Minus Ends Of Microtubules Research Articles

The mechanism of Lis1-mediated regulation of dynein motility and force generation

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated transport towards the minus-ends of microtubules. Recent studies have shown that Lis1 facilitates formation of processive dynein/dynactin/cargo adaptor complexes, but it is not required for subsequent motility. When Lis1 remains bound to the complex, it substantially slows down dynein motility, but the underlying mechanism is not well understood. To address this question, we studied the motility and force generation of yeast cytoplasmic dynein in the presence of Lis1.

Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end.

Reconstitution of DDB-kinesin bidirectional transport in vitro

Robust bidirectional transport in neurons requires motors from the kinesin-1, 2 and 3 superfamilies, which move toward microtubule plus-ends, and cytoplasmic dynein, which moves toward microtubule minus-ends. While the motility parameters of single kinesin and dynein motors, such as stall force, rates of attachment and detachment, and force-velocity relationships, are becoming well-understood, it remains unclear how these characteristics coalesce in teams of motors working cooperatively or antagonistically to transport cargo.

Preference of camsap3 to expanded microtubules contributes to minus end stabilization

CAMSAPs (calmodulin-regulated spectrin-associated proteins) are a family of microtubule binding proteins (MAPs) that have functions in the tracking, decoration and stabilization of the microtubule minus end. Recently, CAMSAPs have been also suggested to act as microtubule nucleators, highlighting their importance in microtubule network organization. Nevertheless, little is known about the molecular basis that allows CAMSAPs to stabilize/nucleate microtubules. Recent structural studies have shown that microtubules can take diverse conformations depending on their binding states with nucleotides, proteins and chemical agents.

Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors.

PurposeCytoplasmic dynein-1 (henceforth dynein) moves cargo in conjunction with dynactin toward the minus end of microtubules. The dynein heavy chain, DYNC1H1, comprises the backbone of dynein, a retrograde motor. Deletion of Dync1h1 abrogates dynein function. The purpose of this communication is to demonstrate effects of photoreceptor dynein inactivation during late postnatal development and in adult retina.MethodsWe mated Dync1h1F/F mice with iCre75 and Prom1-CreERT2 mice to generate conditional rod and tamoxifen-induced knockout in rods and cones, respectively. We documented retina degeneration with confocal microscopy at postnatal day (P) 10 to P30 for the iCre75 line and 1 to 4 weeks post tamoxifen induction (wPTI) for the Prom1-CreERT2 line. We performed scotopic and photopic electroretinography (ERG) at P16 to P30 in the iCre75 line and at 1-week increments in the Prom1-CreERT2 line. Results were evaluated statistically using Student's t-test, two-factor ANOVA, and Welch's ANOVA.ResultsCre-induced homologous recombination of Dync1h1F/F mice truncated DYNC1H1 after exon 23. rodDync1h1−/− photoreceptors degenerated after P14, reducing outer nuclear layer (ONL) thickness and combined inner segment/outer segment (IS/OS) length significantly by P18. Scotopic ERG a-wave amplitudes decreased by P16 and were extinguished at P30. Cones were stable under rod-knockout conditions until P21 but inactive at P30. In tamDync1h1−/− photoreceptors, the IS/OS began shortening by 3wPTI and were nearly eliminated by 4wPTI. The ONL shrank significantly over this interval, indicating rapid photoreceptor degeneration following the loss of dynein.ConclusionsOur results demonstrate dynein is essential for the secretory pathway, formation of outer segments, and photoreceptor maintenance.

In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor.

Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. We used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. We therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. We also devised and validated a nanobody-based proximity biotinylation strategy that can be used to define the interactome of any GFP-tagged protein.

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15.

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule cross-linker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster.

Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. We identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. We propose that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.

Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

Cytoplasmic dynein is activated by the dynactin complex, cargo adapters and LIS1 (Lissencephaly 1). How this process is regulated invivo remains unclear. The dynein motor ring contains six AAA+ (ATPases associated with diverse cellular activities) domains. Here, we used the filamentous fungus Aspergillus nidulans to examine whether ATP hydrolysis at AAA3 regulates dynein activation in the context of other regulators. In fungal hyphae, early endosomes undergo dynein-mediated movement away from the microtubule plus ends near the hyphal tip. Dynein normally accumulates at the microtubule plus ends. The early endosomal adaptor Hook protein, together with dynactin, drives dynein activation to cause its relocation to the microtubule minus ends. This activation process depends on LIS1, but LIS1 tends to dissociate from dynein after its activation. In this study, we found that dynein containing a mutation-blocking ATP hydrolysis at AAA3 can undergo LIS1-independent activation, consistent with our genetic data that the same mutation suppresses the growth defect of the A.nidulans LIS1-deletion mutant. Our data also suggest that blocking AAA3 ATP hydrolysis allows dynein activation by dynactin without the early endosomal adaptor. As a consequence, dynein accumulates at microtubule minus ends whereas early endosomes stay near the plus ends. Dynein containing a mutation-blocking ATP binding at AAA3 largely depends on LIS1 for activation, but this mutation abnormally prevents LIS1 dissociation upon dynein activation. Together, our data suggest that the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

Modeling processive motion of kinesin‐13 MCAK and kinesin‐14 Cik1‐Kar3 molecular motors

Kinesin-13 MCAK, which is composed of two identical motor domains, can undergo unbiased one-dimensional diffusion on microtubules. Kinesin-14 Cik1-Kar3, which is composed of a Kar3 motor domain and a Cik1 motor homology domain with no ATPase activity, can move processively toward the minus end of microtubules. Here, we present a model for the diffusion of MCAK homodimer and a model for the processive motion of Cik1-Kar3 heterodimer. Although the two dimeric motors show different domain composition, in the models it is proposed that the two motors use the similar physical mechanism to move processively. With the models, the dynamics of the two dimers is studied analytically. The theoretical results for MCAK reproduce quantitatively the available experimental data about diffusion constant and lifetime of the motor bound to microtubule in different nucleotide states. The theoretical results for Cik1-Kar3 reproduce quantitatively the available experimental data about load dependence of velocity and explain consistently the available experimental data about effects of the exchange and mutation of the motor homology domain on the velocity of the heterodimer. Moreover, predicted results for other aspects of the dynamics of the two dimers are provided.

Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.

Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization

Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C.elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex ɣ-tubulin ring complex (γ-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.

Atomistic molecular dynamics simulations of tubulin heterodimers explain the motion of a microtubule

Microtubules are essential parts of the cytoskeleton that are built by polymerization of tubulin heterodimers into a hollow tube. Regardless that their structures and functions have been comprehensively investigated in a modern soft matter, it is unclear how properties of tubulin heterodimer influence and promote the self-assembly. A detailed knowledge of such structural mechanisms would be helpful in drug design against neurodegenerative diseases, cancer, diabetes etc. In this work atomistic molecular dynamics simulations were used to investigate the fundamental dynamics of tubulin heterodimers in a sheet and a short microtubule utilizing well-equilibrated structures. The breathing motions of the tubulin heterodimers during assembly show that the movement at the lateral interface between heterodimers (wobbling) dominates in the lattice. The simulations of the protofilament curvature agrees well with recently published experimental data, showing curved protofilaments at polymerization of the microtubule plus end. The tubulin heterodimers exposed at the microtubule minus end were less curved and displayed altered interactions at the site of sheet closure around the outmost heterodimers, which may slow heterodimer binding and polymerization, providing a potential explanation for the limited dynamics observed at the minus end.

A release-and-capture mechanism generates an essential non-centrosomal microtubule array during tube budding

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

Mud binds the kinesin-14 Ncd in Drosophila

Maintenance of proper mitotic spindle structure is necessary for error-free chromosome segregation and cell division. Spindle assembly is controlled by force-generating kinesin motors that contribute to its geometry and bipolarity, and balancing motor-dependent forces between opposing kinesins is critical to the integrity of this process. Non-claret dysjunctional (Ncd), a Drosophila kinesin-14 member, crosslinks and slides microtubule minus-ends to focus spindle poles and sustain bipolarity. However, mechanisms that regulate Ncd activity during mitosis are underappreciated. Here, we identify Mushroom body defect (Mud), the fly ortholog of human NuMA, as a direct Ncd binding partner. We demonstrate this interaction involves a short coiled-coil domain within Mud (MudCC) binding the N-terminal, non-motor microtubule-binding domain of Ncd (NcdnMBD). We further show that the C-terminal ATPase motor domain of Ncd (NcdCTm) directly interacts with NcdnMBD as well. Mud binding competes against this self-association and also increases NcdnMBD microtubule binding in vitro. Our results describe an interaction between two spindle-associated proteins and suggest a potentially new mode of minus-end motor protein regulation at mitotic spindle poles.

Structure and Mechanics of Dynein Motors.

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.

Microtubule-associated proteins and motors required for ectopic microtubule array formation in Saccharomyces cerevisiae.

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

Reconstitution of the recombinant human γ-tubulin ring complex.

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.