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

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.

Download Full-text

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.

Download Full-text

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

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.

Download Full-text

Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

10.1101/855940 ◽

2019 ◽

Cited By ~ 1

Author(s):

Punam Sonar ◽

Wiphu Youyen ◽

Augustine Cleetus ◽

Pattipong Wisanpitayakorn ◽

Iman S. Mousavi ◽

...

Keyword(s):

Single Molecule ◽

Motor Coordination ◽

Intraflagellar Transport ◽

Kinetic Properties ◽

C Elegans ◽

Slow Velocity ◽

Single Molecule Studies ◽

Fast Transport ◽

And Function

SummaryThe construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors ‘line up’ in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other’s way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

Download Full-text

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Nature Communications ◽

10.1038/s41467-021-22861-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sabrina Dietz ◽

Miguel Vasconcelos Almeida ◽

Emily Nischwitz ◽

Jan Schreier ◽

Nikenza Viceconte ◽

...

Keyword(s):

Quantitative Proteomics ◽

Wild Type ◽

C Elegans ◽

Double Stranded Dna ◽

Telomere Sequence ◽

Proteomics Approach ◽

Telomeric Protein ◽

Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

Download Full-text

In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans

Nature Communications ◽

10.1038/ncomms5974 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 26

Author(s):

Hong Zhan ◽

Ramunas Stanciauskas ◽

Christian Stigloher ◽

Kevin K. Dizon ◽

Maelle Jospin ◽

...

Keyword(s):

Calcium Channels ◽

Single Molecule ◽

Single Molecule Imaging ◽

C Elegans

Download Full-text

TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

10.1101/2020.11.19.390005 ◽

2020 ◽

Author(s):

Jaap van Krugten ◽

Noémie Danné ◽

Erwin J.G. Peterman

Keyword(s):

Single Molecule ◽

Transmembrane Proteins ◽

Intraflagellar Transport ◽

Single Molecule Tracking ◽

C Elegans ◽

Normal Diffusion ◽

Cellular Signal ◽

Underlying Mechanisms ◽

And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

Download Full-text

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

10.1101/2020.08.19.256388 ◽

2020 ◽

Author(s):

Sabrina Dietz ◽

Miguel Vasconcelos Almeida ◽

Emily Nischwitz ◽

Jan Schreier ◽

Nikenza Viceconte ◽

...

Keyword(s):

Quantitative Proteomics ◽

Binding Proteins ◽

Protein Complexes ◽

Wild Type ◽

C Elegans ◽

Double Stranded Dna ◽

Telomere Sequence ◽

Proteomics Approach

AbstractTelomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

Download Full-text

A Generalized Kinetic Model for Coupling between Stepping and ATP Hydrolysis of Kinesin Molecular Motors

International Journal of Molecular Sciences ◽

10.3390/ijms20194911 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4911 ◽

Cited By ~ 4

Author(s):

Xie ◽

Guo ◽

Chen

Keyword(s):

Kinetic Model ◽

Atpase Activity ◽

Single Molecule ◽

Rate Constants ◽

Molecular Motors ◽

Atp Hydrolysis ◽

Power Production ◽

Wild Type ◽

Chemomechanical Coupling ◽

Generalized Kinetic Model

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.

Download Full-text

Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia

Science Translational Medicine ◽

10.1126/scitranslmed.aao3003 ◽

2018 ◽

Vol 10 (436) ◽

pp. eaao3003 ◽

Cited By ~ 58

Author(s):

Luis A. Carvajal ◽

Daniela Ben Neriah ◽

Adrien Senecal ◽

Lumie Benard ◽

Victor Thiruthuvanathan ◽

...

Keyword(s):

Tumor Suppressor ◽

Single Molecule ◽

Cellular Proliferation ◽

Wild Type ◽

Suppressor Activity ◽

Mouse Double Minute ◽

Double Minute ◽

Wild Type P53

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled α-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell–enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

Download Full-text

The Centrosomal, Putative Tumor Suppressor Protein TACC2 Is Dispensable for Normal Development, and Deficiency Does Not Lead to Cancer

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6403-6409.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6403-6409 ◽

Cited By ~ 28

Author(s):

Michael M. Schuendeln ◽

Roland P. Piekorz ◽

Christian Wichmann ◽

Youngsoo Lee ◽

Peter J. McKinnon ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Cycle Progression ◽

Tumor Development ◽

Coiled Coil ◽

Physiological Role ◽

Tumor Suppressor Protein ◽

Wild Type ◽

Mouse Development

ABSTRACT TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.

Download Full-text