RESUMO
Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. We investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in kinesin light chains (klc-1 and klc-2) and motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocities in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whilst anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whilst the klc single mutant life-span was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two KLCs playing distinct roles.
RESUMO
Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.
Assuntos
Proteínas de Ciclo Celular , Dineínas do Citoplasma , Complexo Dinactina , Proteínas Associadas aos Microtúbulos , Animais , Cálcio/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
The endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed an analysis workflow for dynamics of established tubules in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active dynamics were observed. Clear differences in dynamic behaviour were observed for established tubules at different positions within the cell using itemset mining. We found that tubules with activity-driven fluctuations were more likely to be located away from the cell periphery and a population of peripheral tubules with no signs of active motion was found.
Assuntos
Retículo Endoplasmático/fisiologia , Fibroblastos/fisiologia , Pulmão/fisiologia , Microtúbulos/fisiologia , Animais , Chlorocebus aethiops , Fibroblastos/citologia , Humanos , Pulmão/citologia , Células VeroRESUMO
In this paper, we formulate the space-dependent variable-order fractional master equation to model clustering of particles, organelles, inside living cells. We find its solution in the long-time limit describing non-uniform distribution due to a space-dependent fractional exponent. In the continuous space limit, the solution of this fractional master equation is found to be exactly the same as the space-dependent variable-order fractional diffusion equation. In addition, we show that the clustering of lysosomes, an essential organelle for healthy functioning of mammalian cells, exhibit space-dependent fractional exponents. Furthermore, we demonstrate that the non-uniform distribution of lysosomes in living cells is accurately described by the asymptotic solution of the space-dependent variable-order fractional master equation. Finally, Monte Carlo simulations of the fractional master equation validate our analytical solution. This article is part of the theme issue 'Transport phenomena in complex systems (part 1)'.
Assuntos
Lisossomos , Animais , Análise por Conglomerados , DifusãoRESUMO
Intracellular transport is predominantly heterogeneous in both time and space, exhibiting varying non-Brownian behavior. Characterization of this movement through averaging methods over an ensemble of trajectories or over the course of a single trajectory often fails to capture this heterogeneity. Here, we developed a deep learning feedforward neural network trained on fractional Brownian motion, providing a novel, accurate and efficient method for resolving heterogeneous behavior of intracellular transport in space and time. The neural network requires significantly fewer data points compared to established methods. This enables robust estimation of Hurst exponents for very short time series data, making possible direct, dynamic segmentation and analysis of experimental tracks of rapidly moving cellular structures such as endosomes and lysosomes. By using this analysis, fractional Brownian motion with a stochastic Hurst exponent was used to interpret, for the first time, anomalous intracellular dynamics, revealing unexpected differences in behavior between closely related endocytic organelles.
Assuntos
Fenômenos Bioquímicos/fisiologia , Transporte Biológico/fisiologia , Movimento/fisiologia , Redes Neurais de Computação , Vesículas Transportadoras/metabolismo , Humanos , Modelos Biológicos , Movimento (Física)RESUMO
Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the ARF activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.
Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Polimerização , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/química , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Membrana/química , Camundongos , Células NIH 3T3 , Peptídeos/metabolismo , Domínios Proteicos , Pseudópodes/metabolismoRESUMO
The endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 µm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 µm, and provides the tubule's persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.
Assuntos
Retículo Endoplasmático/metabolismo , Imagem Molecular , Biomarcadores , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Microscopia de Fluorescência , Imagem Molecular/métodosRESUMO
Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Genes Supressores de Tumor , Cinesinas/fisiologia , Células HeLa , HumanosRESUMO
Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/fisiologia , Receptores ErbB/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Nexinas de Classificação/metabolismo , Vesículas Transportadoras/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/genéticaRESUMO
Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.
Assuntos
Dineínas do Citoplasma/metabolismo , Cinesinas/antagonistas & inibidores , Mitose/fisiologia , Fuso Acromático/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Centríolos/fisiologia , Dineínas do Citoplasma/genética , Complexo Dinactina , Feminino , Células HEK293 , Células HeLa , Humanos , Cinetocoros , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno , Fuso Acromático/genética , Xenopus laevisRESUMO
Particle tracking experiments with high speed digital microscopy yield the positions and trajectories of lipid droplets inside living cells. Angular correlation analysis shows that the lipid droplets have uncorrelated motion at short time scales (τ < 1 ms) followed by anti-persistent motion for lag times in the range of 1 ⩽ τ ⩽ 10 ms. The angular correlation at longer time scales, τ > 10 ms, becomes persistent, indicating directed movement. The motion at all time scales is associated with the lipid droplets being tethered to and driven along the microtubule network. The point at which the angular correlation changes from anti-persistent to persistent motion corresponds to the cross over between sub-diffusive and super diffusive motion, as observed by mean square displacement analysis. Correct analysis of the angular correlations of the detector noise is found to be crucial in modelling the observed phenomena.
Assuntos
Lipídeos/análise , Microtúbulos/metabolismo , Modelos Biológicos , Movimento (Física) , Algoritmos , Linhagem Celular , Difusão , Humanos , Metabolismo dos Lipídeos , Microscopia , ProbabilidadeRESUMO
The first-passage-probability can be used as an unbiased method for determining the phases of motion of individual organelles within live cells. Using high speed microscopy, we observe individual lipid droplet tracks and analyze the motor protein driven motion. At short passage lengths (<10(-2)µm), a log-normal distribution in the first-passage-probability as a function of time is observed, which switches to a Gaussian distribution at longer passages due to the running motion of the motor proteins. The mean first-passage times (
Assuntos
Modelos Biológicos , Transporte Biológico Ativo , Sobrevivência Celular , ProbabilidadeRESUMO
Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85-98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150(Glued) to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150(Glued) remain membrane-bound. Long dynein-dependent movements occur via bursts at up to â¼8 µms(-1) that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.
Assuntos
Dineínas/metabolismo , Endossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Complexo Dinactina , Dineínas/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Reação em Cadeia da Polimerase , Interferência de RNA , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
The organization and function of eukaryotic cells rely on the action of many different molecular motor proteins. Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules, and these events are needed, not just at the single-cell level, but are vital for correct development. In the present paper, I review recent progress on understanding dynein's mechanochemistry, how it is regulated and how it binds to such a plethora of cargoes. The importance of a number of accessory factors in these processes is discussed.
Assuntos
Dineínas do Citoplasma/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Transporte Biológico/fisiologia , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The first passage probability (FPP), of trafficked intracellular particles reaching a displacement L, in a given time t or inverse velocity S = t/L, can be calculated robustly from measured particle tracks. The FPP gives a measure of particle movement in which different types of motion, e.g. diffusion, ballistic motion, and transient run-rest motion, can readily be distinguished in a single graph, and compared with mathematical models. The FPP is attractive in that it offers a means of reducing the data in the measured tracks, without making assumptions about the mechanism of motion. For example, it does not employ smoothing, segmentation or arbitrary thresholds to discriminate between different types of motion in a particle track. In contrast to conventional mean square displacement analysis, FPP is sensitive to a small population of trafficked particles that move long distances (> or = 5 microm), which are thought to be crucial for efficient long range signaling in theories of network dynamics. Taking experimental data from tracked endocytic vesicles, and calculating the FPP, we see how molecular treatments affect the trafficking. We show the FPP can quantify complicated movement which is neither completely random nor completely deterministic, making it highly applicable to trafficked particles in cell biology.
Assuntos
Modelos Teóricos , Algoritmos , Difusão , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Probabilidade , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Organelas/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mitose , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that approximately 50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.
Assuntos
Dineínas/fisiologia , Retículo Endoplasmático/fisiologia , Cinesinas/fisiologia , Movimento/fisiologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Citoplasma/metabolismo , Corrente Citoplasmática/fisiologia , Complexo Dinactina , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Células VeroRESUMO
The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.
Assuntos
Dineínas/metabolismo , Dineínas/fisiologia , Endossomos/metabolismo , Morfogênese , Endossomos/fisiologia , Fator de Crescimento Epidérmico/farmacocinética , Células HeLa , Humanos , Microinjeções , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Transporte Proteico , Receptores da Transferrina/metabolismo , Transdução de Sinais , Transfecção , Transferrina/farmacocinética , Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.