The Experts below are selected from a list of 999 Experts worldwide ranked by ideXlab platform
William O. Hancock - One of the best experts on this subject based on the ideXlab platform.
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Neck-Linker Length is a Critical Determinant of Kinesin Processivity
Biophysical Journal, 2020Co-Authors: Shankar Shastry, William O. HancockAbstract:The Kinesin neck-linker domain is a key mechanical element underlying processive Kinesin motility. Not only is neck-linker docking thought to be the dominant conformational change in the Kinesin hydrolysis cycle, chemomechanical communication between the two head domains must necessarily be transmitted through the two neck-linker domains and their shared coil-coil. Hence, the length of the neck-linker is expected to have a strong influence on Kinesin run length, a quantitative measure of processivity. Across different Kinesin families, motors with longer neck-linker domains, such as Kinesin-2 are generally less processive than Kinesin-1, which has the shortest neck-linker domain among N-terminal Kinesins. However, there is disagreement in the literature as to whether artificially extending the Kinesin-1 neck-linker alters the motor run length. Using single molecule TIRF analysis to visualize GFP-tagged motors in 80 mM PIPES buffer, we find that lengthening the Kinesin-1 neck-linker by three amino acids results in a five-fold reduction in run length. Consistent with this, when the Kinesin-2 neck linker was matched to the effective length of Kinesin-1 by deleting three residues and substituting an alanine for a proline, the Kinesin-2 run length nearly matched that of Kinesin-1. These results demonstrate that run length scales with neck linker length for both Kinesin-1 and Kinesin-2 and is sufficient to account for differences in processivity. In addition, we find that adding positive charge to neck linker inserts enhances processivity, providing a possible explanation for the lack of dependence of run length on neck-linker length observed by others. Our data is consistent with the hypothesis that increasing neck linker compliance reduces processivity by disrupting front head gating and potentially provides a unifying principle across Kinesin families - longer neck-linkers lead to less processive motors.
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Kinesin 2 from c reinhardtii is an atypically fast and auto inhibited motor that is activated by heterotrimerization for intraflagellar transport
Current Biology, 2020Co-Authors: Punam Sonar, William O. Hancock, Willi L Stepp, Wiphu Youyen, Augustine Cleetus, Pattipong Wisanpitayakorn, Sayed I Mousavi, Erkan Tuzel, Zeynep OktenAbstract:Summary 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, on average, 10 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 fully activating FLA8/10 for IFT in vivo. Finally, we linked 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 both complexes 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 work mostly 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.
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Kinesin 2 from c reinhardtii is an atypically fast and auto inhibited motor that is activated by heterotrimerization for intraflagellar transport
bioRxiv, 2019Co-Authors: Punam Sonar, William O. Hancock, Willi L Stepp, Wiphu Youyen, Augustine Cleetus, Pattipong Wisanpitayakorn, Iman S Mousavi, Erkan Tuezel, Zeynep OektenAbstract:Summary The 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.
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Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport.
Biophysical Journal, 2018Co-Authors: Qingzhou Feng, Geng-yuan Chen, Keith J Mickolajczyk, William O. HancockAbstract:Abstract Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including Kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple Kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of Kinesin-1 and Kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than Kinesin-1 at the single-molecule level, addition of Kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the Kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of Kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that Kinesin-2 motors detach and rebind to the microtubule much more frequently than does Kinesin-1. Finally, compared to cargo transported by two Kinesin-1, cargo transported by two Kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which Kinesin-1 motors step effectively against loads whereas Kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by Kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.
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Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport
bioRxiv, 2017Co-Authors: Qingzhou Feng, Geng-yuan Chen, Keith J Mickolajczyk, William O. HancockAbstract:Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including Kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple Kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of Kinesin-1 and Kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than Kinesin-1 at the single-molecule level, addition of Kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the Kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of Kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that Kinesin-2 motors detach and rebind to the microtubule much more frequently than do Kinesin-1. Finally, cargo transported by Kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which Kinesin-1 motors step effectively against loads while Kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by Kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.
Rachel H Giles - One of the best experts on this subject based on the ideXlab platform.
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mobility of the von hippel lindau tumour suppressor protein is regulated by Kinesin 2
Experimental Cell Research, 2008Co-Authors: Dorus A Mans, Martijn P Lolkema, Moniek Van Beest, Laura G M Daenen, Emile E Voest, Rachel H GilesAbstract:Abstract The von Hippel–Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex Kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal Kinesin-2 complex, which slightly differs from renal Kinesin-2. To investigate the role of Kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3β-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to Kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent Kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.
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Mobility of the von Hippel–Lindau tumour suppressor protein is regulated by Kinesin-2
Experimental Cell Research, 2008Co-Authors: Dorus A Mans, Martijn P Lolkema, Moniek Van Beest, Laura G M Daenen, Emile E Voest, Rachel H GilesAbstract:Abstract The von Hippel–Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex Kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal Kinesin-2 complex, which slightly differs from renal Kinesin-2. To investigate the role of Kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3β-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to Kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent Kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.
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the von hippel lindau tumour suppressor interacts with microtubules through Kinesin 2
FEBS Letters, 2007Co-Authors: Martijn P Lolkema, Dorus A Mans, Moniek Van Beest, Emile E Voest, Cristel Snijckers, Mascha Van Noort, Rachel H GilesAbstract:Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the Kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with Kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by Kinesin-2.
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The von Hippel–Lindau tumour suppressor interacts with microtubules through Kinesin‐2
FEBS Letters, 2007Co-Authors: Martijn P Lolkema, Dorus A Mans, Moniek Van Beest, Emile E Voest, Cristel Snijckers, Mascha Van Noort, Rachel H GilesAbstract:Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the Kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with Kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by Kinesin-2.
Zeynep Okten - One of the best experts on this subject based on the ideXlab platform.
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Kinesin 2 from c reinhardtii is an atypically fast and auto inhibited motor that is activated by heterotrimerization for intraflagellar transport
Current Biology, 2020Co-Authors: Punam Sonar, William O. Hancock, Willi L Stepp, Wiphu Youyen, Augustine Cleetus, Pattipong Wisanpitayakorn, Sayed I Mousavi, Erkan Tuzel, Zeynep OktenAbstract:Summary 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, on average, 10 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 fully activating FLA8/10 for IFT in vivo. Finally, we linked 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 both complexes 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 work mostly 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.
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Reconstitution reveals motor activation for intraflagellar transport
Nature, 2018Co-Authors: Mohamed A. A. Mohamed, Willi L Stepp, Zeynep OktenAbstract:The human body represents a notable example of ciliary diversification. Extending from the surface of most cells, cilia accomplish a diverse set of tasks. Predictably, mutations in ciliary genes cause a wide range of human diseases such as male infertility and blindness. In Caenorhabditis elegans sensory cilia, this functional diversity appears to be traceable to the differential regulation of the Kinesin-2-powered intraflagellar-transport (IFT) machinery. Here we reconstituted the first, to our knowledge, functional multi-component IFT complex that is deployed in the sensory cilia of C. elegans. Our bottom-up approach revealed the molecular basis of specific motor recruitment to the IFT trains. We identified the key component that incorporates homodimeric Kinesin-2 into its physiologically relevant context, which in turn allosterically activates the motor for efficient transport. These results will enable the molecular delineation of IFT regulation, which has eluded understanding since its discovery more than two decades ago. Reconstitution of a functional intraflagellar transport complex in Caenorhabditis elegans provides insight into the recruitment and activation of the Kinesin-2 motor protein.
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Kinesin 2 motors adapt their stepping behavior for processive transport on axonemes and microtubules
EMBO Reports, 2017Co-Authors: Willi L Stepp, Zeynep Okten, Georg Merck, Felix MuellerplanitzAbstract:Abstract Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long‐range transport processes in vivo . In all organisms studied so far, the Kinesin‐2 family is essential for long‐range transport on axonemes. Intriguingly, in higher eukaryotes, Kinesin‐2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric Kinesin‐2 motors distinguish between axonemes and microtubules. Unlike canonical Kinesin‐1, Kinesin‐2 takes directional, off‐axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of Kinesin‐2 to side‐track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A‐ and B‐tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co‐evolved the heterodimeric Kinesin‐2 with the ciliary machinery to work on the specialized axonemal surface for two‐way traffic.
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Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules
EMBO Reports, 2017Co-Authors: Willi L Stepp, Georg Merck, Felix Mueller-planitz, Zeynep OktenAbstract:Abstract Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long‐range transport processes in vivo . In all organisms studied so far, the Kinesin‐2 family is essential for long‐range transport on axonemes. Intriguingly, in higher eukaryotes, Kinesin‐2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric Kinesin‐2 motors distinguish between axonemes and microtubules. Unlike canonical Kinesin‐1, Kinesin‐2 takes directional, off‐axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of Kinesin‐2 to side‐track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A‐ and B‐tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co‐evolved the heterodimeric Kinesin‐2 with the ciliary machinery to work on the specialized axonemal surface for two‐way traffic.
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Path-Finding on the Microtubule
Biophysical Journal, 2013Co-Authors: Zeynep OktenAbstract:In long-range transport of cargo, prototypical Kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the Kinesin-2 class on freely suspended microtubules, mimicking cargo transport as accurately as possible. In stark contrast to Kinesin-1, heterodimeric Kinesin-2 motors from diverse organisms displayed an astounding range of rotational pitches, expanding the list of torque generating Kinesins to include processive representatives of the Kinesin-2 family. We provide direct evidence that neck region of Kinesin determines the torque generating properties by reversibly manipulating the properties of the neck of the human Kinesin-1 and the heterodimeric Kinesin-2 from sea urchin. Disrupting the stability of the neck by inserting flexible peptide stretches results in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduces the spiraling of the motor up to the point of protofilament tracking.
Jonathan M. Scholey - One of the best experts on this subject based on the ideXlab platform.
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Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery
FEBS Journal, 2017Co-Authors: Bram Prevo, Jonathan M. Scholey, Erwin J.g. PetermanAbstract:Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde Kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric Kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric Kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric Kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric Kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.
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cilium assembly delivery of tubulin by Kinesin 2 powered trains
Current Biology, 2013Co-Authors: Jonathan M. ScholeyAbstract:The Kinesin-2-driven anterograde transport of intraflagellar transport (IFT) trains has long been suspected to deliver cargo consisting of tubulin subunits for assembly at the axoneme tip. Important new work identifies the tubulin binding site on IFT trains that is responsible for this cargo transport.
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Kinesin 2 a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions
Annual Review of Cell and Developmental Biology, 2013Co-Authors: Jonathan M. ScholeyAbstract:Kinesin-2 was first purified as a heterotrimeric, anterograde, microtubule-based motor consisting of two distinct Kinesin-related subunits and a novel associated protein (KAP) that is currently best known for its role in intraflagellar transport and ciliogenesis. Subsequent work, however, has revealed diversity in the oligomeric state of different Kinesin-2 motors owing to the combinatorial heterodimerization of its subunits and the coexistence of both heterotrimeric and homodimeric Kinesin-2 motors in some cells. Although the functional significance of the homo- versus heteromeric organization of Kinesin-2 motor subunits and the role of KAP remain uncertain, functional studies suggest that cooperation between different types of Kinesin-2 motors or between Kinesin-2 and a member of a different motor family can generate diverse patterns of anterograde intracellular transport. Moreover, despite being restricted to ciliated eukaryotes, Kinesin-2 motors are now known to drive diverse transport events outside ...
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Measuring rates of intraflagellar transport along Caenorhabditis elegans sensory cilia using fluorescence microscopy.
Methods in Enzymology, 2013Co-Authors: Ingrid Brust-mascher, Guangshuo Ou, Jonathan M. ScholeyAbstract:Intraflagellar transport (IFT), the Kinesin-2 and IFT-dynein-dependent bidirectional movement of multisubunit protein complexes called IFT-particles and associated cargo molecules along ciliary axonemes, is thought to be essential for the assembly and maintenance of virtually all eukaryotic cilia and flagella. Transport assays that allow measurements of the rates of movement of specific, fluorescently tagged, functional components of the IFT machinery, including motors, IFT particle subunits, and putative cargo, were first developed in Caenorhabditis elegans sensory cilia, and they have proved to be an important and valuable tool for dissecting the molecular mechanisms of IFT. We describe how these transport assays are performed in our laboratory and summarize the information that has been obtained by using them concerning the mechanisms of action and regulation of the motors that drive IFT, the composition and organization of the IFT-particles, and the identification of IFT-dynein subunits and ciliary tubulin isotypes as likely cargo proteins of Kinesin-2-driven anterograde IFT.
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Kinesin 2 motors transport ift particles dyneins and tubulin subunits to the tips of caenorhabditis elegans sensory cilia relevance to vision research
Vision Research, 2012Co-Authors: Jonathan M. ScholeyAbstract:Abstract The sensory outer segments (OS) of vertebrate retinal photoreceptors, which detect photons of light, resemble the distal segments of Caenorhabditis elegans sensory cilia, which detect chemical ligands that influence the chemotactic movements of the animal. Based on fluorescence microscopy assays performed in sensory cilia of living, transgenic “wild type” and mutant C. elegans, combined with in vitro motility assays using purified motors, we have proposed that two types of Kinesin-2 motor, heterotrimeric Kinesin-II and homodimeric OSM-3, cooperate to build amphid and phasmid sensory cilia on chemosensory neurons. Specifically, we propose that these motors function together in a redundant manner to build the axoneme core (aka middle segments (MS)), whereas OSM-3 alone serves to build the distal segments (DS). Furthermore, our data suggest that these motors accomplish this by driving two sequential steps of anterograde transport of cargoes consisting of IFT-particles, retrograde dynein motors, and ciliary tubulin subunits, from the transition zone to the tips of the axonemal microtubules (MTs). Homologs of Kinesin-II (KIF3) and OSM-3 (KIF17) are also proposed to contribute to the assembly of vertebrate photoreceptors, although how they do so is currently unclear. Here I review our work on Kinesin-2 motors, intraflagellar transport (IFT) and cilium biogenesis in C. elegans sensory cilia, and comment on its possible relevance to current research on vertebrate photoreceptor cilia assembly and function.
David S Williams - One of the best experts on this subject based on the ideXlab platform.
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live cell imaging evidence for the ciliary transport of rod photoreceptor opsin by heterotrimeric Kinesin 2
The Journal of Neuroscience, 2012Co-Authors: Deepti Trivedi, Emilie Colin, Carrie M Louie, David S WilliamsAbstract:Primary cilia detect extracellular signals through membrane receptors and channels. The outer segment of a vertebrate photoreceptor cell represents the most elaborate of all primary cilia, containing extraordinarily large amounts of the visual receptor protein, opsin. Because of its high abundance, opsin represents a potential model system for the study of ciliary membrane receptors, including their transport. Here, we have analyzed the movement of ciliary opsin to test whether the highly conserved intraflagellar transport (IFT), as driven by heterotrimeric Kinesin-2, is required. Results show that opsin can enter and move along the primary cilium of a nonphotoreceptor cell (an hTERT-RPE1 epithelial cell), suggesting that it can co-opt the basic anterograde motor system of cilia. Fluorescence recovery after photobleaching analysis of cilia of hTERT-RPE1 cells showed that the movement of ciliary opsin was comparable to that of the IFT protein, IFT88. Moreover, the movement of opsin in these cilia, as well as in cilia of mouse rod photoreceptor cells, was reduced significantly when KIF3A, the obligate motor subunit of heterotrimeric Kinesin-2, was deficient. These studies therefore provide evidence from live-cell analysis that the conserved heterotrimeric Kinesin-2 is required for the normal transport of opsin along the ciliary plasma membrane.
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dysfunction of heterotrimeric Kinesin 2 in rod photoreceptor cells and the role of opsin mislocalization in rapid cell death
Molecular Biology of the Cell, 2010Co-Authors: David Jimeno, David S Williams, Vanda S Lopes, Kornnika Khanobdee, Xiaodan Song, Bryan Chen, Steven NusinowitzAbstract:: Due to extensive elaboration of the photoreceptor cilium to form the outer segment, axonemal transport (IFT) in photoreceptors is extraordinarily busy, and retinal degeneration is a component of many ciliopathies. Functional loss of heterotrimeric Kinesin-2, a major anterograde IFT motor, causes mislocalized opsin, followed by rapid cell death. Here, we have analyzed the nature of protein mislocalization and the requirements for the death of Kinesin-2-mutant rod photoreceptors. Quantitative immuno EM showed that opsin accumulates initially within the inner segment, and then in the plasma membrane. The light-activated movement of arrestin to the outer segment is also impaired, but this defect likely results secondarily from binding to mislocalized opsin. Unlike some other retinal degenerations, neither opsin-arrestin complexes nor photoactivation were necessary for cell loss. In contrast, reduced rod opsin expression provided enhanced rod and cone photoreceptor survival and function, as measured by photoreceptor cell counts, apoptosis assays, and ERG analysis. The cell death incurred by loss of Kinesin-2 function was almost completely negated by Rho⁻/⁻. Our results indicate that mislocalization of opsin is a major cause of photoreceptor cell death from Kinesin-2 dysfunction and demonstrate the importance of accumulating mislocalized protein per se, rather than specific signaling properties of opsin, stemming from photoactivation or arrestin binding.
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analysis of Kinesin 2 function in photoreceptor cells using synchronous cre loxp knockout of kif3a with rho cre
Investigative Ophthalmology & Visual Science, 2006Co-Authors: David Jimeno, Concepcion Lillo, Lawrence S B Goldstein, Leonard Feiner, Karen Teofilo, Eric A Pierce, David S WilliamsAbstract:Many genes that are important in ocular function and disease have been studied with traditional gene-targeting strategies in the mouse. However, some genes that function in the eye are also critical for normal development and homeostasis of the animal and are thus not amenable to simple knockout techniques.1,2 The development of conditional knockout strategies has enabled the study of these other genes. The use of CRE and other recombinases for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development.3,4 In a previous study, we used Cre-loxP mutagenesis to test for motor transport by Kinesin-2 in photoreceptor cells.5 Vertebrate photoreceptor cells include two distal compartments: an inner segment, which contains much of the cellular machinery, and an outer segment, which is a specialized sensory cilium dedicated to phototransduction. The outer segment is linked to the inner segment by a connecting cilium, which is analogous to the transition zone of a primary cilium.6 Traffick-ing between the inner and outer segments occurs along the connecting cilium and the axoneme of the outer segment and is essential for the function and viability of the cells. Large amounts of phototransductive proteins, including the visual receptor, opsin, are transported in an anterograde direction as part of the continuous renewal of the outer segment.7 Moreover, at least three proteins, arrestin, transducin, and recoverin, redistribute between the inner and outer segments according to ambient lighting.8-14 Kinesin-2 is a likely candidate to provide motor transport along the connecting cilium and axoneme of photoreceptor cells, based on its role in the movement of proteins along cilia and flagella (“intraflagellar transport”) in a variety of organisms, from single cell flagellates to mammals.15,16 Moreover, the motor subunits of Kinesin-2, KIF3A, and KIF3B, have been detected in the photoreceptor connecting cilium.17-20 In the previous study, mice were generated in which a region of the Kif3a gene was flanked by loxP sites and thus could be excised in the presence of CRE. CRE was introduced into the photoreceptor cells by way of an IRBP-Cre transgene, whose expression was restricted primarily to the photoreceptor cells.5 With this strategy, excision of the Kif3a gene occurred in photoreceptor cells, beginning after the second post-natal week. The consequential removal of KIF3A from the photoreceptor cells not only perturbed the flow of protein to the outer segment, but also killed some of the photoreceptor cells. Although this study demonstrated a requirement for Kinesin-2 in photoreceptor cell protein transport and viability, gene excision was incomplete and asynchronous across each retina, and its extent varied among different animals, thus limiting the usefulness of this approach. In particular, these animals were not suitable for any type of biochemical study. In the present study, we first set out to establish a more robust expression of Cre—one that would effect widespread and synchronous recombination across the retina and thus would be more useful for the study of Kif3a and other genes in photoreceptor cells. We settled on a line of RHO-Cre transgenic mice that fulfills these criteria and have characterized the expression and effects of this transgene. We have also used this line to study further the requirement of KIF3A in photoreceptor cells, and especially the time course of the change in gene expression in relation to the ensuing effects on the photore-ceptor cells. Of note, we found that an abnormal accumulation of opsin is the primary cellular defect, occurring when all other aspects of cellular organization appear normal.
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Kinesin 2 and photoreceptor cell death requirement of motor subunits
Experimental Eye Research, 2006Co-Authors: David Jimeno, Concepcion Lillo, Elizabeth A Roberts, Lawrence S B Goldstein, David S WilliamsAbstract:Kinesin-2 function is essential for photoreceptor cell viability. The removal of one of the Kinesin-2 motor proteins, KIF3A, by photoreceptor-specific conditional mutagenesis, has been shown to cause rapid photoreceptor cell degeneration. We have explored the possibility that the genes encoding the Kinesin-2 motor proteins (KIF3A, KIF3B, and KIF3C)are linked to retinal disease, by examining retinas of knockout mice. We conclude that the reduced KIF3A and KIF3B in heterozygous animals, or the complete absence of KIF3C in homozygous animals does not affect photoreceptor cell survival. Photoreceptor cell death seems to be limited to conditions that, if systemic, are embryonic lethal, indicating that reduced function of the Kinesin-2 motor genes is unlikely to underlie inherited retinal degeneration.
