Search

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly. [ABSTRACT FROM AUTHOR]

Kinesin is a motor protein, comprised of two heavy and two light chains that transports cargo along the cytoskeletal microtubule filament network. The heavy chain has a neck domain connecting the ATPase motor head responsible for walking along microtubules, with the stalk and subsequent tail domains that bind cargo. The neck domain consists of a coiled coli homodimer with about five heptad repeats, preceded by a linker region that joins to the ATPase head. Here we report 1H, 15N, and 13C NMR assignments and a solution structure for the kinesin neck domain from rat isoform Kif5c. The calculation of the NMR structure of the homodimer was facilitated by unambiguously assigning sidechain NOEs between heptad a and d positions to interchain contacts, since these positions are too far apart to give sidechain contacts in the monomers. The dimeric coiled coil NMR structure is similar to the previously described X-ray structure, whereas the linker region is disordered in solution but contains a short segment with β-strand propensity— the β-linker. Only the coiled coil is protected from solvent exchange, with ∆G values for hydrogen exchange on the order of 4–6 kcal/mol. The high stability of the hydrogen-bonded α-helical structure makes it unlikely that unzippering of the coiled coil is involved in kinesin walking. Rather, the linker region serves as a flexible hinge between the kinesin head and neck. [ABSTRACT FROM AUTHOR]

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that, during entry into dendrites, axonally destined cargos move with a retrograde bias toward the cell body, while dendritically destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysine-rich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axons-dendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates.

Neuronal migration is a critical step during the formation of functional neural circuits in the brain. Newborn neurons need to move across long distances from the germinal zone to their individual sites of function; during their migration, they must often squeeze their large, stiff nuclei, against strong mechanical stresses, through narrow spaces in developing brain tissue. Recent studies have clarified how actomyosin and microtubule motors generate mechanical forces in specific subcellular compartments and synergistically drive nuclear translocation in neurons. On the other hand, the mechanical properties of the surrounding tissues also contribute to their function as an adhesive support for cytoskeletal force transmission, while they also serve as a physical barrier to nuclear translocation. In this review, we discuss recent studies on nuclear migration in developing neurons, from both cell and mechanobiological viewpoints.

Abstract Background Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Results Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. Conclusions The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Neurons are incredibly reliant on their cytoskeletal transport machinery. During development the cytoskeleton is the primary driver of growth and remodelling. In mature neurons the cytoskeleton keeps all components in a constant state of movement, allowing both supply of newly synthesized proteins to distal locations as well as the removal of aging proteins and organelles for recycling or degradation. This process is most challenging within axons as large distances need to be covered between synthesis and degradation, but it is essential as the lifetime of any single protein is much shorter than the lifetime of the neuron and its synapses. However, the transport machinery itself also has to be actively transported, recycled and degraded in order to localise properly and perform within neurons. This review provides an overview of the lifecycle of cytoskeletal components in neurons, focusing on its spatial organisation over time in the axon. [ABSTRACT FROM AUTHOR]

In the present report, we used highly elongated Drosophila bristle cells to dissect the role of dynein heavy chain (Dhc64C) in Golgi organization. We demonstrated that whereas in the bristle "somal" region Golgi units are composed of cis-, medial, and trans-Golgi compartments (“complete Golgi”), the bristle shaft contains Golgi satellites that lack the trans-Golgi compartment (hereafter referred to as “incomplete Golgi”) and which are static and localized at the base area. However, in Dhc64C mutants, the entire bristle shaft was filled with complete Golgi units containing ectopic trans-Golgi components. To further understand Golgi bristle organization, we tested the roles of microtubule (MT) polarity and the Dhc-opposing motor, kinesin heavy chain (Khc). For our surprise, we found that in Khc and Ik2Dominant-negative (DN) flies in which the polarized organization of MTs is affected, the bristle shaft was filled with complete Golgi, similarly to what is seen in Dhc64C flies. Thus, we demonstrated that MTs and the motor proteins Dhc and Khc are required for bristle Golgi organization. However, the fact that both Dhc64C and Khc flies showed similar Golgi defects calls for an additional work to elucidate the molecular mechanism describing why these factors are required for bristle Golgi organization. [ABSTRACT FROM AUTHOR]

Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia in humans and several domestic animal species. Typical clinical findings are chronic recurrent infections of the respiratory tract and fertility problems. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by PCD. The parents were unaffected suggesting autosomal recessive inheritance. Linkage and homozygosity mapping defined critical intervals comprising ~118 Mb. Whole genome sequencing of one case and comparison to 601 control genomes identified a disease associated frameshift variant, c.43delA, in the NME5 gene encoding a sparsely characterized protein associated with ciliary function. Nme5-/- knockout mice exhibit doming of the skull, hydrocephalus and sperm flagellar defects. The genotypes at NME5:c.43delA showed the expected co-segregation with the phenotype in the Alaskan Malamute family. An additional unrelated Alaskan Malamute with PCD and hydrocephalus that became available later in the study was also homozygous mutant at the NME5:c.43delA variant. The mutant allele was not present in more than 1000 control dogs from different breeds. Immunohistochemistry demonstrated absence of the NME5 protein from nasal epithelia of an affected dog. We therefore propose NME5:c.43delA as the most likely candidate causative variant for PCD in Alaskan Malamutes. These findings enable genetic testing to avoid the unintentional breeding of affected dogs in the future. Furthermore, the results of this study identify NME5 as a novel candidate gene for unsolved human PCD and/or hydrocephalus cases. [ABSTRACT FROM AUTHOR]

The mitotic spindle has long been known to play a crucial role in mitosis, orchestrating the segregation of chromosomes into two daughter cells during mitosis with high fidelity. Intracellular forces generated by the mitotic spindle are increasingly well understood, and recent work has revealed that the efficiency and the accuracy of mitosis is ensured by the scaling of mitotic spindle size with cell size. However, the role of the spindle in cancer progression has largely been ignored. Two recent studies point toward the role of mitotic spindle evolution in cancer progression through extracellular force generation. Cancer cells with lengthened spindles exhibit highly increased metastatic potential. Further, interpolar spindle elongation drives protrusive extracellular force generation along the mitotic axis to allow mitotic elongation, a morphological change that is required for cell division. Together, these findings open a new research area studying the role of the mitotic spindle evolution in cancer metastasis. [ABSTRACT FROM AUTHOR]

Immunological synapse (IS) formation between a T cell and an antigen-presenting cell is accompanied by the reorientation of the T cell centrosome toward the interface. This polarization response is thought to enhance the specificity of T cell effector function by enabling the directional secretion of cytokines and cytotoxic factors toward the antigen-presenting cell. Centrosome reorientation is controlled by polarized signaling through diacylglycerol (DAG) and protein kinase C (PKC). This drives the recruitment of the motor protein dynein to the IS, where it pulls on microtubules to reorient the centrosome. Here, we used T cell receptor photoactivation and imaging methodology to investigate the mechanisms controlling dynein accumulation at the synapse. Our results revealed a remarkable spatiotemporal correlation between dynein recruitment to the synaptic membrane and the depletion of cortical filamentous actin (F-actin) from the same region, suggesting that the two events were causally related. Consistent with this hypothesis, we found that pharmacological disruption of F-actin dynamics in T cells impaired both dynein accumulation and centrosome reorientation. DAG and PKC signaling were necessary for synaptic F-actin clearance and dynein accumulation, while calcium signaling and microtubules were dispensable for both responses. Taken together, these data provide mechanistic insight into the polarization of cytoskeletal regulators and highlight the close coordination between microtubule and F-actin architecture at the IS. [ABSTRACT FROM AUTHOR]

We report the development and characterization of a method, named reversible association with motor proteins (RAMP), for manipulation of organelle positioning within the cytoplasm. RAMP consists of coexpressing in cultured cells (i) an organellar protein fused to the streptavidin-binding peptide (SBP) and (ii) motor, neck, and coiled-coil domains from a plus-end–directed or minus-end–directed kinesin fused to streptavidin. The SBP–streptavidin interaction drives accumulation of organelles at the plus or minus end of microtubules, respectively. Importantly, competition of the streptavidin–SBP interaction by the addition of biotin to the culture medium rapidly dissociates the motor construct from the organelle, allowing restoration of normal patterns of organelle transport and distribution. A distinctive feature of this method is that organelles initially accumulate at either end of the microtubule network in the initial state and are subsequently released from this accumulation, allowing analyses of the movement of a synchronized population of organelles by endogenous motors. [ABSTRACT FROM AUTHOR]

LDC3/Dynarrestin, an aminothiazole derivative, is a recently developed small molecule, which binds protein tyrosine phosphatase interacting protein 51 (PTPIP51). PTPIP51 interacts with various proteins regulating different signaling pathways leading to proliferation and migration. Her2 positive breast cancer cells (SKBR3) express high levels of PTPIP51. Therefore, we investigated the effects of LDC3/Dynarrestin on PTPIP51 and its interactome with 12 different proteins of various signal pathways including the interaction with dynein in SKBR3 cells. The localization and semi-quantification of PTPIP51 protein and the Tyr176 phosphorylated PTPIP51 protein were evaluated. Protein-protein-interactions were assessed by Duolink proximity ligation assays. Interactions and the activation of signal transduction hubs were examined with immunoblots. LDC3/Dynarrestin led to an increased PTPIP51 tyrosine 176 phosphorylation status while the overall amount of PTPIP51 remained unaffected. These findings are paralleled by an enhanced interaction of PTPIP51 with its crucial kinase c-Src and a reduced interaction with the counteracting phosphatase PTP1B. Furthermore, the treatment results in a significantly augmented interaction of PTPIP51/14-3-3β and PTPIP51/Raf1, the link to the MAPK pathway. Under the influence of LDC3/Dynarrestin, the activity of the MAPK pathway rose in a concentration-dependent manner as indicated by RTK assays and immunoblots. The novel small molecule stabilizes the RelA/IκB/PTPIP51 interactome and can abolish the effects caused by TNFα stimulation. Moreover, LDC3/Dynarrestin completely blocked the Akt signaling, which is essential for tumor growth. The data were compared to the recently described interactome of PTPIP51 in LDC3/Dynarrestin treated non-cancerous keratinocyte cells (HaCaT). Differences were identified exclusively for the mitochondrial-associated ER-membranes (MAM) interactions and phospho-regulation related interactome of PTPIP51.LDC3/Dynarrestin gives the opportunity/possibility to influence the MAPK signaling, NFkB signaling and probably calcium homeostasis in breast cancer cells by affecting the PTPIP51 interactome. [ABSTRACT FROM AUTHOR]

Whole cell responses involve multiple subcellular processes (SCPs). To understand how balance between SCPs controls the dynamics of whole cell responses we studied neurite outgrowth in rat primary cortical neurons in culture. We used a combination of dynamical models and experiments to understand the conditions that permitted growth at a specified velocity and when aberrant growth could lead to the formation of dystrophic bulbs. We hypothesized that dystrophic bulb formation is due to quantitative imbalances between SCPs. Simulations predict redundancies between lower level sibling SCPs within each type of high level SCP. In contrast, higher level SCPs, such as vesicle transport and exocytosis or microtubule growth characteristic of each type need to be strictly coordinated with each other and imbalances result in stalling of neurite outgrowth. From these simulations, we predicted the effect of changing the activities of SCPs involved in vesicle exocytosis or microtubule growth could lead to formation of dystrophic bulbs. siRNA ablation experiments verified these predictions. We conclude that whole cell dynamics requires balance between the higher-level SCPs involved and imbalances can terminate whole cell responses such as neurite outgrowth. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein-1 (hereafter dynein) is an essential cellular motor that drives the movement of diverse cargos along the microtubule cytoskeleton, including organelles, vesicles and RNAs. A longstanding question is how a single form of dynein can be adapted to a wide range of cellular functions in both interphase and mitosis. Recent progress has provided new insights - dynein interacts with a group of activating adaptors that provide cargo-specific and/or function-specific regulation of the motor complex. Activating adaptors such as BICD2 and Hook1 enhance the stability of the complex that dynein forms with its required activator dynactin, leading to highly processive motility toward the microtubule minus end. Furthermore, activating adaptors mediate specific interactions of the motor complex with cargos such as Rab6-positive vesicles or ribonucleoprotein particles for BICD2, and signaling endosomes for Hook1. In this Cell Science at a Glance article and accompanying poster, we highlight the conserved structural features found in dynein activators, the effects of these activators on biophysical parameters, such as motor velocity and stall force, and the specific intracellular functions they mediate. [ABSTRACT FROM AUTHOR]

Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of axonal regrowth, loss of dynein dramatically impaired axonal regeneration and also reduced injury-induced Schwann cell remodeling. Chimeras between wild type and dynein mutant embryos demonstrate that dynein function in neurons is sufficient to promote axonal regrowth. Finally, by simultaneously monitoring actin and microtubule dynamics in regenerating axons we find that dynein appears dispensable to initiate axonal regrowth, but is critical to stabilize microtubules, thereby sustaining axonal regeneration. These results reveal two previously unappreciated roles for dynein during peripheral nerve regeneration, initiating injury induced Schwann cell remodeling and stabilizing axonal microtubules to sustain axonal regrowth. [ABSTRACT FROM AUTHOR]

The nuclear division takes place in the daughter cell in the basidiomycetous budding yeast Cryptococcus neoformans. Unclustered kinetochores gradually cluster and the nucleus moves to the daughter bud as cells enter mitosis. Here, we show that the evolutionarily conserved Aurora B kinase Ipl1 localizes to the nucleus upon the breakdown of the nuclear envelope during mitosis in C. neoformans. Ipl1 is shown to be required for timely breakdown of the nuclear envelope as well. Ipl1 is essential for viability and regulates structural integrity of microtubules. The compromised stability of cytoplasmic microtubules upon Ipl1 depletion results in a significant delay in kinetochore clustering and nuclear migration. By generating an in silico model of mitosis, we previously proposed that cytoplasmic microtubules and cortical dyneins promote atypical nuclear division in C. neoformans. Improving the previous in silico model by introducing additional parameters, here we predict that an effective cortical bias generated by cytosolic Bim1 and dynein regulates dynamics of kinetochore clustering and nuclear migration. Indeed, in vivo alterations of Bim1 or dynein cellular levels delay nuclear migration. Results from in silico model and localization dynamics by live cell imaging suggests that Ipl1 spatio-temporally influences Bim1 or/and dynein activity along with microtubule stability to ensure timely onset of nuclear division. Together, we propose that the timely breakdown of the nuclear envelope by Ipl1 allows its own nuclear entry that helps in spatio-temporal regulation of nuclear division during semi-open mitosis in C. neoformans. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) or kala-azar, the most severe form of leishmaniasis, can lead to death if not properly diagnosed and treated. Correct identification of infected patients and reservoirs is vital for controlling the spread of leishmaniasis. Current diagnostic kits for leishmaniasis show high sensitivity and specificity, but can also result in false negatives and cross reactions with related parasitic infections. New diagnostic methods with greater accuracy are urgently needed for diagnosis of leishmaniasis. In this study, we aimed to uncover a new highly effective antigen for the diagnosis of visceral leishmaniasis in dogs and humans, aiming to improve the accuracy compared with those of current methods of diagnosis. Initially, in-silico epitope prediction analyses identified several potential B-cell epitopes in the repetitive region of Leishmania infantum kinesin, which co-localized with predicted structural disordered regions, suggesting high potential for antigenicity. Based on this analysis, 8.5 genomic motifs, which encode the repetitive sequence of 39 degenerate amino acids, were selected for recombinant expression. BLASTn analysis of this repetitive region indicated that it is absent in the T. cruzi parasite, which is closely related to Leishmania, indicating the specificity of this region. This potentially antigenic protein, named recombinant kinesin degenerated derived repeat (rKDDR), was recombinantly expressed in Escherichia coli BL21-Star using the pET28a-TEV expression vector. We then evaluated the performance of rKDDR in correctly diagnosing Leishmania infection and compared this new assay with currently used diagnostic tests for leishmaniasis. rKDDR showed greater sensitivity and specificity in correctly diagnosing leishmaniasis both in human (sensitivity 92.86% and specificity 100%) and canine (sensitivity 88.54% and specificity 97.30%) sera compared with those of rK39 (human: sensitivity 90.48% and specificity 97.92%; canine: sensitivity 78.13% and specificity 90.09%). In addition, the rKDDR-ELISA outperformed the EIE-LVC kit, which is the serologic kit recommended by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis. These results indicate that rKDDR is a highly promising candidate for diagnosis of visceral leishmaniasis, and is more accurate than the currently used gold-standard antigens. [ABSTRACT FROM AUTHOR]

Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies. [ABSTRACT FROM AUTHOR]

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates. [ABSTRACT FROM AUTHOR]

Intracellular transport of organelles is fundamental to cell function and health. The mounting evidence suggests that this transport is in fact anomalous. However, the reasons for the anomaly is still under debate. We examined experimental trajectories of organelles inside a living cell and propose a mathematical model that describes the previously reported transition from sub-diffusive to super-diffusive motion. In order to explain super-diffusive behaviour at long times, we introduce non-Markovian detachment kinetics of the cargo: the rate of detachment is inversely proportional to the time since the last attachment. Recently, we observed the non-Markovian detachment rate experimentally in eukaryotic cells. Here we further discuss different scenarios of how this effective non-Markovian detachment rate could arise. The non-Markovian model is successful in simultaneously describing the time averaged variance (the time averaged mean squared displacement corrected for directed motion), the mean first passage time of trajectories and the multiple peaks observed in the distributions of cargo velocities. We argue that non-Markovian kinetics could be biologically beneficial compared to the Markovian kinetics commonly used for modelling, by increasing the average distance the cargoes travel when a microtubule is blocked by other filaments. In turn, sub-diffusion allows cargoes to reach neighbouring filaments with higher probability, which promotes active motion along the microtubules. [ABSTRACT FROM AUTHOR]

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly. [ABSTRACT FROM AUTHOR]

Widespread success of the intracellular bacterium Wolbachia across insects and nematodes is due to efficient vertical transmission and reproductive manipulations. Many strains, including wMel from Drosophila melanogaster, exhibit a specific concentration to the germplasm at the posterior pole of the mature oocyte, thereby ensuring high fidelity of parent-offspring transmission. Transport of Wolbachia to the pole relies on microtubules and the plus-end directed motor kinesin heavy chain (KHC). However, the mechanisms mediating Wolbachia’s association with KHC remain unknown. Here we show that reduced levels of the host canonical linker protein KLC results in dramatically increased levels of Wolbachia at the oocyte’s posterior, suggesting that KLC and some key associated host cargos outcompete Wolbachia for association with a limited amount of KHC motor proteins. Consistent with this interpretation, over-expression of KHC causes similarly increased levels of posteriorly localized Wolbachia. However, excess KHC has no effect on levels of Vasa, a germplasm component that also requires KHC for posterior localization. Thus, Wolbachia transport is uniquely KHC-limited because these bacteria are likely outcompeted for binding to KHC by some host cargo/linker complexes. These results reveal a novel host-symbiont interaction that underscores the precise regulation required for an intracellular bacterium to co-opt, but not disrupt, vital host processes. [ABSTRACT FROM AUTHOR]

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei undergoes life cycle form transitions from trypomastigotes to epimastigotes in the insect vector by re-positioning the mitochondrial genome and re-locating the flagellum and flagellum-associated cytoskeletal structures. The mechanism underlying these dramatic morphology transitions remains poorly understood. Here we report the regulatory role of the orphan kinesin KIN-E in controlling trypanosome morphology transitions. KIN-E localizes to the flagellum and is enriched at the flagellar tip, and this localization depends on the C-terminal m-calpain domain III-like domains. Depletion of KIN-E in the trypomastigote form of T. brucei causes major morphology changes and a gradual increase in the level of EP procyclin, generating epimastigote-like cells. Mechanistically, through its C-terminal importin α-like domain, KIN-E targets FLAM3, a flagellar protein involved in morphology transitions, to the flagellum to promote elongation of the flagellum attachment zone and positioning of the flagellum and flagellum-associated cytoskeletal structure, thereby maintaining trypomastigote cell morphology. Our findings suggest that morphology transitions in trypanosomes require KIN-E-mediated transport of FLAM3 to the flagellum. [ABSTRACT FROM AUTHOR]

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based “assisted-diffusion” behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a behavior of potential importance for physiological cargoes as well. [ABSTRACT FROM AUTHOR]

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth. [ABSTRACT FROM AUTHOR]

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10–/–mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10–/–mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1. [ABSTRACT FROM AUTHOR]

Protein-protein interactions (PPIs) formed between short linear motifs and globular domains play important roles in many regulatory and signaling processes but are highly underrepresented in current protein-protein interaction databases. These types of interactions are usually characterized by a specific binding motif that captures the key amino acids shared among the interaction partners. However, the computational proteome-level identification of interaction partners based on the known motif is hindered by the huge number of randomly occurring matches from which biologically relevant motif hits need to be extracted. In this work, we established a novel bioinformatic filtering protocol to efficiently explore interaction network of a hub protein. We introduced a novel measure that enabled the optimization of the elements and parameter settings of the pipeline which was built from multiple sequence-based prediction methods. In addition, data collected from PPI databases and evolutionary analyses were also incorporated to further increase the biological relevance of the identified motif hits. The approach was applied to the dynein light chain LC8, a ubiquitous eukaryotic hub protein that has been suggested to be involved in motor-related functions as well as promoting the dimerization of various proteins by recognizing linear motifs in its partners. From the list of putative binding motifs collected by our protocol, several novel peptides were experimentally verified to bind LC8. Altogether 71 potential new motif instances were identified. The expanded list of LC8 binding partners revealed the evolutionary plasticity of binding partners despite the highly conserved binding interface. In addition, it also highlighted a novel, conserved function of LC8 in the upstream regulation of the Hippo signaling pathway. Beyond the LC8 system, our work also provides general guidelines that can be applied to explore the interaction network of other linear motif binding proteins or protein domains. [ABSTRACT FROM AUTHOR]

Kinesin1 plays a major role in neuronal transport by recruiting many different cargos through its kinesin light chain (KLC). Various structurally unrelated cargos interact with the conserved tetratricopeptide repeat (TPR) domain of KLC. The N-terminal capping helix of the TPR domain exhibits an atypical sequence and structural features that may contribute to the versatility of the TPR domain to bind different cargos. We determined crystal structures of the TPR domain of both KLC1 and KLC2 encompassing the N-terminal capping helix and show that this helix exhibits two distinct and defined orientations relative to the rest of the TPR domain. Such a difference in orientation gives rise, at the N-terminal part of the groove, to the formation of one hydrophobic pocket, as well as to electrostatic variations at the groove surface. We present a comprehensive structural analysis of available KLC1/2-TPR domain structures that highlights that ligand binding into the groove can be specific of one or the other N-terminal capping helix orientations. Further, structural analysis reveals that the N-terminal capping helix is always involved in crystal packing contacts, especially in a TPR1:TPR1’ contact which highlights its propensity to be a protein–protein interaction site. Together, these results underline that the structural plasticity of the N-terminal capping helix might represent a structural determinant for TPR domain structural versatility in cargo binding. [ABSTRACT FROM AUTHOR]

Motor proteins generally have a two-way coupling between the ATP hydrolysis site, the lever movement and the binding affinity for their track, which allows them to perform efficient stepping. Here we explore the minimal requirements for directed motility based on simpler schemes in which the binding/unbinding from the track is decoupled from the ATPase cycle. We show that a directed power stroke alone is not sufficient for motility, but combined with an asymmetry in force-induced unbinding rates it can generate stepping. The energetic efficiency of such stepping is limited to approximately 20%. We conclude that the allosteric coupling between the ATP hydrolysis and the track binding is not strictly necessary for motility, but it greatly improves its efficiency. [ABSTRACT FROM AUTHOR]

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein is a giant ATP-driven molecular motor that proceeds to the minus end of the microtubule (MT). Dynein hydrolyzes ATP in a ring-like structure, containing 6 AAA+ (ATPases associated with diverse cellular activities) modules, which is ~15 nm away from the MT binding domain (MTBD). This architecture implies that long-distance allosteric couplings exist between the AAA+ ring and the MTBD in order for dynein to move on the MT, although little is known about the mechanisms involved. Here, we have performed comprehensive molecular simulations of the dynein motor domain based on pre- and post- power-stroke structural information and in doing so we address the allosteric conformational changes that occur during the power-stroke and recovery-stroke processes. In the power-stroke process, the N-terminal linker movement was the prerequisite to the nucleotide-dependent AAA1 transition, from which a transition cascade propagated, on average, in a circular manner on the AAA+ ring until it reached the AAA6/C-terminal module. The recovery-stroke process was initiated by the transition of the AAA6/C-terminal, from which the transition cascade split into the two directions of the AAA+ ring, occurring both clockwise and anti-clockwise. In both processes, the MTBD conformational change was regulated by the AAA4 module and the AAA5/Strut module. (201 words< 300 words) [ABSTRACT FROM AUTHOR]

Dynactin is a dynein-regulating protein that increases the processivity of dynein movement on microtubules. Recent studies have shown that a tripartite complex of dynein–dynactin–Bicaudal D2 is essential for highly processive movement. To elucidate the regulation of dynein motility by dynactin, we focused on two isoforms (A and B) of dynactin 1 (DCTN1), the largest subunit of dynactin that contains both microtubule- and dynein-binding domains. The only difference between the primary structures of the two isoforms is that DCTN1B lacks the K-rich domain, a cluster of basic residues. We measured dynein motility by single molecule observation of recombinant dynein and dynactin. Whereas the tripartite complex containing DCTN1A exhibited highly processive movement, the complex containing DCTN1B dissociated from microtubules with no apparent processive movement. This inhibitory effect of DCTN1B was caused by reductions of the microtubule-binding affinities of both dynein and dynactin, which was attributed to the coiled-coil 1 domain of DCTN1. In DCTN1A, the K-rich domain antagonized these inhibitory effects. Therefore, dynactin has two antagonistic domains and promotes or suppresses dynein motility to accomplish correct localization and functions of dynein within a cell. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Kif5A is a neuronally-enriched isoform of the Kinesin-1 family of cellular transport motors. 23 separate mutations in the motor domain of Kif5A have been identified in patients with the complicated form of hereditary spastic paraplegia (HSP). We performed in vitro assays on dimeric recombinant Kif5A with HSP-causing mutations in the Switch I domain, which participates in the coordination and hydrolysis of ATP by kinesin. We observed a variety of significantly reduced catalytic and mechanical activities as a result of each mutation, with the shared phenotype from each that motility was significantly reduced. Substitution of Mn2+ for Mg2+ in our reaction buffers provides a dose-dependent rescue in both the catalytic and ensemble mechanical properties of the S203C mutant. This work provides mechanistic insight into the cause of HSP in patients with these mutations and points to future experiments to further dissect the root cause of this disease. [ABSTRACT FROM AUTHOR]

We have used an insertional mutagenesis approach to generate new C. reinhardtii motility mutants. Of 56 mutants isolated, one is a new allele at the ODA3 locus, called oda3-6. Similar to the previously characterized oda3 alleles, oda3-6 has a slow-jerky swimming phenotype and reduced swimming speed. The oda3-6 mutant fails to assemble the outer dynein arm motor and outer dynein arm—docking complex (ODA-DC) in the ciliary axoneme due to an insertion in the 5’ end of the DCC1 gene, which encodes the DC1 subunit of the ODA-DC. Transformation of oda3-6 with the wild-type DCC1 gene rescues the mutant swimming phenotype and restores assembly of the ODA-DC and the outer dynein arm in the cilium. This is the first oda3 mutant to be characterized at the molecular level and is likely to be very useful for further analysis of DC1 function. [ABSTRACT FROM AUTHOR]

Intraflagellar transport (IFT) particles or trains are composed of IFT-A and IFT-B complexes. To assess the working mechanism of the IFT-A complex in IFT and ciliogenesis, we have analyzed ift43 mutants of Chlamydomnonas in conjunction with mutants of the other IFT-A subunits. An ift43 null mutant or a mutant with a partial deletion of the IFT43 conserved domain has no or short flagella. The mutants accumulate not only IFT-B but also IFT-Ain the short flagella, which is in contrast to an ift140 null mutant. The IFT43 conserved domain is necessary and sufficient for the function of IFT43. IFT43 directly interacts with IFT121 and loss of IFT43 results in instability of IFT-A. A construct with a partial deletion of the IFT43 conserved domain is sufficient to rescue the instability phenotype of IFT-A, but results in diminishing of IFT-A at the peri-basal body region. We have further provided evidence for the direct interactions within the IFT-A complex and shown that the integrity of IFT-A is important for its stability and cellular localization. Finally, we show that both IFT43 and IFT140 are involved in mobilizing ciliary precursors from the cytoplasmic pool during flagellar regeneration, suggesting a novel role of IFT-A in transporting ciliary components in the cytoplasm to the peri-basal body region. [ABSTRACT FROM AUTHOR]

Background: Numerous studies have shown Kinesin family member 20A (KIF20A) may play a critical role in the development and progression of cancer. However, the clinical value of KIF20A in nasopharyngeal carcinoma (NPC) is unknown. Here, we investigated the expression pattern of KIF20A in NPC and its correlation with clinicopathological features of patients. Methods: Real-time PCR and Western blotting were used to quantify KIF20A expression in NPC cell lines and clinical specimens compared with normal controls. KIF20A protein expression was also examined in archived paraffin embedded tumor samples from 105 patients with pathologically confirmed NPC by immunohistochemistry (IHC). Statistical analyses were applied to assess the associations between KIF20A expression and the clinicopathological features and survival outcomes. Effects on migration and invasion were assessed by wound healing and transwell invasion assays after KIF20A silencing. Results: KIF20A was significantly overexpressed at both the mRNA and protein levels in NPC cell lines and human tumor tissues. 45/105 (42.9%) of NPC specimens expressed high levels of KIF20A among the KIF20A detectable cases. Statistical analysis revealed that high KIF20A expression was significantly associated with gender (P = 0.046), clinical stage (P<0.001), T category (P = 0.022), N category (P<0.001), distant metastasis (P = 0.001) and vital status (P = 0.001). Moreover, Higher KIF20A expression patients had shorter overall survival (OS) and progression-free survival (PFS) (P = 0.001 and P = 0.001; log-rank test). In multivariate analysis, KIF20A was an independent prognostic factor for OS and PFS in the entire cohort (P = 0.033, P = 0.008). Knock down of KIF20A expression significantly suppressed NPC cell’s migration and invasion. Conclusions: KIF20A is overexpressed and may serve as an independent prognostic biomarker in NPC. Targeting KIF20A reduces migration and invasion of NPC cells. [ABSTRACT FROM AUTHOR]

Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo-?or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1. [ABSTRACT FROM AUTHOR]

Kymographs or space-time plots are widely used in cell biology to reduce the dimensions of a time-series in microscopy for both qualitative and quantitative insight into spatio-temporal dynamics. While multiple tools for image kymography have been described before, quantification remains largely manual. Here, we describe a novel software tool for automated multi-peak tracking kymography (AMTraK), which uses peak information and distance minimization to track and automatically quantify kymographs, integrated in a GUI. The program takes fluorescence time-series data as an input and tracks contours in the kymographs based on intensity and gradient peaks. By integrating a branch-point detection method, it can be used to identify merging and splitting events of tracks, important in separation and coalescence events. In tests with synthetic images, we demonstrate sub-pixel positional accuracy of the program. We test the program by quantifying sub-cellular dynamics in rod-shaped bacteria, microtubule (MT) transport and vesicle dynamics. A time-series of E. coli cell division with labeled nucleoid DNA is used to identify the time-point and rate at which the nucleoid segregates. The mean velocity of microtubule (MT) gliding motility due to a recombinant kinesin motor is estimated as 0.5 μm/s, in agreement with published values, and comparable to estimates using software for nanometer precision filament-tracking. We proceed to employ AMTraK to analyze previously published time-series microscopy data where kymographs had been manually quantified: clathrin polymerization kinetics during vesicle formation and anterograde and retrograde transport in axons. AMTraK analysis not only reproduces the reported parameters, it also provides an objective and automated method for reproducible analysis of kymographs from in vitro and in vivo fluorescence microscopy time-series of sub-cellular dynamics. [ABSTRACT FROM AUTHOR]

Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as ‘FPKATD’ and ‘Y/FTNEKL’ without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids’ pattern in different proteins. [ABSTRACT FROM AUTHOR]

In this study we provide evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor/dASCIZ. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. We also confirm a previously documented function of Digitor/dASCIZ as a regulator of Dynein light chain/Cut up expression. Using transgenic expression of a mCitrine-labeled Digitor construct, we show that Digitor/dASCIZ is a nuclear protein that is localized to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggest the possibility that Digitor/dASCIZ plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, we have characterized a P-element insertion that is likely to be a true null Digitor/dASCIZ allele resulting in complete pupal lethality when homozygous, indicating that Digitor/dASCIZ is an essential gene. Phenotypic analysis of the mutant provided evidence that Digitor/dASCIZ plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor/dASCIZ null mutant larvae there was greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor/dASCIZ decreased the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. We show that an early developmental consequence of the absence of Digitor/dASCIZ is reduced third instar larval brain size although overall larval development appeared otherwise normal at this stage. While Digitor/dASCIZ mutant larvae initiate pupation, all mutant pupae failed to eclose and exhibited various defects in metamorphosis such as impaired differentiation, incomplete disc eversion, and faulty apoptosis. Altogether we provide evidence that Digitor/dASCIZ is a nuclear protein that performs multiple roles in Drosophila larval and pupal development. [ABSTRACT FROM AUTHOR]

Intracellular transport is an essential function in eucaryotic cells, facilitated by motor proteins—proteins converting chemical energy into kinetic energy. It is understood that motor proteins work in teams enabling unidirectional and bidirectional transport of intracellular cargo over long distances. Disruptions of the underlying transport mechanisms, often caused by mutations that alter single motor characteristics, are known to cause neurodegenerative diseases. For example, phosphorylation of kinesin motor domain at the serine residue is implicated in Huntington’s disease, with a recent study of phosphorylated and phosphomimetic serine residues indicating lowered single motor stalling forces. In this article we report the effects of mutations of this nature on transport properties of cargo carried by multiple wild-type and mutant motors. Results indicate that mutants with altered stall forces might determine the average velocity and run-length even when they are outnumbered by wild type motors in the ensemble. It is shown that mutants gain a competitive advantage and lead to an increase in the expected run-length when the load on the cargo is in the vicinity of the mutant’s stalling force or a multiple of its stalling force. A separate contribution of this article is the development of a semi-analytic method to analyze transport of cargo by multiple motors of multiple types. The technique determines transition rates between various relative configurations of motors carrying the cargo using the transition rates between various absolute configurations. This enables a computation of biologically relevant quantities like average velocity and run-length without resorting to Monte Carlo simulations. It can also be used to introduce alterations of various single motor parameters to model a mutation and to deduce effects of such alterations on the transport of a common cargo by multiple motors. Our method is easily implementable and we provide a software package for general use. [ABSTRACT FROM AUTHOR]

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα–LGN–NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation. [ABSTRACT FROM AUTHOR]

Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased ‘search-and-capture’ mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process. The model is used to optimize the spatial field of drift in simulations, by comparison to experimental motility statistics. In a second step, this optimized gradient is used to determine the location of immobilized dynein motors and MT polymerization parameters, since these are hypothesized to generate the gradient of forces needed to move MTOCs. We compare these scenarios to self-organized mechanisms by which asters have been hypothesized to find the cell-center- MT pushing at the cell-boundary and clustering motor complexes. By minimizing the error between simulation outputs and experiments, we find a model of “pulling” by a gradient of dynein motors alone can drive the centripetal motility. Interestingly, models of passive MT based “pushing” at the cortex, clustering by cross-linking motors and MT-dynamic instability gradients alone, by themselves do not result in the observed motility. The model predicts the sensitivity of the results to motor density and stall force, but not MTs per aster. A hybrid model combining a chromatin-centered immobilized dynein gradient, diffusible minus-end directed clustering motors and pushing at the cell cortex, is required to comprehensively explain the available data. The model makes experimentally testable predictions of a spatial bias and self-organized mechanisms by which MT asters can find the center of a large cell. [ABSTRACT FROM AUTHOR]

A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. [ABSTRACT FROM AUTHOR]