RESUMEN
Differential stability of kinetochore-microtubule attachments at low versus high tension is critical for accurate chromosome segregation. Miller et al. find that a TOG domain microtubule-binding protein imparts intrinsic tension selectivity to kinetochore-microtubule attachments.
Asunto(s)
Segregación Cromosómica , Cinetocoros , Microtúbulos/genética , Huso Acromático/metabolismoRESUMEN
Dense packaging of genomic DNA is crucial for organismal survival, as DNA length always far exceeds the dimensions of the cells that contain it. Organisms, therefore, use sophisticated machineries to package their genomes. These systems range across kingdoms from a single ultra-powerful rotary motor that spools the DNA into a bacteriophage head, to hundreds of thousands of relatively weak molecular motors that coordinate the compaction of mitotic chromosomes in eukaryotic cells. Recent technological advances, such as DNA proximity-based sequencing approaches, polymer modelling and in vitro reconstitution of DNA loop extrusion, have shed light on the biological mechanisms driving DNA organization in different systems. Here, we discuss DNA packaging in bacteriophage, bacteria and eukaryotic cells, which, despite their extreme variation in size, structure and genomic content, all rely on the action of molecular motors to package their genomes.
RESUMEN
Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdc20/metabolismo , Cinetocoros/metabolismo , Activación Transcripcional , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas Cdc20/genética , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/metabolismoRESUMEN
Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.
Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Genes de Helminto , Técnicas Genéticas , Proteínas Fluorescentes Verdes/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Mutación , Proteolisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Distribución TisularRESUMEN
The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.
Asunto(s)
Núcleo Celular/genética , Complejo Dinactina/genética , Dineínas/genética , Microtúbulos/genética , Animales , Caenorhabditis elegans/genética , Centrosoma/metabolismo , Dineínas/química , Edición Génica , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Dominios Proteicos , Huso Acromático/genética , Tubulina (Proteína)/genética , Tirosina/genética , Cigoto/crecimiento & desarrollo , Cigoto/metabolismoRESUMEN
Förster Resonance Energy Transfer (FRET) is the phenomenon of non-radiative transfer of electronic excitations from a donor fluorophore to an acceptor, mediated by electronic dipole-dipole coupling. The transfer rate and, as a consequence, efficiency depend non-linearly on the distance between the donor and the acceptor. FRET efficiency can thus be used as an effective and accurate reporter of distance between two fluorophores and changes thereof. Over the last 50 years or so, FRET has been used as a spectroscopic ruler to measure conformations and conformational changes of biomolecules. More recently, FRET has been combined with microscopy, ultimately allowing measurement of FRET between a single donor and a single acceptor pair. In this review, we will explain the physical foundations of FRET and how FRET can be applied to biomolecules. We will highlight the power of the different FRET approaches by focusing on its application to the motor protein kinesin, which undergoes several conformational changes driven by enzymatic action, that ultimately result in unidirectional motion along microtubule filaments, driving active transport in the cell. Single-molecule and ensemble FRET studies of different aspects of kinesin have provided numerous insights into the complex chemomechanical mechanism of this fascinating protein.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Cinesinas/análisis , Cinesinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Humanos , Microtúbulos/metabolismoRESUMEN
The KMN (Knl1/Mis12/Ndc80) network at the kinetochore, primarily known for its role in chromosome segregation, has been shown to be repurposed during neurodevelopment. Here, we investigate the underlying neuronal mechanism and show that the KMN network promotes the proper axonal organization within the C. elegans head nervous system. Postmitotic degradation of KNL-1, which acts as a scaffold for signaling and has microtubule-binding activities at the kinetochore, led to disorganized ganglia and aberrant placement and organization of axons in the nerve ring - an interconnected axonal network. Through gene-replacement approaches, we demonstrate that the signaling motifs within KNL-1, responsible for recruiting protein phosphatase 1, and activating the spindle assembly checkpoint are required for neurodevelopment. Interestingly, while the microtubule-binding activity is crucial to KMN's neuronal function, microtubule dynamics and organization were unaffected in the absence of KNL-1. Instead, the NDC-80 microtubule-binding mutant displayed notable defects in axon bundling during nerve ring formation, indicating its role in facilitating axon-axon contacts. Overall, these findings provide evidence for a noncanonical role for the KMN network in shaping the structure and connectivity of the nervous system in C. elegans during brain development.
Asunto(s)
Axones , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cinetocoros , Proteínas Asociadas a Microtúbulos , Microtúbulos , Neuronas , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Axones/metabolismo , Axones/fisiología , Cinetocoros/metabolismo , Neuronas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Sistema Nervioso/metabolismo , Huso Acromático/metabolismo , Proteínas del Citoesqueleto/metabolismo , Segregación Cromosómica , Transducción de SeñalRESUMEN
Dendrites are intricately designed neuronal compartments that play a vital role in the gathering and processing of sensory or synaptic inputs. Their diverse and elaborate structures are distinct features of neuronal organization and function. Central to the generation of these dendritic arbors is the neuronal cytoskeleton. In this review, we delve into the current progress toward our understanding of how dendrite arbors are generated and maintained, focusing on the role of the actin and microtubule cytoskeleton.
Asunto(s)
Actinas , Dendritas , Microtúbulos , Citoesqueleto/fisiología , NeuronasRESUMEN
Multiple microtubule-directed activities concentrate on chromosomes during mitosis to ensure their accurate distribution to daughter cells. These activities include couplers and dynamics regulators localized at the kinetochore, the specialized microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and to mitotic chromatin. Here, we describe an in vivo reconstruction approach in which the effect of removing the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. This approach revealed that the kinetochore dynein module, comprised of the minus end-directed motor cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes and to remodel outer kinetochore composition following microtubule attachment; by contrast, the kinetochore dynein module is unable to support chromosome congression. The chromosome-autonomous action of kinetochore dynein, in the absence of the other major microtubule-directed factors on chromosomes, rotates and orients a substantial proportion of chromosomes such that their sister chromatids attach to opposite spindle poles. In tight coupling with orientation, the kinetochore dynein module drives removal of outermost kinetochore components, including the dynein motor itself and spindle checkpoint activators. The removal is independent of the other major microtubule-directed activities and kinetochore-localized protein phosphatase 1, suggesting that it is intrinsic to the kinetochore dynein module. These observations indicate that the kinetochore dynein module has the ability coordinate chromosome biorientation with attachment state-sensitive remodeling of the outer kinetochore that facilitates cell cycle progression.
RESUMEN
During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules.1,2 One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin.3 Here, we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-CHCP-4 act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-CHCP-4 resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore.4,5 This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans: centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.
Asunto(s)
Proteínas de Caenorhabditis elegans , Cinetocoros , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis , Proteínas de Complejo Poro Nuclear/metabolismo , Huso Acromático/metabolismoRESUMEN
Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Morfogénesis , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , MitosisRESUMEN
One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.
Asunto(s)
Microscopía Fluorescente/métodos , Nanotecnología/métodos , Fluorescencia , Colorantes Fluorescentes/químicaRESUMEN
Cilia are built and maintained by intraflagellar transport (IFT), driving IFT trains back and forth along the ciliary axoneme. How IFT brings about the intricate ciliary structure and how this structure affects IFT are not well understood. We identify, using single-molecule super-resolution imaging of IFT components in living C. elegans, ciliary subdomains, enabling correlation of IFT-train dynamics to ciliary ultra-structure. In the transition zone, IFT dynamics are impaired, resulting in frequent pauses. At the ciliary base and tip, IFT trains show intriguing turnaround dynamics. Surprisingly, deletion of IFT motor kinesin-II not only affects IFT-train dynamics but also alters ciliary structure. Super-resolution imaging in these mutant animals suggests that the arrangement of IFT trains with respect to the axonemal microtubules is different than in wild-type animals. Our results reveal a complex, mutual interplay between ciliary ultrastructure and IFT-train dynamics, highlighting the importance of physical cues in the control of IFT dynamics.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cilios/ultraestructura , Imagen Individual de Molécula/métodos , Animales , Transporte BiológicoRESUMEN
The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.
Asunto(s)
Caenorhabditis elegans/embriología , División Celular , Técnicas Citológicas/métodos , Embrión no Mamífero/citología , Alelos , Animales , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , Ingeniería Genética , Microtúbulos/metabolismo , Mutación/genética , Interferencia de ARN , Reproducibilidad de los Resultados , TransgenesRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/metabolismo , Chlamydomonas/metabolismo , Cilios/metabolismo , Flagelos/metabolismo , Animales , Axonema/metabolismo , Axonema/ultraestructura , Cuerpos Basales/metabolismo , Cuerpos Basales/ultraestructura , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Chlamydomonas/genética , Chlamydomonas/ultraestructura , Cilios/ultraestructura , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Flagelos/ultraestructura , Regulación de la Expresión Génica , Cinesinas/química , Cinesinas/genética , Cinesinas/metabolismo , Multimerización de Proteína , Transducción de Señal , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. Whereas cytoplasmic dynein 1 has been widely studied, IFT dynein has received far less attention. Here, we use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. We find that while the kinesin composition of motor ensembles varies along the track, the amount of dynein remains relatively constant. Remarkably, this does not result in directionality changes of cargo along the track, as has been reported for other opposite-polarity, tug-of-war motility systems. At the single-molecule level, IFT-dynein trajectories reveal unexpected dynamics, including diffusion at the base, and pausing and directional switches along the cilium. Stochastic simulations show that the ensemble IFT-dynein distribution depends upon the probability of single-motor directional switches. Our results provide quantitative insight into IFT-dynein dynamics in vivo, shedding light on the complex functioning of dynein motors in general.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Imagen Individual de Molécula , Animales , Transporte Biológico , Proteínas Fluorescentes Verdes/metabolismo , Cinesinas/metabolismo , Modelos Biológicos , Procesos EstocásticosRESUMEN
During cell division, genome inheritance is orchestrated by microtubule attachments formed at kinetochores of mitotic chromosomes. The primary microtubule coupler at the kinetochore, the Ndc80 complex, is regulated by Aurora kinase phosphorylation of its N-terminal tail. Dephosphorylation is proposed to stabilize kinetochore-microtubule attachments by strengthening electrostatic interactions of the tail with the microtubule lattice. Here, we show that removal of the Ndc80 tail, which compromises in vitro microtubule binding, has no effect on kinetochore-microtubule attachments in the Caenorhabditis elegans embryo. Despite this, preventing Aurora phosphorylation of the tail results in prematurely stable attachments that restrain spindle elongation. This premature stabilization requires the conserved microtubule-binding Ska complex, which enriches at attachment sites prior to anaphase onset to dampen chromosome motion. We propose that Ndc80-tail dephosphorylation promotes stabilization of kinetochore-microtubule attachments via the Ska complex and that this mechanism ensures accurate segregation by constraining chromosome motion following biorientation on the spindle.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Anafase , Animales , Cromosomas/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Eliminación de Gen , Complejos Multiproteicos/química , Fosforilación , Unión Proteica , Polos del Huso/metabolismoRESUMEN
Dynamic processes are ubiquitous and essential in living cells. To properly understand these processes, it is imperative to measure them in a time-dependent way and analyze the resulting data quantitatively, preferably with automated tools. Kymographs are single images that represent the motion of dynamic processes and are widely used in live-cell imaging. Although they contain the full range of dynamics, it is not straightforward to extract this quantitative information in a reliable way. Here we present two complementary, publicly available software tools, KymographClear and KymographDirect, that have the power to reveal detailed insight in dynamic processes. KymographClear is a macro toolset for ImageJ to generate kymographs that provides automatic color coding of the different directions of movement. KymographDirect is a stand-alone tool to extract quantitative information from kymographs obtained from a wide range of dynamic processes in an automated way, with high accuracy and reliability. We discuss the concepts behind these software tools, validate them using simulated data, and test them on experimental data. We show that these tools can be used to extract motility parameters from a diverse set of cell-biological experiments in an automated and user-friendly way.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Movimiento Celular , Procesamiento de Imagen Asistido por Computador/instrumentación , Simulación de Dinámica Molecular , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Inside the cell, vital processes such as cell division and intracellular transport are driven by the concerted action of different molecular motor proteins. In C. elegans chemosensory cilia, 2 kinesin-2 family motor proteins, kinesin-II and OSM-3, team up to drive intraflagellar transport (IFT) in the anterograde direction, from base to tip, whereas IFT dynein hitchhikes toward the tip and subsequently drives IFT in the opposite, retrograde direction, thereby recycling both kinesins. While it is evident that at least a retrograde and an anterograde motor are necessary to drive IFT, it has remained puzzling why 2 same-polarity kinesins are employed. Recently, we addressed this question by combining advanced genome-engineering tools with ultrasensitive, quantitative fluorescence microscopy to study IFT with single-molecule sensitivity.(1,2) Using this combination of approaches, we uncovered a differentiation in kinesin-2 function, in which the slower kinesin-II operates as an 'importer', loading IFT trains into the cilium before gradually handing them over to the faster OSM-3. OSM-3 subsequently acts as a long-range 'transporter', driving the IFT trains toward the tip. The two kinesin-2 motors combine their unique motility properties to achieve something neither motor can achieve on its own; that is to optimize the amount of cargo inside the cilium. In this commentary, we provide detailed insight into the rationale behind our research approach and comment on our recent findings. Moreover, we discuss the role of IFT dynein and provide an outlook on future studies.
RESUMEN
A study of kinesin-1 has shed new light on how motor proteins are able to move along microtubules inside cells.