Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation.

ABSTRACT: We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy transfer. Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells. Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia. Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

SUBMITTER: Espenel C

PROVIDER: S-EPMC3824023 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation.

Espenel Cedric C Acharya Bipul R BR Acharya Bipul R BR Kreitzer Geri G

The Journal of cell biology 20131104 3

We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy ...[more]

PMID: 24189273

Similar Datasets

Sensitive Fluorescent Biosensor Reveals Differential Subcellular Regulation of PKC.

Project description:The protein kinase C (PKC) family of serine/threonine kinases, which consist of three distinctly regulated subfamilies, have long been established as critical for a variety of cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs such as the ER, lysosome, and Par signaling complexes, is unclear. Here, we present a sensitive Excitation Ratiometric (ExRai) C Kinase Activity Reporter (ExRai-CKAR2) that enables the detection of minute changes in subcellular PKC activity. Using ExRai-CKAR2 in conjunction with an enhanced diacylglycerol (DAG) biosensor capable of detecting intracellular DAG dynamics, we uncover the differential regulation of PKC isoforms at distinct subcellular locations. We find that G-protein coupled receptor (GPCR) stimulation triggers sustained PKC activity at the ER and lysosomes, primarily mediated by Ca2+ sensitive conventional PKC (cPKC) and novel PKC (nPKC), respectively, with nPKC showing high basal activity due to elevated basal DAG levels on lysosome membranes. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or Par-complexes, further enabled us to detect previously inaccessible endogenous atypical PKC (aPKC) activity in 3D organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.

| S-EPMC10996667 | biostudies-literature

The kinesin superfamily protein KIF17: one protein with many functions.

Project description:Kinesins are ATP-dependent molecular motors that carry cargos along microtubules, generally in an anterograde direction. They are classified into 14 distinct families with varying structural and functional characteristics. KIF17 is a member of the kinesin-2 family that is plus end-directed. It is a homodimer with a pair of head motor domains that bind microtubules, a coiled-coil stalk, and a tail domain that binds cargos. In neurons, KIF17 transports N-methyl-D-aspartate receptor NR2B subunit, kainate receptor GluR5, and potassium Kv4.2 channels from cell bodies exclusively to dendrites. These cargos are necessary for synaptic transmission, learning, memory, and other functions. KIF17's interaction with NXF2 enables the transport of mRNA bidirectionally in dendrites. KIF17 or its homolog OSM-3 also mediates intraflagellar transport of cargos to the distal tips of flagella or cilia, thereby aiding in ciliogenesis. In many invertebrate and vertebrate sensory cells, KIF17 delivers cargos that contribute to chemosensory perception and signal transduction. In vertebrate photoreceptors, KIF17 is necessary for outer segment development and disc morphogenesis. In the testis, KIF17 (KIF17b) mediates microtubule-independent delivery of ACT from the nucleus to the cytoplasm and microtubule-dependent transport of Spatial-?, both are presumably involved in spermatogenesis. KIF17 is also implicated in epithelial polarity and morphogenesis, placental transport and development, and the development of specific brain regions. The transcriptional regulation of KIF17 has recently been found to be mediated by nuclear respiratory factor 1 (NRF-1), which also regulates NR2B as well as energy metabolism in neurons. Dysfunctions of KIF17 are linked to a number of pathologies.

| S-EPMC3677786 | biostudies-literature

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms.

Project description:Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.

| S-EPMC2886353 | biostudies-literature

An activity-dependent local transport regulation via degradation and synthesis of KIF17 underlying cognitive flexibility.

Project description:Synaptic weight changes among postsynaptic densities within a single dendrite are regulated by the balance between localized protein degradation and synthesis. However, the molecular mechanism via these opposing regulatory processes is still elusive. Here, we showed that the molecular motor KIF17 was locally degraded and synthesized in an N-methyl-d-aspartate receptor (NMDAR)-mediated activity-dependent manner. Accompanied by the degradation of KIF17, its transport was temporarily dampened in dendrites. We also observed that activity-dependent local KIF17 synthesis driven by its 3' untranslated region (3'UTR) occurred at dendritic shafts, and the newly synthesized KIF17 moved along the dendrites. Furthermore, hippocampus-specific deletion of Kif17 3'UTR disrupted KIF17 synthesis induced by fear memory retrieval, leading to impairment in extinction of fear memory. These results indicate that the regulation of the KIF17 transport is driven by the single dendrite-restricted cycle of degradation and synthesis that underlies cognitive flexibility.

| S-EPMC7744090 | biostudies-literature

The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development.

Project description:Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.

| S-EPMC2362383 | biostudies-literature

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP.

Project description:The biogenesis, maintenance and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. Here, we identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLSs) suggest that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a ciliary-cytoplasmic gradient of the small GTPase Ran, with high levels of GTP-bound Ran (RanGTP) in the cilium. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with the nuclear import protein importin-beta2 in a manner dependent on the CLS and inhibited by RanGTP. We propose that Ran has a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments.

| S-EPMC2896429 | biostudies-literature

An EB1-kinesin complex is sufficient to steer microtubule growth in vitro.

Project description:Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization neurons showed that while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules [1]. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus ends entering branch points be actively directed toward the cell body. A model was proposed in which EB1 tracks the plus ends of microtubules growing into a branch and an associated kinesin-2 motor walks along a static microtubule to steer the plus end toward the cell body. However, the fast plus-end binding dynamics of EB1 [2-5] appear to be at odds with this proposed mechanical function. To test this model in vitro, we reconstituted the system by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus ends and static microtubules. Consistent with in vivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus, EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated this activity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells.

| S-EPMC3963495 | biostudies-literature

Local control of intracellular microtubule dynamics by EB1 photodissociation.

Project description:End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein-protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.

| S-EPMC5826794 | biostudies-literature

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport.

Project description:Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.

| S-EPMC4791134 | biostudies-literature

A biosensor for the activity of the "sheddase" TACE (ADAM17) reveals novel and cell type-specific mechanisms of TACE activation.

Project description:Diverse environmental conditions stimulate protein "shedding" from the cell surface through proteolytic cleavage. The protease TACE [tumor necrosis factor-α (TNFα)--converting enzyme, encoded by ADAM17] mediates protein shedding, thereby regulating the maturation and release of various extracellular substrates, such as growth factors and cytokines, that induce diverse cellular responses. We developed a FRET (fluorescence resonance energy transfer)-based biosensor called TSen that quantitatively reports the kinetics of TACE activity in live cells. In combination with chemical biology approaches, we used TSen to probe the dependence of TACE activation on the induction of the kinases p38 and ERK (extracellular signal-regulated kinase) in various epithelial cell lines. Using TSen, we found that disruption of the actin cytoskeleton in keratinocytes induced rapid and robust TSen cleavage and the accumulation of TACE at the plasma membrane. Cytoskeletal disruption also increased the cleavage of endogenous TACE substrates, including transforming growth factor-α. Thus, TSen is a useful tool for unraveling the mechanisms underlying the spatiotemporal activation of TACE in live cells.

| S-EPMC5012641 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data