽

G. Ou ◽

J. Snow ◽

A. Gunnarson

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Protein Complexes ◽

Intraflagellar Transport ◽

Time Lapse ◽

Protein Fusion ◽

C Elegans ◽

Sensory Cilia ◽

Macromolecular Protein ◽

Green Fluorescent

IFT (intraflagellar transport) assembles and maintains sensory cilia on the dendritic endings of chemosensory neurons within the nematode Caenorhabditis elegans. During IFT, macromolecular protein complexes called IFT particles (which carry ciliary precursors) are moved from the base of the sensory cilium to its distal tip by anterograde IFT motors (kinesin-II and Osm-3 kinesin) and back to the base by retrograde IFT-dynein [Rosenbaum and Witman (2002) Nat. Rev. Mol. Cell Biol. 3, 813–825; Scholey (2003) Annu. Rev. Cell Dev. Biol. 19, 423–443; and Snell, Pan and Wang (2004) Cell 117, 693–697]. In the present study, we describe the protein machinery of IFT in C. elegans, which we have analysed using time-lapse fluorescence microscopy of green fluorescent protein-fusion proteins in concert with ciliary mutants.

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Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

10.1101/2020.05.21.108449 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kade M. Power ◽

Jyothi S. Akella ◽

Amanda Gu ◽

Jonathon D. Walsh ◽

Sebastian Bellotti ◽

...

Keyword(s):

Genetic Screen ◽

Mucociliary Transport ◽

Neuronal Function ◽

Post Translational Modifications ◽

Forward Genetic Screen ◽

C Elegans ◽

Neuronal Cilia ◽

Microtubule Stability ◽

Progressive Degeneration ◽

Downstream Effects

AbstractCiliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

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Sensory cilia act as a specialized venue for regulated EV biogenesis and signaling

10.1101/2021.02.04.429799 ◽

2021 ◽

Cited By ~ 1

Author(s):

Juan Wang ◽

Inna A. Nikonorova ◽

Malan Silva ◽

Jonathon D. Walsh ◽

Peter Tilton ◽

...

Keyword(s):

Fluorescent Protein ◽

Coiled Coil ◽

Super Resolution ◽

Intraflagellar Transport ◽

Sensory Stimulation ◽

Intercellular Signaling ◽

C Elegans ◽

Sensory Cilia ◽

Ciliated Neurons

AbstractExtracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study signaling EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

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Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

Nature Communications ◽

10.1038/s41467-021-24743-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nathan J. VanDusen ◽

Julianna Y. Lee ◽

Weiliang Gu ◽

Catalina E. Butler ◽

Isha Sethi ◽

...

Keyword(s):

Gene Expression ◽

Genetic Screen ◽

Forward Genetics ◽

Epigenetic Mark ◽

Biological Processes ◽

Dynamic Changes ◽

Forward Genetic Screen ◽

High Resource ◽

Organ Systems

AbstractThe forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

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Regulation of Yeast-to-Hyphae Transition inYarrowia lipolytica

mSphere ◽

10.1128/msphere.00541-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 9

Author(s):

Kyle R. Pomraning ◽

Erin L. Bredeweg ◽

Eduard J. Kerkhoven ◽

Kerrie Barry ◽

Sajeet Haridas ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Stress Response ◽

Hyphal Growth ◽

Genetic Screen ◽

Morphological Transition ◽

Histidine Kinases ◽

Content Type ◽

Forward Genetic Screen ◽

Response To Stress

ABSTRACTThe yeastYarrowia lipolyticaundergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All thesmoothmutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5′-CCCCT-3′) and upregulation of genes with cell cycle box (5′-ACGCG-3′) motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p inSaccharomyces cerevisiae. We confirmed that theY. lipolyticamsn2(Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2enables hyphal growth insmoothstrains. The cell cycle box is bound by the Mbp1p/Swi6p complex inS. cerevisiaeto regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1or Ylswi6homologs decreased hyphal growth and that deletion of either Ylmbp1or Ylswi6promotes hyphal growth insmoothstrains. A second forward genetic screen for reversion to hyphal growth was performed with thesmooth-33mutant to identify additional genetic factors regulating hyphal growth inY. lipolytica. Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1and Ylnik1and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1. Together, these results demonstrate thatY. lipolyticatransitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCEMany yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition inYarrowia lipolytica. We confirmed that the transcription factor Ylmsn2is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1and Ylnik1as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest thatY. lipolyticatransitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.

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UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans

10.1101/2021.01.27.428454 ◽

2021 ◽

Author(s):

Kelly H. Oh ◽

Mia Krout ◽

Janet E. Richmond ◽

Hongkyun Kim

Keyword(s):

Active Zone ◽

Calcium Influx ◽

Genetic Screen ◽

Scaffolding Protein ◽

Tight Coupling ◽

Forward Genetic Screen ◽

Precise Control ◽

C Elegans ◽

Vesicle Exocytosis ◽

Active Zones

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

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